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We focused on the role of Uric Acid (UA) as a possible determinant of Heart Failure (HF) related issues in Acute Coronary Syndromes (ACS) patients. Main outcomes were acute HF and cardiogenic shock at admission, secondary outcomes were the use of Non Invasive Ventilation (NIV) and the admission Left Ventricular Ejection Fraction (LVEF). We consecutively enrolled 1269 ACS patients admitted to the cardiological Intensive Care Unit of the Niguarda and San Paolo hospitals (Milan, Italy) from June 2016 to June 2019. Median age was 68 (first-third quartile 59–77) years and males were 970 (76%). All the evaluated outcomes occurred more frequently in the hyperuricemic subjects (UA higher than 6 mg/dL for females and 7 mg/dL for males, n = 292): acute HF 35.8 vs 11.1% (p < 0.0001), cardiogenic shock 10 vs 3.1% (p < 0.0001), NIV 24.1 vs 5.1% (p < 0.0001) and lower admission LVEF (42.9±12.8 vs 49.6±9.9, p < 0.0001). By multivariable analyses, UA was confirmed to be significantly associated with all the outcomes with the following Odds Ratio (OR): acute HF OR = 1.119; 95% CI 1.019;1.229; cardiogenic shock OR = 1.157; 95% CI 1.001;1.337; NIV use OR = 1.208; 95% CI 1.078;1.354; LVEF β = -0.999; 95% CI -1.413;-0.586. We found a significant association between UA and acute HF, cardiogenic shock, NIV use and LVEF. Due to the cross-sectional nature of our study no definite answer on the direction of these relationship can be drawn and further longitudinal study on UA changes over time during an ACS hospitalization are needed.

The soccer athlete has a greater incidence of overuse and acute knee injuries and as a consequence is at most risk for chondropenia and osteoarthritis. Chondropenia conceptually defines the complex nature of the multivariable processes over time including acute and chronic injury, modulators and the aging process. The role of the Sports Medicine team is to prevent injury, restore the joint and ultimately return the athlete to sport while preventing Osteoarthritis. The clinical consequences of full thickness Articular Cartilage defects are pain, swelling, mechanical symptoms athletic and functional disability and osteoarthitis. In the soccer athlete it is articular cartilage that confers the highest levels of performance. It is the fact that any partial or full thickness loss results in loss of day to day resilience and a spectrum of soreness, stiffness, pain and swelling and most importantly. These injuries may be career- ending In this group of high demand participants any increase in activity and loading beyond the articular cartilage threshold for injury, results in a clinical overuse response with the potential negative adaptive consequences of chondropenia and an increased risk of developing osteoarthritis. Lesions to articular cartilage are common acutely but are associated with ACL injuries and long-term chondropenia and Osteoarthitis. The challenge of ACL injury reduction, articular cartilage repair and regeneration and osteoarthitis prevention in the soccer athlete continues to be significant despite significant recent advances. The purpose of this lecture is to present the current concepts with respect to the Athlete's Articular Cartilage the histological, biochemical and clinical implications and contemporary treatments from a historical and an evolutionary prospective as applied during or after their competitive years., Use and abuse of joints, as well as joint trauma can influence the development of osteoarthritis (OA). Soccer is a sport that combines high joint loading and a considerable risk for injuries, especially knee injuries. Since soccer is the most popular sports activity in the world with about 40 million participants it is a useful model for studying OA. An increased risk of hip and knee OA has been shown in former top level soccer players regardless of injuries. There has been much focus on sports related knee injuries and the risk of developing posttraumatic knee OA early in life after these injuries. A knee injury in sports is often associated with an anterior cruciate ligament (ACL) injury, and this injury has been studied extensively both concerning the short and long term effects. The incidence of an ACL injury is almost 1 injury per 1000 of the physical active population1. According to the national ACL registers in the Scandinavian countries some 50% of the injured patients will require an ACL reconstruction. In the US this figure is higher and up to 200 000 reconstructions are performed each year. The indications for surgical intervention are to ensure a return to preinjury activity level or to reduce the risk of osteoarthritis. However, there is not much scientific data supporting either the ability to return to sport or reducing the risk of OA with surgery. Many of the studies have an uncontrolled design and other methodological shortcomings making the interpretation difficult. Top level athletes are usually not taking part in the studies. On group level most studies on ACL reconstruction ends up with an activity level of 6–7 on the Tegner scale compared to a preinjury level of 9. A literature research on OA after ACL injury showed that regardless of treatment about 50% of the patients had radiographic OA 15 years after the injury2. However there is a wide range, from 10–90 %. Many studies point out that an associated meniscus tear increases the risk for OA. The meniscus problem is complex since it is not fully understood if the meniscus tear is entirely due to a trauma or if it is a part of an OA process. If it is a part of the OA process, it is not surprising that it is associated with a higher risk of OA. A reduced risk for secondary meniscus tears has been reported after ACL reconstruction compared to non reconstructed patients. This has also been suggested as an important factor in favour for surgery. However, the lowest prevalence of OA after ACL injury has been noted in a cohort of 100 non-surgically treated patients, and especially in the subgroup of patients without meniscus injury. At follow up more than 75 % had stayed with the non operative treatment3. The 22 who underwent surgery during the follow up had severe problems with instability or a repairable meniscus tear. In the whole group the activity level was 4.5 (median) after 15 years which is comparable to an uninjured population at the same age (45 years). In a recent report from the Danish ACL registry the activity level was 5 on the Tegner scale one year after ACL reconstruction. The mean age was below 30 years of age. Obviously the risk for OA differs among patients after an ACL injury. The risk seems to be influenced by the trauma itself. Compression forces differ depending on the type of trauma, and in a report from 1995 it was shown that ACL injury after soccer had more associated meniscus injuries than knees with an ACL injury after alpine skiing4. Many studies report that bone marrow edemas (BME) are associated with an ACL injury, depending on the MR technique in up to 90% of the cases. The BME may act as a footprint of the trauma. With quantified MR technique one study indicate that a greater trauma is associated with a lager BME. The study showed that a larger BME was related to compression fractures, mainly laterally located in the knee joint. Structural cartilage changes were also seen, and these changes were more pronounced in the surgically treated patients compared to non surgical treated5. With the contrast enhanced MR technique, there are data showing persistent GAG loss in the cartilage up to 2 years after an ACL injury, which verifies the results from studies on joint fluid markers6. A low GAG content could indicate that the matrix is not normalized and probably have a reduced capacity of load absorption, thus making it more vulnerable much longer after a trauma or a surgical procedure than was known previously. An early return to high demanding sports could in this perspective be quite harmful for the cartilage. A normal time frame at present, regarding return to sports after an ACL injury is 6 months, while these findings eventually indicate that the cartilage is not ready for high demanding sports until much later in the course after an injury. In the published studies with a lower prevalence of OA it seems to have been a very controlled rehabilitation or even a slow return to demanding activities. If there will be a fast return to sports activities in combination with persisting biomechanical abnormalities and neuromuscular deficits, this may strongly contribute to the OA risk after an ACL injury. Although it seems clear that some patients do benefit from an ACL reconstruction, there is no generally accepted treatment algorithm based on evidence from the literature. A problem is that the natural course is not known. There is a hope that the new technically demanding double bundle reconstruction shall be the solution. It seems like this technique will decrease the rotational instability compared to the traditional single bundle technique. This fact may not improve the patient satisfaction in the short run, but may, theoretically, have positive impact on the risk of OA. The main problem after an ACL injury certainly is the high risk of OA. It is known that females have an increased risk to sustain an ACL tear. This means that many of the young girls with ACL tears already as teenagers will have OA very early in life with well known consequences. There are possibilities to reduce the risk of OA after a sports related knee injury, the main solution today is probably not surgery. For the future it seems extremely important to continue the work on ACL injury prevention, but also to find methods for the identification of the patients at risk to develop early OA after such an injury. The increased understanding of the post trauma OA process and its determinants is of greatest importance to be able to find methods to reduce the risk., The field of cartilage repair suffers from a lack of randomized studies and thus a consensus on treatment or product efficacy. Unfortunately, while post-operative rehabilitation is recognized as a critical component for achieving outcome success in cartilage repair, it too suffers from little or no comparative data, suggesting that greater efforts are needed in studying rehabilitation concurrent with cartilage repair. This is particularly important for cartilage repair clinical trials being used to meet regulatory requirements, since regulatory agencies are now expecting to see standardized rehabilitation data in their submissions. Such standardization and control of rehabilitation offers unique challenges, particularly in multicenter studies of large or varied geographic scale. Therefore, the design of studies should make careful consideration in the choice, implementation and follow-up of post-operative rehabilitation. Physiotherapy has not been well-studied with regards to cartilage repair, and in some cases orthopaedic surgeons have developed their own preferred programs with little or no scientific basis. But for the most part, patients are relegated to follow programs established by the therapist themselves. Therefore, the standardization of the rehabilitation within a trial must begin with the selection of the appropriate therapeutic modalities and post-operative timeframe, such as the period of restricted weightbearing, or the use of continuous passive motion (CPM). Furthermore, clinical trials which compare cartilage repair therapies that differ in their repair mechanisms- as with an osteochondral graft device versus microfracture-might call for different programs altogether. Close consultation between the investigators and physiotherapists in designing the program is needed. Selection and training of physiotherapist is a critical step in successfully implementing a standardized study-wide rehabilitation program. International studies offer the challenge of multiple languages and may need to employ therapists with differing educational backgrounds including athletic, physical and rehabilitation therapists, for example, who have appropriate experience. Direct training and communication with all physiotherapists is critical, especially with regards to treatment, lesion size and location or other unique study components. Furthermore, the logistics and financial management of such a relationship with the clinic or hospital providing the rehabilitation service needs to be well established in advance of patient treatment. The objective of a solid standardized trial rehabilitation program should try to avoid the situation where compliance becomes a function of willingness or ability to pay. Patients should be well informed regarding the importance of compliance to both the health of their knee as well as the clinical study. Encouragement should come from the investigator, the clinical site as well as the recruited physiotherapists. A randomized clinical trial for cartilage repair is an excellent opportunity to also understand the influence of rehabilitation on repair outcomes. Even simple variables regarding the patient compliance, the progression of range of motion and weightbearing, could be informative as well as more complex data obtained from functional tests. The extent of the data and the method of collection should be established with the physiotherapists prior to treatment. Finally, consideration should be made as to the role that physiotherapy plays within a study protocol. Regulatory requirements have not been clear regarding the level of statistical analysis and correlation to treatment outcomes which must be carried out. This issue should be clarified-particularly if poor rehabilitation compliance is to be viewed as a protocol deviation, and negatively affects the final sample size in study analyses. The need for standardized rehabilitation in cartilage repair clinical trials which will support regulatory submissions now requires a more concerted effort to be put into the management and tracking of post-treatment care. A well-implemented program with experienced physiotherapists will certainly serve to normalize cartilage repair outcome data, while at the same time focusing more attention on the specific rehabilitation modalities currently used but for which there is little scientific support. And although regulatory rules do not explicitly describe the analyses required for rehabilitation data, any correlations to repair outcomes obtained during these regulated trials will bring valuable insight for the clinical practice of cartilage repair., Rehabilitation is generally accepted as being an important component of cartilage repair surgery. However, the degree to which the rehabilitation process has the potential to influence the outcome of cartilage repair surgery, either positively or negatively, has not been fully elucidated to date. Can the rehabilitation process influence the quality and composition of the cartilage repair tissue; optimise repair tissue maturation; maximise repair tissue durability; increase patient satisfaction; and/or facilitate faster return to work and activity for patients? These are just a selection of the many questions to be asked with a view to being answered. The primary goals of a cartilage repair rehabilitation programme are the local adaptation and remodelling of the repair tissue; a return to function; and the reduction in the risk of further degeneration in later life.[1] Cartilage repair tissue maturation is a long process that has been shown to take more than 18 months to be completed.[2] Consequently, the postoperative rehabilitation programme needs to balance getting each patient back to function as quickly as possible whilst respecting the time that it takes for the body to return to homeostasis and the repair tissue to adapt. The evolution of cartilage repair procedures over the last twenty years has resulted in an ever-increasing range of surgical procedures with chondral repair tissues that vary in the timescales needed to accept loading. Despite this variability in timescales each person undergoing cartilage repair progresses through the three rehabilitative phases of: protection, function and activity. These are not discrete phases and at any one point therapy may be split over more than one phase. Differing therapeutic interventions and modalities can be utilised to work with the patient to help them progress through the postoperative period of protection, to assist in the restoration of function and prepare and support the patient for a return to activity. Rehabilitation protocols are the frameworks that guide patients and their therapists in the formulation of the individual rehabilitation programme. Cartilage repair patients are a very heterogeneous group and postoperative rehabilitation programmes need to be individualised based on the surgical procedure, the nature and history of the lesion and the individual patient characteristics. The translation of emerging basic science to the clinical environment necessitates an understanding of the fundamental principles of the biology of healing, clinical biomechanics, exercise programming and cartilage maturation timescales. The challenge for the therapist is to apply these principles to construct an individualised rehabilitation programme that has a load bearing-mechanical match to the status of the repair tissue at any postoperative point in time. Factors that have been shown to influence the rehabilitation process and therefore need to be incorporated into the rehabilitation programme design process include: lesion size; lesion location; concomitant procedures; preoperative duration of symptoms; age; preoperative baseline condition; and individual patient motivation and goals. In considering the importance of the rehabilitation process to the overall outcome there are areas of controversy, a situation that is compounded by the historical poor reporting of rehabilitation in published studies on articular cartilage repair.[3] Graft delamination is frequently cited as one of the main concerns following autologous chondrocyte implantation (ACI) procedures and, where this has materialised, it is often anecdotally attributed in single case reports to over zealous rehabilitation and/or too early return to sports activity. Consequently, initial rehabilitation protocols tended to be focused on minimising the risk of graft delamination by placing restrictions on weight bearing and range of movement with the aim of protecting the graft from deleterious forces. This was reflected in rehabilitation protocols advising restrictions on weight bearing and ranges of movement that sometimes lasted in excess of 12 weeks. [1] However, the most common adverse events following ACI that are currently reported in the literature are not always graft failure and delamination. Recent studies have reported arthrofibrosis/joint adhesions and graft hypertrophy as being the most common serious adverse event.[4, 5] Additionally, the STAR study reported that the most frequent surgical intervention performed in the first 6 months after ACI was lysis of adhesions.[4] This relatively high incidence of arthrofibrosis and joint adhesions could well be at least partially attributed to postoperative rehabilitation guidance that was too restrictive regarding joint mobility. An additional consideration is that therapists with limited experience of working with cartilage repair patients may well take even more of a conservative approach in their implementation of the rehabilitation protocols. In 2008 Ebert et al. published the results of a study on a traditional vs. an accelerated approach to post-operative rehabilitation following matrix-induced autologous chondrocyte implantation (MACI).[6] The traditional approach entailed the first 6 postoperative weeks being non-weight bearing whereas the accelerated approach incorporated earlier partial weight bearing and a more graduated series of progressions to full weight bearing. One of the key findings of their study was that regardless of the rehabilitation protocol employed, no patient suffered any adverse effect to the implant. We have been indoctrinated by the ‘form follows functionÂ’ argument but how relevant is that to cartilage repair? Do chondrocytes need the biomechanical stimuli and if so in what form, over what time period, with what progressions? These are the questions that therapists are asking of basic science and where the therapists are looking for the translation of the science into the clinical arena. Lateral integration of the new cartilage repair tissue has been identified as a chronic problem in cartilage repair.[7] Physical demands need to be placed on the neocartilage repair tissue but there is a delicate balance between exposing the repair tissue to sufficient loading to stimulate chondral matrix production and minimising the exposure to levels and types of loading that could lead to mechanical failure or poor integration of the repair tissue. Recent research in an animal model has shown that rehabilitative joint motion in the form of continuous passive motion stimulates chondrocyte PRG4 metabolism.[8] However, it is not know whether rehabilitative joint motion in the form of active motion in vivo has similar effects on PRG4 metabolism. There is a higher incidence of graft hypertrophy following ACI in periosteal and patella ACI repairs. One explanation given for the patella repairs exhibiting higher levels of hypertrophy is that higher patellar shear forces provide stimuli for hypertrophy.[9] Minimisation of shear forces requires not only a precise knowledge of the repair location but also the ability to select and adapt rehabilitative exercises to suit the location of the repair and the joint arthrokinematics. [10] The counter argument that could be postulated is that early postoperative range of movement restrictions and reduced weight bearing could facilitate the graft hypertrophy. Finally, but by no means least, rehabilitation is important to the patients themselves. The regular input from therapists during the long rehabilitation process provides a valuable personal support mechanism. The therapist has an important role to fulfil in not only guiding (progressing and/or regressing) the rehabilitation but also in keeping the patient motivated and adherent to their rehabilitation throughout its duration. In conclusion, there is still limited direct evidence to support the importance of rehabilitation in cartilage repair but that is increasing. There is a need for further research to evaluate optimal rehabilitative practice for existing cartilage repair procedures and to determine the key factors that can be optimised to improve the overall results for current patient cohorts. Concurrently there is also a need to track new surgical developments to anticipate future rehabilitation requirements as issues such as whether or not mesenchymal stem cell based cartilage repairs will need different rehabilitation protocols to chondrocyte repairs emerge., ICRS Rehabilitation and Sports Committee Injuries of the articular cartilage surfaces of the knee are observed with increasing frequency in athletes. Particularly participation in pivoting sports such as football, basketball, and soccer has been associated with a rising number of sports-related articular cartilage injuries with higher injury rates at the competitive and professional level. Injuries of the articular cartilage surface of the knee in the athlete can often occur in association with other acute injuries such as ligament or meniscal injuries, traumatic patellar dislocations, and osteochondral injuries and have been described in up to 50% of athletes undergoing anterior cruciate ligament reconstruction. Besides acute traumatic injury, articular cartilage injury can develop in the high-impact athletic population from chronic pathologic joint loading patterns such as joint instability or axis deviation. While intact articular cartilage adjusts to the increasing weightbearing activity in athletes by increasing cartilage volume and thickness in a linear dose-response relationship, recent studies indicate that this dose-response curve reaches a threshold and that activity beyond this threshold can result in maladaptation and injury of articular cartilage. High-impact joint loading above this threshold has been shown to decrease cartilage protoglycan content and to increase levels of degradative enzymes and chondrocyte apoptosis. Over time the integrity of the functional weight bearing unit is lost and a chondropenic response is initiated that can include loss of articular cartilage volume and stiffness, elevation of contact pressures, and development or progression of articular cartilage defects. The limited spontaneous repair following acute or chronic articular cartilage injury is well documented. Recent reports demonstrated that hyaline cartilage defects in athletes resulted in significant pain and swelling and were associated with marked life-style changes and limitation of athletic activity. Some long-term data in athletes with isolated severe chondral or osteochondral damage in the weightbearing condyles showed a 75% initial return to sport initially, but a significant decline of athletic activity was observed over time with development of radiographic evidence of osteoarthritis in the 45–60% of athletes 14–34 years after the injury. These results are supported by the up to 12 fold increased risk of knee osteoarthritis in high-impact athletes established by the National Institute of Health (NIH) and other independent studies. Untreated articular cartilage defects have been shown to result in significantly worse long-term joint function. The high demands on the joint surfaces in athletes make treatment of articular cartilage injuries and restoration of the injured joint surfaces critically important to facilitate continued athletic participation and to maintain a physically active lifestyle. The documented detrimental effect of high-impact articular loading in the athletic population requires cartilage surface restoration that can effectively withstand the significant mechanical joint stresses generated during high-impact, pivoting sports. Besides reducing pain, increasing mobility and improving knee function, the ability to return the athlete to sport and to continue to perform at the pre-injury athletic level presents one of the most important parameters for a successful outcome from articular cartilage repair in this challenging population. Treatment of articular cartilage injuries in the athletic population has traditionally presented a significant therapeutic challenge. However, development of new surgical techniques has created considerable clinical and scientific enthusiasm for articular cartilage repair. Based on the source of the cartilage repair tissue, these new surgical techniques can generally be categorized into three groups: marrow stimulation based techniques, osteochondral transplantation techniques, and cell-based repair techniques. Several studies have evaluated the microfracture technique specifically in the athletic population. These studies included recreational and professional athletes with follow-up ranging from 2–6 years. Activity scores and knee function scores increased significantly after microfracture in these athletes. Athletes were able to successfully return to high-impact, pivoting sports including football, soccer, alpine skiing, basketball, rugby, and tennis. Return to sports was reported at an average of 6.5–10 months. However, there was marked variability in the ability to return to sport after microfracture. The ability to return to athletics at the preoperative level also varied significantly. Return to professional and competitive level sports was much better than to recreational athletics. A recent study showed that while return to sports participation after microfracture can be achieved rapidly, performance and playing time will increase gradually to full participation. Return to high-impact sports after microfracture has been found to be higher in younger athletes with small lesion size, shorter preoperative symptoms, and without prior surgical intervention. A decline of initial improvement of postoperative sports participation was observed in some studies after microfracture in athletes and occurred between 24–37 months, however, activity levels and functional scores were still better than at baseline. Osteochondral mosaicplasty has also been specifically evaluated in athletes. Up to 95% good or excellent results with improved functional scores and MRI rating have been reported. Return to full athletic activity was reported in 61–93% of athletes at an average of 6.5 months. Longer preoperative symptoms and increased athlete age resulted in delayed return to sport after mosaicplasty. Preoperative radiographic or clinical evidence of joint degeneration predicted a return to sport at a lower level or even retirement from competitive sports following mosaicplasty. Prospective randomized comparison of mosaicplasty and microfracture in athletes reported significantly better results with mosaicplasty at an average of 36 months. While studies have evaluated autologous osteochondral transfer in athletes, no specific information has been reported on this technique using allograft in this population. Autologous chondrocyte transplantation has recently been evaluated in the demanding athletic population. Good to excellent results were demonstrated in 72–96% with significant improvement of activity score. Best results were obtained with single cartilage lesions of the medial femoral condyle. Return to high impact-athletics was higher in younger, competitive athletes, with short preoperative intervals while return in recreational athletes was less predictable. The time to return to sport was shorter in competitive level athletes. Athletes often returned to the same skill level and a high portion of returning athletes maintained their ability to perform 52 months after chondrocyte implantation. Return to athletics was better with fewer prior surgeries but return to sports was successful also with autologous cartilage transplantation as a salvage procedure. Combined pathology such as malalignment, ligamentous instability, or meniscal injury and deficiency is frequently encountered by the surgeon treating articular cartilage defects in the athletic knee. Surgically addressing these concomitant pathologies is critical for an effective and durable articular cartilage repair. Recent data demonstrated that isolated or combined adjuvant procedures have no significant negative effect on the ability to return to athletics after microfracture, mosaicplasty, or autologous chondrocyte transplantation. In conclusion, articular cartilage repair in athletes is aimed at returning the athlete to the pre-injury level of athletic participation without increased risk for long-term arthritic degeneration. Several surgical techniques have been shown to improve function and athletic activity after articular cartilage repair in this population but the rate of improvement and ability to return to athletic activity is dependent on several factors. The choice of repair technique should be tailored to individual patient and lesion characteristics using an established treatment algorithm. Long-term studies in this population will determine the efficacy of articular cartilage repair to reverse chondropenia and to prevent development of secondary arthritic degeneration., Introduction Chondropathies of the acetabulum and the femoral head are a frequent cause of pain and functional limitation. The incidence of acetabular cartilage damage is estimated to be of 74% in a total of 736 hip arthroscopies. Furthermore there is an association between cartilage damage and lesions of the acetabular labrum in 81% of the cases. Currently, treatment of hip cartilage pathologies is based exclusively on arthroscopic debridement, microfractures, multiple femoral head perforations or fibrine glue injection for chondral delamination. The purpose of this study was to report the results obtained in treating hip chondropathies using the arthroscopic ACT or AMIC technique. A comparison between the two techniques and results was made to evaluate advantages and disadvantages of these two procedures, Materials and Methods A controlled retrospective randomized study was carried out on 182 patients affected by a hip chondropathy of 3rd and 4th degree, according to the Outerbridge classification, extended 2cm2 or more. 120 of these patients underwent arthroscopic autologous chondrocyte transplantation (ACT), while the other 62 underwent arthroscopic autologous matrix induced chondroplasty (AMIC). The surgical treatment, in those cases treated by ACT, was always carried out in two steps. The first was a diagnostic arthroscopy used to evaluate the chondral damage and to take a cartilage biopsy from the area surrounding the pulvinar. In the second step the transplant was implanted by arthroscopy. On the contrary the AMIC procedure was carried out as a one step procedure. Once the chondral defect was located, the area was cleaned and microfractures were performed. Than the collagen menbrane was applied to cover the defect. In that cases treated with the ACT procedure, the chondrocyte culture was carried out on a polymer scaffold, which is a reabsorbable composite material of polyglactin 910 and poly-p-dioxanone, in 65 cases and on a Hyaluronic acid scaffold in 55 cases. In all the cases treated with the AMIC procedure a suine collagen membrane, added with autologous growth factors, was applied to cover the chondral defect. The two groups were similar in age, gender, degree and location of the pathology. The mean follow-up was 23.8 months (36 to 12) in the group of patiets treated with the ACT procedure and 22.6 (36 to 12) in the group of patients treated with the AMIC procedure. The mean size of the defects was 2.6 cm2 (2.0 – 4.8) in the ACT group and 2.8 cm2 (2.0 – 5.0) in the AMIC group. All the patients were assessed before and after the procedure with the Harris Hip Score (HHS). Postoperatively all the patients underwent physiokinesitherapy Exercises began from the 1st postoperative day. Patients were discharged on the 2nd day and were subject to both active and passive physiotherapy to regain complete range of motion without putting any weight on the articulation for 4 weeks. Partial load was allowed after 4 weeks, when exercises on a gym bike and swimming were recommended. After 7 weeks, crutches were no longer required and the patients were allowed to return to normal work activity. Jogging was allowed only after 6 months, while a complete return to sports activities was recommended only one year after the surgical procedure. Results The mean preoperative HHS in the group of patients treated with the ACT procedure was 52 (32 – 60), similar to that of the patients treated with the AMIC procedure that was 48 (28 – 56). Mean post operative HHS results in both groups were also similar: ACT = 86 (58 – 92); AMIC = 88 (56 – 98), showing no significant difference. In both groups, unsatisfactory results were recorded in those patients suffering from a cartilage defect on the femoral head or where standard x-rays showed a reduced or compromised articular space. Discussion Knee arthroscopy has for some time now been able to show the present of chondral lesions and has allowed for the development of the current surgical techniques used for treating these lesions. Even hip arthroscopy, although considerably less common, has allowed for chondropathies in this area to be detected. The therapeutic approach is different, however, since the hip is a deep articulation surrounded by large muscular masses that make surgical access difficult. Hip arthrotomy exposes the articulation to the serious risk of aseptic necrosis of the femoral head, along with being a significantly invasive procedure. The arthroscopic approach to the treating hip chondropathies, therefore, solves the serious problem regarding arthrotomy. The AMIC procedure have several advantages compared to the ACT. Fist of all it is a one step procedure, with no need to expose the patient to a second operation. The other advantage is that there is no need for a logistic support to the procedure, having no external laboratory support. Considering that the post operative results obtained with the two procedures showed no significant differences, the AMIC appears to be much less invasive and more cost effective compared to the ACT. The cartilage defects located on the acetabulum can be treated with athroscopic ACT or AMIC procedure. This study shows the effectiveness of the AMIC procedure respect to the ACT., ChondroCelect, a cell therapy product for the repair of damaged cartilage, has been developed according to the requirements of a medicinal product. This encompasses the definition of quality attributes for the manufacturing and release of the product, preclinical demonstration of safety and proof of concept, as well as demonstration of clinical safety and efficacy through a controlled clinical trial. Development of cell therapy products according to the medicinal product requirements represent a series of challenges, and necessitates a specific translation of these requirements adapted to the nature of these products and their clinical evaluation. Manufacturing of medicinal products requires demonstration of consistency of manufacturing as well as the establishment of stringent product release criteria. Given their complex and biological nature, cell therapy products represent a more difficult type of products to manufacture and characterize when compared to chemical or other biological products. Moreover, autologous products represent an additional challenge since they consist each time of a unique product derived from a defined autologous source, with inherent biological variability. Demonstration of manufacturing consistency of autologous cell products requires therefore a well-defined manufacturing process and knowledge of its associated variability. Definition of the product release criteria necessitates a good understanding of the variability ranges of the product quality attributes, whilst meanwhile clearly establishing the minimal criteria to ensure product safety and quality. Testing of a cell therapy product in non-clinical models has also its particular challenges and limitations. Two specific issues are to be noted in this respect. The first is the relevance or representativity of the animal model. Indeed, whilst large animal models exist for studying cartilage repair by cell implantation, the differences in cartilage biology as well as the difficulty to control the post-operative rehabilitation represent major limitations towards its relevance for the situation in humans. The second potential issue relates to the fact that the medicinal product itself, i.e. human cartilage cells, is of xenogenic nature to the animal and can thus elicit an immune response. Despite these inherent limitations, the non-clinical models can provide important information on the proof of concept of the therapy (by using autologous animal cells) as well to document key safety aspects like cell dispersion and adverse events. A key aspect in the development of medicinal products, being it classical drugs, biologicals like monoclonal antibodies or vaccines, or advanced therapy medicinal products for cell therapy, is the demonstration of safety and efficacy in well-controlled clinical trials. The gold standard of such trials is a prospective, randomized, controlled clinical trial, comparing the new product to an established therapy or placebo. With respect to performing such trials in the field of cartilage repair, a series of particular issues can be recognized. A first one is the blinding of the patient's treatment. Indeed, ACI is often compared to first-line treatment microfracture; given the two vs one step procedure, respectively, it is obvious to both patient and physician to which treatment arm one was allocated. A second relates to the long duration of obtaining stable tissue repair. Cartilage repair clinical studies can generally enroll only a limited number of patients, and maintaining all patients in the study over years periods represent a major challenge. This impacts consequently the calculations of long term efficacy and safety, and their statistical power and significance. Thirdly, cartilage repair recommendations follow a series of possible treatment algorithms, and generating cohorts of similar patient groups for the respective arms of a comparative trial might not be in line with these recommendations. For instance, patients with small lesions would rather be treated with microfracture than ACI, which will not be the case when obligatory enrolling similar patient groups in typical comparative trials. Fourthly, the definition of the most relevant clinical read-outs for a given trial, the validation of the measurement of these outcomes, the statistical criteria to be applied (non-inferiority vs superiority), as well as the clinical relevance of a given statistical results are non-negligible challenges when designing and running cartilage repair clinical trials. Lastly, documentation of the safety profile is a critical hallmark in developing medicinal products. In the case of ACI products, adverse events due to the surgical intervention are common and expected. Such events are not necessarily related to the medicinal product itself, i.e. the cells, and make definition of the product safety profile more difficult than for classical medicinal products. The development of ChondroCelect has anticipated the above regulatory expectations and requirements. The manufacturing process has extensively been elaborated, and product quality attributes have been defined based on the regulatory requirements for medicinal products. Non-clinical evaluation has demonstrated the proof of concept of good cartilage repair as well as the non-clinical safety profile. A prospective, randomized, controlled clinical trial comparing ACI with ChondroCelect to microfracture has demonstrated its clinical efficacy, i.e. clinical superiority of ChondroCelect over microfracture at 36 months, as well as its safety profile. This comprehensive data package for ChondroCelect has been submitted to the European Medicines Agency (EMEA) for evaluation of the product following the centralized procedure. The registration fle is currently being reviewed and the experience gained during the review will exemplify the current regulatory expectations cartilage cell therapy medicinal products are expected to meet., Introduction New legislation has come into operation in the European Union (EU) - Regulation (EC) No 1394/2007[i] on Advanced Therapy Medicinal Products (ATMPs) in Europe. ATMPs are defined as gene and cell therapy medicinal products and tissue engineered products. This long awaited legislation provides clarity on the legal and regulatory framework for this novel class of product, and especially for tissue engineered products, which are considered as medicinal products. Regulation (EC) No 1394/2007 on Advanced Therapies The Regulation defines a Tissue engineered product (TEP) as a product that contains or consist of engineered cells or tissues that is presented as having properties for, or is used in or administered to humans with a view to regenerating, repairing or replacing a human tissue. Engineering is defined as substantial manipulation of the cells or tissues so that biological characteristics, physiological function or structural properties for the intended regeneration, repair or replacement are achieved, or when the cells or tissues are not intended to be used for the same essential function or functions in the recipient as in the donor (art. 2 of the Regulation). Many of the products commonly referred to as ‘Regenerative medicines’ would fall within the definition of a TEP. Other keypoints from the Regulation are that All marketing authorization applications for ATMPs will be reviewed via the centralized procedure and will get a marketing authorization valid for the entire EU (art. 8);The requirements for combined ATMPs (ATMPs containing, as anintegral part, a medical device or implantable medical device) and the involvement of notified bodies of medical devices have been clarified (art. 6 and 9);A new expert Committee, the Committee for Advanced Therapies, with expertise specific to ATMPs will be established (art 20 – 23);Incentives are provided to companies developing ATMPs, including: fee reductions for Scientific advice (art. 16), scientific recommendation on ATMP classification (art. 17) and evaluation and certification of quality and non-clinical data (this certification procedure is available for Small and Medium Size enterprises only) (art. 18);ATMPs legally on the markets of Member States will have to comply with the new legislation by 2011 or 2012 (for tissue engineered products) (art. 29);Follow-up of safety and efficacy, risk management as well as traceability is considered to be a crucial aspect of the regulation, protecting public health and their confidence in advanced therapies (art. 14 and 15);In well defined conditions, Member States can authorize custom-made ATMPs for an individual patients on their national market (art. 28). Regulation (EC) No 1394/2007 entered into operation on 30 December 2008. Implementation of the Regulation on Advanced Therapies Over the last two years, EMEA has been actively implementing the new Regulation on Advanced Therapies. These activities include, but are not limited to: Setting up of the Committee for Advanced therapies (CAT), including the development of the CAT Rules of Procedure;Development of procedures for the evaluation on ATMPs, including the procedure on the involvement of notified bodies in the evaluation of combined ATMPs.Development of procedures for the classification of ATMPs and for the Certification procedure;Scientific contribution to the European Commission on the revision of Annex I to Directive 2001/83/EC (technical requirements for ATMPs), GCP and GMP for ATMPs and Traceability;Development of a Guideline on Post marketing follow-up and risk management plans for ATMPs[ii]. Scientific guidelines in the field of gene and cell therapy and tissue engineering are developed by EMEA Working Parties. Multidisciplinary guidelines covering the pharmaceutical development, non-clinical testing and clinical investigations of ATMPs and guidelines on aspects specific to one of the classes of ATMPs are already in place. Additional, specific scientific guidelines are under development. All scientific and procedural guidelines are published on the EMEA Website for public consultation before finalization. The Committee for Advanced Therapies The Committee for Advanced Therapies (CAT) is an expert Committee composed of: Five members (or co-opted members) from the Committee for Human Medicinal Products (CHMP) and 5 alternates to those membersOne member and one alternate from the Member States not represented by the members/alternates appointed by CHMP;Two members and two alternates representing clinicians;Two members and two alternates representing patient organizations. Following expertise, relevant to ATMPs, is represented in the CAT: medical devices, tissue engineering, gene therapy, cell therapy, biotechnology, surgery, pharmacovigilance, risk management and ethics. CAT will be the main Committee involved in the initial evaluation of applications for marketing authorization for ATMPs: they will be responsible for the scientific review and will prepare a draft opinion on the quality, safety and efficacy of an ATMPs for final approval by CHMP. Other main activities of the CAT will be: Scientific evaluation of quality and non-clinical data for the Certification ProcedureContribution to scientific advices for ATMPsScientific recommendation on ATMP classification The CAT held it inaugural meeting on 15–16 January 2009[iii]. Under the chairmanship of Dr Christian Schneider[iv], it meets on a monthly basis (11 times a year) for a two day meeting, generally set in the week before the CHMP meeting. Authorisation of cartilage repair products in Europe Cell-based products for cartilage repair, such as chondrocytes in suspension or imbedded in three-dimensional structures, are currently under development, in clinical trials or even in clinical use in Europe. These products fulfill the EU definition of a tissue engineered product: the expansion of cell obtained from a biopsy in culture flasks or in a fermentor is considered as a substantial manipulation step. As a consequence, a formal marketing authorization application will have to be submitted and an approval obtained before they can be put on the market. Companies developing such products should consult the EMEA Guideline on Cell-based medicinal products[v], which provides guidance on the quality requirements, the non-clinical testing and the clinical trial requirements for cell therapy and tissue engineered products. It is acknowledged that the non-clinical and clinical testing for a tissue engineered products might pose challenges to developers and authorities (for example shall the primary endpoint for a clinical trial of a cartilage repair product be based on a structural repair, or can this be based on a surrogate endpoint, such a MRI?). The newly established CAT will be a very valuable forum for such discussion and for interactions with applicants (developers) of tissue engineered products. The approval of clinical trials is the responsibility of the member state where the trial will be conducted. This is outside of the scope of the Regulation on Advanced Therapies. Nevertheless, it is highly recommended that developers already in this stage of development consult the above mentioned guideline and/or seek for scientific advice from the EMEA. If the developer is a Small and Medium size Enterprise, he might ask for an evaluation of the quality and non-clinical data by the CAT, for certification. Regarding cell-based cartilage repair product currently on the member state markets, it should be stressed that these products will have to comply with the above mentioned Regulation by end of 2012 at the latest. This implies that a formal marketing authorization application will have to be submitted at least one year before, demonstrating quality, safety and efficacy of the product. It should be noted that clinical data generate from the use of this product (not arising from controlled trials) might not be sufficient to demonstrate the clinical efficacy and safety of the product. Any company having such product on the market in one of the EU member states should contact the EMEA[vi]., ACI-The First generation of Autologous Chondrocyte Implantation Cartilage has a limited capacity for self-repair after trauma which has led to many different surgical attempts to improve the repair of injured articular cartilage surfaces during the 5–6 decades. The first example of clinical cartilage tissue engineering was performed in 1987 when a knee with an articular cartilage defect on the femoral condyle was treated by implanting the patient's own chondrocytes that had been expanded in vitro and then implanted into the defect in combination with a covering mechanical membrane—the periosteum. This, technology is either termed autologous chondrocyte transplantation (ACT) or autologous chondrocyte implantation (ACI). Today, there exist many modifications of the technique, from the first generation to now second and third generations of chondrocyte implantation. This paper discusses the first generation of ACI, the scientific base and results. The articular chondrocytes are responsible for the unique features of articular cartilage, they keep the cartilage alive, and they alone maintain it and regulate it. Therefore it seems rational to use true committed chondrocytes to repair a cartilaginous defect. From a piece of arthroscopically harvested cartilage, chondrocytes can be isolated by enzymatic digestion and in in vitro culture expanded 20–50 times the initial amount of cells. The cells are cultured in so called monolayer and during that time the cells dedifferentiate. The dedifferentiated chondrocytes have a similarity to primitive mesenchymal cells and an implantation of a high density of those in vitro expanded primitive immature chondrocytes could imitate the prechondrogeneic cell condensation and cartilage formation In 1982 Lars Peterson and co-workers developed a rabbit model to treat cartilage defects in the rabbit patella with autologous chondrocytes (Peterson et al, 1984) The same rabbit model has since then been used and further developed by our group at the Göteborg University (Brittberg et al, 1996). The cultured cells are injected into a premade cartilage defect in the patella of the rabbit and covered with a flap of periosteum, functioning as a biological membrane. This method resulted in a high degree of healed rabbit patellar defects and the repair tissue had a similarity to the original cartilaginous tissue. Furthermore, in the rabbit work, the patellar defects were treated with autologous chondrocytes together with a covering periosteal graft on one side and the contra-lateral side was treated with periosteum alone. The defects were deep, reaching down to the calcified zone but with no opening of the subchondral space. In a defect of this type without any treatment, there was an intrinsic repair of 29% of the total defect area, primarily by what we call matrix flow from mitotic activity at the edges of the defect (Brittberg et al, 1996). This level of repair should be compared with the mean repair area of 30% one year after periosteal grafting alone and significantly different from the 87% repair area with chondrocytes and periosteum. More than 20 year's ago, in October 1987 the technique was first used to treat patients with chronic disabling symptoms of the knee joint with cultured cartilage cells from their own cartilage. The first 23 patients (mean age 27) were presented as pilot study in the New England Journal of Medicine in 1994(Brittberg et al, 1994). Those patients had local deep cartilage injuries that had been treated with conventional methods without any healing and 16 defects were located on the so called femoral surface and 7 on the patella. In all, 16 patients had ..good or ..excellent.. knee function at mean three years postoperatively. The best results were found in the femoral group compared with the patella patients that were less successful. The technique appeared to be most successful in patients that had injuries on the femoral surfaces producing a single, localized deep cartilage lesion. This is important to note as opposed to the gradual wear and tear of advancing age. The disappointing outcome of the patella group might have resulted from mechanical misalignments of the patella that were not corrected at the time of transplant surgery. In a long-term durability study (Peterson et al 2002, Brittberg et al, 2003), the degree of patients still belonging to the group of good-excellent was 84 %. Notably, all ACI failures occurred in the first two years and patients showing good to excellent improvement at two years had a high percentage of good results at long-term follow-up. Results are available with up to 16 years' follow-up, and more than 80% of the patients have shown improvement with relatively few complications (Jones and Peterson, 2006). Since 1987 more than 30000 patients worldwide have been treated with ACI techniques. In most published randomised studies (Horas et al 2003, Dozin et al 2005, Knutsen et al 2007, there are no differences found between the different cartilage repairs methods evaluated. In all those studies significant improvement of patient's symptomatology have been found. However, in a recent study by Saris et al. 2008, one year after treatment, ACI with periosteum was associated with a tissue regenerate that was histologically superior to that after microfracture. However, the use of first generation ACI today has become less common because different variants of chondrocyte transplantation have appeared. Due to repeat problems with hypertrophy of the periosteum needing second surgeries for trimming of grafts, risk of uneven distribution of cells and an open surgery change of the initial technique has been required. Second generation ACI has been presented with a collagen membrane used instead of the periosteum (Russlies et al, 2002) and third generation with cells on a carrier(MACI) (Zheng et al, 2007) or with cells precultured in a 3-D scaffold such in hyalyronic acid (Hyalograft –C) (Marcacci et al, 2005). Even the Saris et al- study with ACI + periosteum was different compared to the first generation of ACI as they used a well characterized chondrocyte cell population (called ChondroCelect). This presentation describes the history of chondrocytes used for cartilage repair with a periosteal membrane; the first generation of ACI, more than 20 year's experience and start of cartilage tissue engineering plus an outlook of the evolution of the technique for the future., The management of chondral lesions is still a challenging problem for the orthopaedic surgeon due to the cartilage's poor capacity to heal. The challenge to restore the articular cartilage surface is a multidimensional task faced by both basic scientists in the laboratory and orthopaedic surgeons in the operating room. In the last 30 years, different techniques to address articular cartilage injuries and defects have emerged as valid therapeutic options. While many treatments are mostly directed to the recruitment of bone marrow cells to obtain potential cartilage precursors and allow to form a reparative tissue, the bioengineered approach aims to regenerate the damaged tissue, restoring a biologically and biomechanically valid hyaline-like cartilage surface. The clinical use of first generation autologous chondrocyte transplantation reported encouraging clinical results, especially in the femoral condyle. However, they have to be weighed against the number of problems that can be observed with the standard ACI methods, such us surgical complexity and biological problem related to the cell culture. To address these problems, the so-called second generation ACI techniques have been developed. The second generation ACI used a tissue-engineering technology to create a cartilage-like tissue in a three-dimensional culture system with the attempt to address all the concerns related to the cell culture and the surgical technique. Essentially, the concept is based on the use of biodegradable polymers as temporary scaffolds for the in vitro growth of living cells and their subsequent transplantation onto the defect site. Whereas chondrocytes in two-dimensional cell cultures alter their phenotype and dedifferentiate to fibroblast, cells that no longer posses the capacity to produce collagen type II and proteglycans, the use of three-dimensional scaffolds has been shown to favour the maintenance of a chondrocyte differentiated phenotype. Scaffolds composed of synthetic or natural materials in a variety of physical forms (fibers, meshes, gels) have been applied to cartilage tissue engineering. Commonly used synthetic materials are the polylactides, like polylactic (PLA) and polyglicolic (PGA) acids. Natural materials used to produce scaffolds include agarose, alginate, hyaluronic acid, gelatin, fibrin glue, collagen derivatives and acellular collagen matrix; they have impeccable biocompatibility, can be processed in a reliable and reproducible way and may enhance cell performance. Matrixes mainly used in clinical practice in Europe are collagen or hyaluronic acid based. In the USA there is not FDA approval for matrix-assisted chondrocyte transplantation in human application, yet. The use of a three-dimensional scaffold with open surgery already permits a reduction of joint exposure because it avoids periosteal harvesting and suturing. Furthermore, the easy handling of some of the scaffolds allowed to develop arthroscopic implantation techniques. Autologous chondrocyte transplantation on a three-dimentional matrix was introduced in clinical practice from 1998–1999, so it is very difficult to obtain medium or long-term clinical findings. The clinical outcome is documented for most of the scaffolds at short term follow up, whereas only a few papers report results at follow-up greater than three years. 2ND GENERATION TECHNIQUES In 1998 Behrens et al performed the first transplantation of auotologous chondrocytes using a porcine collagen I/III matrix (Chondro-Gide) as a scaffold. The collagen membrane was utilized as substrate for the so-called matrix-associated autologous chondrocyte transplantation (MACT, MACI®). Since the introduction of the MACI® technique in 1998, more than 3000 patients have been treated across Europe and Australia1. This simple surgical technique obviates periosteal harvest, is generally suture free, is less invasive than traditional treatment methods and allows an early mobilization of the joint. All MACI® studies showed significant improvement in each of the different scoring methods employed at short term follow up, and results have been confirmed at 5 years follow up, with 81.8% of good or excellent results obtained in the knee objective evaluation2. In 1999 a hyaluronic acid based scaffold was introduced into clinical practice in a number of European countries for the treatment of full-thickness cartilage defects. This scaffold, entirely based on the benzylic ester of hyaluronic acid (HYAFF® 11), consists of a network of 20-μ m-thick fibers with interstices of variable sizes and has been demonstrated to be an optimal physical support to allow cell-cell contacts, cluster formation and extracellular matrix deposition. Seeded on the scaffold the cells are able to re-differentiate and retain a chondrocytic phenotype even after a long period of in vitro expansion in monolayer culture3. The cells expanded and then seeded onto the scaffold create the tissue-engineered product Hyalograft C®, that can be implanted by press-fitting directly into the lesion, avoiding suturing to surrounding cartilage and obviating the need for a periosteal flap. No implant related complications were reported, and even in cases where more than 2 patches were used with overlapping of the grafts, no symptoms related to overgrowth or hypertrophy were observed. The features of this device have also permitted the development of an arthroscopic approach, reducing patient morbidity, surgical time and recovery and complications related to open surgery. Some papers report a satisfactory clinical outcome at medium term follow up: Nehrer4 report interesting results at 5 years follow up and Marcacci5 and Kon6 demonstrated a high percentage of subjective and objective improvement at 2 years, maintained at the 4 years follow up. Despite the promising results of this bioengineered approach, there is no agreement about the effective superiority of Second Generation ACI on the other classic techniques. Therefore, our group decided to compare microfracture with arthroscopic second-generation ACI7. In this recently published study better clinical results, assessed with objective and subjective IKDC scores at medium-term follow-up, were found in the group treated with arthroscopic autologous chondrocyte transplantation. Also, no decrease in the resumption of sports activity from 2 to 5 years was observed in patients treated with autologous chondrocyte transplantation, whereas the decrease in sports activity was detected in the group treated with microfracture from 2- to 5-year follow-up. The clinical results obtained using Hyalograft C® are shown to be better and more lasting than the ones obtained with this classic bone marrow stimulation technique. We also applied this scaffold for the treatment of patellofemoral articular cartilage lesions via minimally invasive or arthroscopic approach, and we obtained lower (with respect to femoral condyle cartilage lesions) but satisfactory results even in this complex location at 2 and 5 years of follow up8,9. Another bioengineered product, Bioseed C®, has been introduced into the clinical practice from 2001: it combines autologous chondrocytes with the tissue development-promoting properties of gel-like matrices in an initially mechanically stable bioresorbable polymer scaffold. The cartilage tissue engineering graft Bioseed C® is a polyglactin/poly-p-dioxanon fleece with a standard sizing of 2cm × 3cm or 2cm × 1cm. Autologous chondrocytes are expanded ex vivo and therefore loaded on a 2 mm thick porous scaffold using a fibrin glue to distribute cells, providing a three-dimensional environment and obtaining the bioengineerized tissue for cartilage replacement. To ensure the fixation a transosseus fixation technique is available, moreover in posttraumatic or degenerative defects without intact surrounding cartilage, with high endpoint fixation strength. Thus, the rationale of this fixation approach is to allow a safer and shortened rehabilitation period, without endangering the success of the procedure. Ossendorf applied this bioengineered tissue for the treatment of chondral knee defects and reports a low failure rate and interesting promising results at 2 years follow up10. Numerous other cartilage substitutes have been studied and applied in clinical trials. CaReS® is composed by autologous chondrocytes seeded on 3D collagen type-I gel. Cells are isolated from the patients biopsy, mixed with the collagen gel and after the complete gelling and two-weeks culturing in patient's serum cultivation medium the chondrocyte-loaded gel is available for transplantation. Diameter and thickness of the transplant can be chosen individually depending on the nature of the defect. The transplantation is performed by mini-open technique and a thin layer of fibrin glue is applied to the defect bottom and marginal ridge to secure the graft stability. Clinical results published on a limited number of patients at 2 and 3 years follow up are encouraging, even in patello-femoral defects. Cartipatch® (TBF Banque de tissues, France) is an autologous chondrocyte implant on hydrogel composed of Agarose and Alginate. This vegetal origin hydrogel, mixed with isolated autologous cell suspension, can be modulated at 37° C into complex shape implants which solidify at approximately 25° C. Alginate provides matrix elasticity, making it easy to handle in the O.R. The transplantation is performed by mini-open technique using a dedicated instrumentation. The clinical phase II multi-centre trial was started in France in 2002 and presently Encouraging clinical and histological results are reported in the first 20 patients analyzed at 3–24 months. A different scaffold strategy involves the development of a bifasic scaffold. Novocart 3D® is a autologous chondrocyte implant on collagen-based bifasic scaffold, where a specific protective dense layer was developed to cover the collagen sponge to prevent synovial cells from invasion and improve the mechanical properties of the structure. The transplantation is performed by mini-open technique using a dedicated instrumentation and resorbable mini-pins can be used for the fixation of the graft. The implant is applied in clinical practice from 2003 and promising clinical results have been reported in literature in the first patients treated at short term follow up. Finally, some clinical studies report the utilize of autologous chondrocytes cultured on fibrin glue with significant better performance respect to the chondroabrasion technique, and some case reports report the utilize of autologous chondrocytes cultured on Atelocollagen gel. In this case, this technique cannot be properly defined as second generation autologous chondrocyte implant, considering that the implant is performed with the use of periosteal flap to cover the chondrocyte culture, but the results reported by Japanese colleagues with this bioengeneered approach are interesting. Conclusions In conclusion, the advantages related to the use of a three-dimensional scaffold seeded with chondrocytes can be summarized as follows: a minimally invasive arthroscopic implantation procedure can be used; there is great stability of the implant; and the cells tend to maintain their original phenotype. On the basis of published results, the autologous chondrocyte implant on three-dimensional scaffolds guarantees results comparable, even better of the traditional ACI technique, but reduces the morbidity of the procedure and avoids the use of a periosteal flap with marked advantages from a biological and surgical point of view. It has to be emphasized that presently none of existing second generation autologous chondrocytes products are indicated in generalized degenerative joint disease, yet. Several improvement are soon expected, as the result of the rapidly growing knowledge on cell culture and chondrocyte behaviour, leading to a more reliable surgical technique and better clinical outcome even in larger degenerative lesions., Autologous osteochondral transfer is a popular surgical technique aiming to provide hyaline or hyaline-like cartilage repair for small and medium sized focal chondral and osteochondral defects of weight bearing surfaces. Initial experimental and clinical experiences with autogenous osteochondral grafting have shown consistent survival of the transplanted hyaline cartilage.1, 2 However, two important problems have been encountered in the process: the donor sites must be taken from surfaces that do not bear much weight, which limits the procurement field, and the use of large grafts can cause incongruity at the recipient site, which permanently alters the biomechanics of the joint and represents limited clinical outcome in the long term.3, 4 Mosaicplasty technique is a new way of autologous osteochondral grafting to respond these practical problems.5 Theoretical considerations to eliminate the above mentioned problems suggested to resurface the defected area by several small diameter cylindrical grafts instead of one big osteochondral block. After some technical preparation cadaver studies were done to develop a precise instrumentation for an ideal and standardized way to manage the problems of optimal graft harvest as well as to standardize a safe and efficient way of a mosaic-like graft implantation. Initially, the mosaicplasty concept was tested in German Shepherd dogs and horses and in cadaver studies. Macroscopic and histological evaluations of the resurfaced areas and the donor sites showed: 1)consistent survival of the transplanted hyaline cartilage2)formation of a composite cartilage layer consisting of ï, / 80% transplanted hyaline cartilage and ï, / 20% fibrocartilage ingrown from the prepared bony base of the defect3)deep matrix integration of the transplanted osteochondral cylinders at the recipient site4)donor site filling to the surface with cancellous bone capped by fibrocartilage by 8–12 weeks.6, 7 Fibrocartilage coverage of the donor holes seemed to be acceptable gliding surface for these less weight bearing areas. Preclinical experimental works and evaluations of initial clinical experiences promoted to develop the present recommendations for the most effective rehabilitation algorithms.8 These considerations involve instructions for postoperative course of mosaicplasties combined with procedures to restore the normal biomechanics, such as femorotibial realignment osteotomies, ACL reconstruction, meniscus surgery, patellofemoral corrections etc. The main features of the proper rehabilitation are the following elements: immediate range of motion exercises to provide proper nutrition of the transplanted graftsfew weeks postoperative non weight bearing period to protect the bony part of the grafts from necrosis or collapse due to extreme initial loadingindividually determined partial loading period after initial non weight bearing to promote fibrocartilage ingrowth between the transplanted grafts and in donor areasfull weight bearing period combined with well adapted proprioception training to promote the tolerance of shear forces These main features of rehabilitation protocols should be individually determined to promote the best repair of the defected area, to have less donor site problem and support effectively the concomitant procedure to restore the biomechanics.9 Autologous osteochondral mosaicplasty involves obtaining small-sized cylindrical osteochondral grafts (2.7, 3.5, 4.5, 6.5, and 8.5 mm in diameter) from the minimal weight-bearing periphery of the femoral condyles at the level of the patellofemoral joint and transplanting them to prepared defect sites on the weight-bearing surfaces. Combinations of different graft sizes allow 80% to 100% defect filling rate. Fibrocartilage grouting, stimulated by abrasion arthroplasty or sharp curettage of the underlying bone at the base of the defect, is expected to complete the new surface. Mosaicplasty can be done as an open procedure, through a miniarthrotomy or –in most of femoral condylar defects - arthroscopically. Arthroscopic implantation of the graft requires optimal location and size of the defect as well as special surgical skill of the operating surgeon.10 Clinical application was begun on February 6, 1992. During the following 17 years, clinical results published by various authors matched the animal results, and since 1995, the procedure has been used with equal success at numerous institutes and clinics throughout the world.11, 12, 13, 14 These results were identical with the authors' follow up. According to our experiences in a series of more than one thousand cases involving several diarthrodial joints with varying function and biomechanical loads, the composite results have been in the good to excellent range with a low complication rate. Emphasizing the age limitations of the procedure (patients should be younger than 50 years), it is not surprising that patients who are older (older than 35 years) have faired less well. More than hundred cases of high professional athletes treated by knee or ankle mosaicplasties gave similar clinical outcome as normal population −83% of them were able to return to high professional sports activity. Beside initial clinical application of this technique for femoral condylar and patellofemoral defects, the use of mosaicplasty was extended to tibial damages as well. After first promising results of knee mosaicplasties ankle application was also started to treat talar osteochondritis dissecans defects.15, 16 This is still the most frequent application outside the knee, but femoral head lesions, capitulum humeri lesions of the elbow and humeral head lesions were also treated as rare indications. Some authors also published mosaicplasties on different small joints. Concerns of donor site morbidity remain integral parts of current evaluations. In cases of complaints related to donor sites usually transient symptoms were observed and only in few patients suffered from long lasting patellofemoral pain. The authors think that the full restoration of the donor site centers on the peripheral position of the donor area and the small size and proper spacing of the individual grafts. These elements allow the joint to reconstitute structurally to reaccept the relatively low loads in these parts of the knee. Excessive bleeding from empty donor sites caused painful intraarticular haemorrhage in a limited number of patients. In spite of the fact that this is a relatively rare complication, many experimental studies were done to test different biodegradable materials to provide an optimal filling of donor tunnels preventing such enormous bleedings.17, 18 From these encouraging results from an increasingly large series and similar results from other centers, it seems that autologous osteochondral mosaicplasty – beside cell culture techniques and biodegradable repair options - may be still a useful alternative treatment for localized full-thickness cartilage damage of the weight bearing surfaces of the knee and other weight bearing synovial joints., History Articular surface damage is a common finding in traumatic knee injuries. The limited ability of articular cartilage defects for spontaneous repair was described by William Hunter in 1743 (1). Because of this, chondral injury can lead to progressive, irreversible degenerative changes in the knee. Microfracture of the subchondral bone is a bone marrow– stimulation technique developed by Steadman et al. for the treatment of chondral defects (2). The main indication of this procedure is full-thickness articular cartilage defects caused by acute trauma or chronic repetitive microtrauma (3). It provides a suitable environment for tissue regeneration with pluripotent mesenchymal stem cells from the subchondral bone marrow. Indications and Contraindications The most common indication for microfracture is full-thickness chondral loss in the weight-bearing surface of the femur or tibia or on the articular surfaces of the patella or trochlear groove (2,3). Also it is indicated for unstable, full-thickness cartilage flaps overlying subchondral bone (4). Although microfracture alone has limited indications for degenerative lesions and osteochondritis dissecans, the most significant contraindication of this technique is a malaligned knee (5). Inflammatory arthropathies, systemic cartilage conditions, and septic or neoplastic disorders are other contraindications (6). Patient not willing to participate in the strict postoperative rehabilitation regimen may also be a relative contraindication. Preoperative Planning Preoperative planning for the patient with knee pain begins with a careful history and physical examination. The patient's social status and expectations for the postoperative course as well as short- and long-term functional outcome should be assessed thoroughly. Chondral lesions can present with pain localized to the involved articular surface or with more diffuse pain. A chondral flap can cause a patient to face popping, clicking, or even a feeling of instability. An effusion is often present with an acute chondral injury. With a chondral lesion located on patellofemoral joint, pain can be elicited with compression of the patella. Ligamentous stability should be tested. Physical examination should also determine patient body weight index because an excessive body weight has been associated with limited functional improvement. All patients presenting with a potential chondral lesion should undergo a series of radiographs including bilateral longstanding radiographs, standard AP and lateral radiographs of both knees, and patellar views to evaluate the patellofemoral joint. For further evaluation, we use magnetic resonance imaging. Surgical Technique (7) We perform a thorough diagnostic arthroscopy involving a systematic evaluation of all areas of the knee including the suprapatellar pouch, the patellofemoral joint, the medial and lateral gutters, the intercondylar notch, the anterior interval, the femoral condyles, the tibia plateaus, the entirety of the medial and lateral menisci, and the posterior compartment. All other necessary intraarticular procedures are performed before the microfracture technique to eliminate the cloudy visualization caused by the fat droplets and blood from the microfracture holes. Regions of full-thickness cartilage loss are thoroughly probed and evaluated for a cartilage flap. At the site of the lesion, any unstable cartilage flaps are debrided with a curette out to the edge of viable and stable cartilage. This is followed by the curettage of the necrotic subchondral bone. The presence of two different subchondral tissues is detected macroscopically in the necrotic area: white necrotic tissue at the superficial zone, and pink cancellous tissue that did not seem necrotic but was found not to be healthy in the deeper zones. The curettage is stopped at the second zone and multiple holes were prepared in that “seminecrotic” area using an arthroscopic awl. The holes are approximately 3 to 4 mm apart (3 or 4 holes per cm2) as suggested by Steadman et al. In regards to the depth of the holes, we prefer 4 mm, but in some cases it may be necessary to make them 6 to 7 mm deep. Treatment is considered adequate when fat droplets are observed flowing from all of the holes. The surgically induced marrow clot provides the basis for the optimal environment for the pluripotent cells to mature into fibrocartilagenous repair tissue. We do not use drain because it may inhibit formation of the surgically induced clot within the defect. A compression dressing and ice is applied. The involved cartilage area is not a handicap for this procedure, and some of our cases had 900 mm2 of necrotic lesion. Although there is no limitation as to the area of the necrosis, the depth is an important factor for the indication. We limit the procedure only to lesions that were no more than 10 mm deep from the joint level. For deeper necrosis, debridement is performed as a salvage procedure until subsequent replacement surgery. When the microfracture is completed, all instruments are removed from the joint and the knee is evacuated of fluid. Passive and active range of motion is started the day of surgery. A continuous passive motion machine is used, if available, for 2 weeks. Patients are encouraged for assisted passive motion when the machine could not be used. Weight bearing is not allowed for 6 to 8 weeks regarding to the size of the lesion, and then the patients are progressed to a aggressive rehabilitation program with full weight bearing. For those patients whose lesion area is smaller than 100 mm2, weight bearing is allowed as tolerated in the early postoperative period. Clinical improvement is not observed in the early postoperative period but patients are satisfied after 6 months. The follow-up clinical examination included assessment of pain, range of motion, effusion, instability, and atrophy of the muscles for all patients. Lysholm and Cincinnati Activity scores are used to evaluate the clinical results. Weight-bearing anteroposterior and lateral radiographs are taken of all knees for radiologic follow-up. These imaging studies are assessed for staging, healing, or worsening of the lesion according to Koshino's criteria. In this staging system, stage-1 knees have a normal appearance. Stage-2 knees have a radiolucent oval shadow in the subchondral area with distal sclerosis. In stage-3, there is a calcified plate with radiolucency surrounded by a definite sclerotic halo and collapse of the subchondral bone. Stage-4 knees have osteophytes and osteosclerosis in both ipsilateral femoral and tibial condyles. Our Experience (7) We performed a retrospective clinical study to evaluate the results of arthroscopic subchondral microfracture treatment for patients with primary osteonecrosis (ON) (group 1) or secondary ON (group 2) of the knee joint. ON of the knee may be either primary (spontaneous), or secondary to steroid therapy or chronic illness, such as systemic lupus erythematosus, vasculitis, and hemoglobinopathies. Primary ON of the knee is typically seen with unilateral involvement in elderly patients. Secondary ON generally occurs in younger patients and the necrotic areas are larger than the lesions seen in spontaneous ON. Bilateral and multifocal involvements are possible. Group 1 included 26 patients (mean age, 48 years) who had spontaneous ON. Group 2 included 15 patients (mean age, 32 years) with ON secondary to inflammatory disease or steroid therapy. Seventy-six percent of the chondral defects were located in the medial femoral condyle. The average defect size in group 1 was 162 mm2 and in group 2 was 362 mm2. After debridement of the necrotic tissues, multiple perforations were placed into the subchondral bone to obtain revascularization. There was an increase in the average Lysholm scores from 57 to 90 in group 1 after 27 months of mean follow-up (P⇠0.05); 71% of patients could participate in strenuous sports with no or minimal limitation. The mean activity level in the group1 according to Cincinnati Knee Rating System was 6 preoperatively and 13.54 postoperatively. For group 2, the average scores showed significant improvement and patient satisfaction after surgery (preoperative and postoperative average Lysholm scores were 41 and 75, respectively, with mean follow-up of 37 months). Average activity level in group 2 increased from 2.67 to 11.73. Control magnetic resonance imaging scans of the cases revealed the continuity of normal cartilage with cartilage-like tissue in the treated areas. However, an increase of the size of ON in the subchondral bone was detected in 27% of the knees. In conclusion, the microfracture technique is safe, simple, and cost-effective, and may be an alternative procedure for treatment of ON of the knee, especially in young patients, before possible subsequent replacement surgery. Arthroscopic articular cartilage repair using a resorbable matrix scaffold TruFit® CB Plug (Smith & Nephew, San Antonio, TX) is a synthetic resorbable biphasic implant which provides a scaffold for tissue repair that is both biologically friendly and biomechanically stable. It is a composite of polylactide-co-glycolide, calcium sulfate and polyglycolide fibers and can be fabricated into products such as granules, blocks, wedges and other preformed shapes. It supports the local migration of chondrogenic or osteogenic cells that ultimately synthesize new ground substance. Preclinical studies demonstrated restoration of hyaline-like cartilage in a goat model with subchondral bony incorporation at 12 months (8). Our Experience Fifteen skeletally mature patients with symptomatic, full-thickness cartilage lesions of the femoral condyles, between 1 and 2.5 cm in diameter, were treated with implantation of bioabsorbable implant. Implants were press-fit into holes drilled into the defect. All patients were evaluated both preoperatively and postoperatively with the Lysholm knee score, International Knee Documentation Committee (IKDC) Standard Evaluation Form, and magnetic resonance imaging of the affected joint. Fifteen patients of a mean age of 39 years were followed-up for a mean time of 9.1 months. The mean Lysholm score significantly improved from 43.6 preoperatively to 87.5 postoperatively (P⇠.001). Excellent or good outcome was accomplished in 12 of the patients (80%). Using the IKDC assessment, 11 of the patients (73%) reported their knee as being normal or nearly normal. Congruency of the articular surface was restored in 13 of 15 patients (87%) who underwent magnetic resonance imaging examination. Abnormal marrow signal in the subchondral bone beneath the region of implant was present in all patients. In conclusion, the TruFit CB implant is an effective and safe method of treating symptomatic full-thickness chondral defects of the femoral condyles in appropriately selected cases. However, further studies with long-term follow-up are needed to determine if the implanted area will maintain structural and functional integrity over time., Since patients requiring joint replacement are heavier, more physically active and live more than 25% longer than several decades ago, other modalities have an increasingly important role in the treatment of cartilage pathology of the knee. Cartilage does not have an inherent reparative capability. The natural history of isolated chondral injuries is not fully delineated. It is however accepted that a chondral injury on a weight bearing area of the knee may lead to the development of degenerative joint disease as well as deterioration in knee functional scores. Cartilage and osteochondral defects of the knee are commonly encountered in orthopaedic practice and their incidence is higher in the young active population. With the increasing understanding of the physiology and biomechanics of the musculoskeletal system as well as the progress made in surgical techniques, this area of orthopaedic surgery has evolved into a specialist field. Hyaline cartilage has superior biomechanical properties and is more durable than fibrocartilage. Of the various treatment modalities, only osteoarticular autograft transfer, osteoarticular allograft, and autologous chondrocyte implantation can provide hyaline repair cartilage. In the management of knee cartilage defects, the main factors that affect technique selection are the diameter of the chondral defect, the depth of the bone defect and the knee alignment. When bone is not involved and the chondral defect is less than 3cm in diameter, microfractures, autologous chondrocyte transplantation, osteochondral autografts or periosteal grafts may be offered as treatment options. Osteochondral defects of less than 3cm in diameter and 1cm in bone depth can be addressed with autologous chondrocyte transplantation, osteochondral autografts or periosteal grafts. Articular defects more than 3cm in diameter and 1cm in bone depth require osteochondral allografts. Allografts are indicated also in cases of uncontained defects but should be reserved only if the lesion is beyond the other modalities. Realignment osteotomy is a useful adjunct to all these techniques, in the case of increased loading in the pathological compartment. For osteochondral defects great than 3cms in diameter and 1cm in depth, osteochondral allografts are a reconstructive solution for young, active patients with large osteoarticular defects of the knee. They are useful where implants or an arthrodesis are not desirable. Although the use of allografts has a risk of disease transmission similar to blood transfusion, there are significant advantages since there is no donor site morbidity, the bone stock can be restored, and the allografts can be adjusted in size and shape to fit the defect exactly. There is an 85% long-term survival rate of osteochondral allografts in carefully selected patients, who present with osteochondritis dissecans or traumatic defects of the knee. This talk discusses the indications and contraindications of osteochondral allografts, outlines the peri-operative management of patients, and reviews our long-term results. Our present surgical technique will also be presented. This talk will also cover our long-term retrieval studies. We examined histologic features of 35 fresh osteochondral allograft specimens retrieved at the time of subsequent graft revision, osteotomy, or TKA. The graft survival time in our samples ranged from 1 to 25 years based on their time to reoperation. Histologic features of early graft failures were lack of chondrocyte viability, and loss of matrix cationic staining. Histologic features of late graft failures were fracture through the graft, active and incomplete remodeling of the graft bone by the host bone, and resorption of the graft tissue by synovial inflammatory activity at graft edges. Histologic features associated with long-term allograft survival included viable chondrocytes, functional preservation of matrix, and complete replacement of the graft bone with the host bone. Given chondrocyte viability, long-term allograft survival depends on graft stability by rigid fixation of host bone to graft bone. With the stable osseous graft base, the hyaline cartilage portion of the allograft can survive and function for 25 years or more. Recently we have started a new programme for posttraumatic hip defects, and preliminary results will be presented., The treatment of large or complex lesions involving articular cartilage is extremely challenging and requires a more carefully considered approach. Large or complex lesions are generally defined by any or all of the following criteria: Greater than 2 centimetersSubchondral bone involvementMultifocal or bipolar lesions or presence of arthritisFailed previous repair treatmentPatellofemoral or tibial lesionsMeniscal or ligamentous deficiencyLimb malalignment Currently available techniques for treatment of these lesions include autologous chondrocyte implantation (ACI) and osteochondral allograft transplantation (OCA). Because many patients have abnormalities of the joint or limb in addition to the articular cartilage lesion, the surgical skill set for appropriate management include techniques for ligament reconstruction, meniscus allografting, osteotomy and patellofemoral realignment procedures. The technique of ACI is well described (1,2). The basis of the procedure involve ex vivo cell manipulation and subsequent delivery of chondrocytes to the injury site with coverage by a periosteal patch to contain the cells and potentially provide growth factors. The so called sandwich procedure has been described to address deeper osteochondral lesions with an underlying bone graft covered by a second periosteal patch. ACI requires 2 separate surgeries, the first to arthroscopically harvest articular cartilage for chondrocyte expansion and culture and the second, requiring an arthrotomy, to prepare the lesion, harvest and suture the periosteal patch and inject cells. The technique of OCA has also been well described (3). The basis of the procedure is transplantation of an intact osteochondral tissue with living chondrocytes and mature hyaline cartilage. Most commonly dowels or plugs 15 −30 mm are utilized, however often shell or small fragment grafts are necessary for lesions that have difficult access or exceptionally large. The procedure is performed through an arthrotomy of variable size Clinical Evaluation Defining the lesion size, location and depth is of primary importance. It is also useful to determine the etiology or pathogenesis of the disease process. Is the lesion the result of Osteochondritis dissecans or osteonecrosis or is the process purely chondral? Is the lesion acute or chronic? What previous treatments have been attempted? Does the lesion correlate with the patient's symptoms? What other background factors are present? Is the patient realistic in his or her goals? Large and complex lesions are often associated with significant joint dysfunction and overall disease burden, which may compromise outcome. It can be very helpful to use some type of scoring system (IKDC, Cincinnati, Lysholm, KOOS, Chondropenia severity score, etc.) to quantify the overall status of the joint. Imaging should be comprehensive, including “cartilage series” radiographs: standing AP, PA flexion, lateral, Patellofemoral view and long standing alignment images (hip-knee-ankle). Missed malalignment is perhaps the most common error of omission in complex cartilage restoration. Magnetic resonance (MRI) is important to evaluate both bone and soft tissues and a relatively recent arthroscopic evaluation may be necessary to rule out occult chondral injuries. In cases of enigmatic pain, a bone scan may be useful. For patients that have a contraindication to MRI, computerized tomography arthrography (intra-articular contrast) is a useful study. Diagnostic Categories The clinical evaluation of the patient described above should lead the surgeon to a diagnosis that falls into one of the following six categories. These characteristic categories help to define treatment and predict outcome. Salvage of previous chondral surgery (microfracture, OAT, ACI)Osteochondritis dissecans (OCD)OsteonecrosisOsteochondral fracture or malunionDegenerative chondral lesionsEstablished osteoarthritis Salvage of Previous Cartilage Surgery These patients may be of any age group and a revision cartilage procedure is perhaps the last chance at biological repair. The critical issue is to determine why the previous treatment failed. Is it a biological, mechanical or technical issue that can be overcome? Is there a need for adjunctive procedure? ACI is generally indicated for chondral surface lesions in younger patients (4) and OCA may be a better choice for older patients or those with compromised subchondral bone (5). Osteochondritis Dissecans OCD represents one of the best indications for cartilage repair. Patients with OCD are generally young (15–30 years old) and have a focal lesion of the medial or lateral femoral condyle (often large) with little or no underlying pathology except occasional malalignment. Type III or IV OCD represents cases where the fragment is not salvageable are ideal for osteochondral allografting as this technique uniquely restores both the osseous and chondral deficit. Autologous chondrocyte implantation is also effective for smaller or shallow lesions (⇠8 mm deep) (6). Autologous bone grafting may be necessary for very deep lesions. Treatment outcome in these patients is excellent with 80–90% success (7,8). Osteonecrosis It is important to distinguish two variants of osteonecrosis (9). Spontaneous osteonecrosis of the knee (SONK) is generally characterized by a focal osseous lesion or cyst which may be a prodrome of early osteoarthritis in a middle aged patient. True osteonecrosis is a more diffuse, multifocal pattern of necrosis which may be idiopathic or often associated with steroid treatment in younger individuals. SONK can be effectively treated with bone grafting of the cystic lesion with or without ACI, or with OCA. Particular attention should be made to correcting malalignment as this is often a predisposing factor. Diffuse osteonecrosis is best treated by small or large fragment allografts, particularly if the patient is young and there is a desire to avoid prosthetic arthroplasty (10). Osteochondral Fracture or Malunion This condition is generally the result of high energy trauma such as motor vehicle accident or falls or occasionally the result of patellar dislocation. The typical case involves malunion of a tibial plateau fracture where joint incongruity results or, on the femoral side, where a non salvageable fragment is associated with a sub-acute or chronic osteochondral injury. In this setting ACI is rarely indicated and OCA is usually the best option as anatomic bone restoration is the most critical component of treatment. Realignment osteotomies are commonly employed as well as meniscal transplantation in the case of tibial plateau grafts (11). Degenerative Chondral Lesions These lesions typically occur in older patients (35–55) years old and represent a chronic disease state, resulting from repetitive trauma and surgery. The distinction between degenerative lesions and osteoarthritis is probably only a matter of degree and represent the same disease continuum. These knees are characterized by multiple lesions with meniscus and ligament compromise. Both ACI and OCA can be utilized but multiple grafts that may be necessary are technically challenging and staged or combination procedures (realignment) are common. Results from treatment with either modality are fair to good, but patients must be willing to accept arthroplasty as a salvage (12,13,14) Established Osteoarthritis Established osteoarthritis in the young adult, characterized by significant joint derangement and radiographic changes such as joint space loss should be considered one of the major unsolved problems in orthopaedics. Prosthetic arthroplasty has not yet demonstrated excellent functionality or durability in this population. Cartilage restoration should not be routinely used except in highly selected cases. When appropriate, osteotomy alone as a first line treatment should be considered. Summary Treatment of large and complex cartilage lesions requires a broader and more thoughtful approach than the more common small focal lesion. Patient evaluation should include a comprehensive clinical assessment, with particular attention to biomechanics of the limb and joint. Diagnostic categories appear to be more useful than lesion size in clinical decision making. Current cartilage restoration options are limited to autologous chondrocyte implantation and osteochondral allografting, with few exceptions. Use of adjunctive procedures is critical to success. Future innovation will provide more tools and better outcomes in treating this difficult clinical entity., Introduction “Doc isn't there something you can put in my knee to keep me playing sports?” Seventy million Americans have osteoarthritis leading to 300,000 artificial joint implants annually. The limitations on the patient, the complications, the short lifespan of the implants, and the cost to society all drive the search for biologic joint replacement rather than artificial joint replacement. The two cartilage components of the knee joint, the meniscus and the articular cartilage were traditionally irreparable and irreplaceable. Both can now be repaired and replaced. The methodology and the source of the cells and tissues that make up this new biologic joint replacement era is the subject of the ICRS meeting. The future may lie in new tissue sources from animals as the barriers to transplantation, immunology, source, sterilization and healing are resolved. The potential for superior outcomes, unlimited availability, and sterility may drive this science. Biological vs. Prosthetic: The true unmet clinical need in osteoarthritic knees is to replace the damaged articular cartilage and the missing meniscus cartilage with new tissue. Artificial materials all eventually fail and all limit activities and motion. The holy grail of biologic knee replacement is normal meniscus replacement and complete retreading of the worn surface with hyaline articular cartilage. The meniscus cartilage can today be partially re-grown through the collagen meniscus scaffold and replaced in arthritic knees with allograft menisci [1]. The re-growing technique is limited to pristine knees and the allograft source is limited to young tissue in short and expensive supply. CURRENT TECHNIQUES OF CARTILAGE REGENERATION Biological articular cartilage replacement may most simply be divided into the cellular regeneration of tissue versus implantation of biologic prostheses. The cellular group includes both endogenous and exogenous cellular stimulation. The endogenous group includes drilling, microfracture stimulation of fibrous repair and the paste graft application of cartilage and bone matrix to morselized arthritic lesions [2]. The exogenous cellular group, autologous cartilage implantation and its variants, depend on cellular expansion followed by remodeling of cartilage. The biologic prostheses group follows the original shell allograft transplantation experience and diversified into OATS and Mosaicplasty. Table I provides a brief summary or current techniques for cartilage repair and replacement. Table 1: Current Techniques for Cartilage Repair and Replacement Cartilage Repair Technique Description Debridement / Chondroplasty Debridement and reshaping of the damaged area of the joint by cutting, scraping, heating, or burring the arthritic joint surface. Potential for superficial cartilage reshaping Microfracture Systematically debriding and perforating an traumatic lesion through surface cartilage layer but not through the subchondral plate. Repair tissue consists of a mixed fibrous repair. Autologous Chondrocyte Implantation (ACI) This open surgical (as opposed to arthroscopic) procedure is performed in a two-stage procedure. The first surgery harvests chondrocytes (live cartilage cells) from the patient's knee. The cells are then cultured, expanded and re-implanted during a second surgery. Periosteal tissue, is harvested and sutured into place over the cartilage repair with cultured chondrocytes injected. No bleeding is induced. Matrix Induced Chondrocyte Implantation (MACI) This arthroscopic procedure is similar to ACI, but uses a bio-absorbable cover instead of periosteal tissue. The cover is a biopolymer matrix layer that requires no suturing. Osteochondral Autograft Transfer (OATS) / Mosaicplasty Osteochondral (articular cartilage + attached bone) plugs are harvested from one area of the patient's knee, and transplanted into the damaged portion of the joint. Osteochondral Allograft Transplantation Articular cartilage and the attached bone is transplanted from a cadaver donor to a damaged region of the recipient's joint. Articular Cartilage Paste Grafting Arthritic lesions are extensively microfractured and morselized through to subchondral bone. A core of cartilage and bone is taken from the intercondylar notch and crushed into a paste. The paste is impacted into the lesion and the progenitor cells contained in the paste and recruited from the bleeding fractured lesion bed aid the healing process. These surgical techniques produced variable degrees of success clinically and are limited to focal, or small to moderately sized, lesions in articular cartilage. Treatment becomes more difficult as the arthritic lesions increase in size, as is the case in severe arthritis. The future must be to provide these tissues on a cost effective off-the-shelf basis customizable for each knee. The tissues must be adequately effective immediately and able to undergo remodeling without significant degeneration over time. They must be able to undergo biologic healing in a degradative chemical environment and a mechanically abnormal physical environment without early tearing or loosening. These goals will most probably be met by transplantation of intact cartilage loaded with progenitor cells. CONCEPTS IN ORTHOPAEDIC XENOGRAFTING The Animal to Human Problem: The effectiveness of cartilage regeneration typically depends on endongenous or exogenously embedded and nurtured cells. This problem is compounded in the xenograft case where animal cells may be implanted into human. The use of animal derived cells in humans is strictly prohibited due to the risk of zoonotic disease transmission by agents such and endogenous retroviruses or in the case of bovine species, spongiform encephalopathy. Although several accellular xenograft bioscaffolds are commercially available for musculoskeletal reconstruction, few studies have addressed the primary host antibody reaction to a xenogeneic cell and matrix surface carbohydrate antigen called the ï,μ-galactosyl epitope (alpha-Gal). Humans and Old World primates lack the alpha-Gal epitope, but all other mammals produce and incorporate ïi-Gal epitopes into cellular and extracellular structures using the alpha1,3-galactosyl transferase enzyme [3]. To compound the problem, humans and Old World primates continuously produce anti-Gal antibodies constituting about 1% of circulating immunoglobulins, and are therefore not immunotolerant towards grafts presenting with alpha-Gal epitopes. Previous studies where grafts were tested in lower order species did not show the rejection response. To date, no decellularization, washing or sterilization processing technique has been shown to remove the Gal epitope leaving xenografts with short residence times when transplanted to humans. Our investigations have characterized the immunological response elicited by porcine tissues and examined alpha-Gal epitope enzymatic cleavage methods as applied to porcine fibrocartilage, articular cartilage, bone, tendon, and soft tissue grafts [4]. Due to the above noted concerns about non-human cells, current strategies for the use of xenogeneic materials in human orthopaedic reconstruction are limited to re-assembled scaffolds and devitalized tissues. The collagen meniscal implant and immunochemically modified porcine patellar tendon are relevant examples of applied research to xenograft development. Collagen Meniscal Implant: The collagen meniscal implant scaffold (CMI) was specifically designed to foster regeneration by optimizing the porosity of the scaffold to be cell migration friendly, to chemically cross link the device with glycosaminoglycans and vapor aldehyde to delay the degradation time and to permit tissue remodeling. The device was also designed to be an off the shelf implantable and customizable at the time of surgery. It met the criteria for safety and implantability in 1993 and subsequently approved in the U.S. in 2008 for meniscus repair [5]. The design process serves as an excellent example for tissue engineering for a defined application and unfortunately the approval process highlights the difficulties facing biologic solutions to joint problems. Immunochemically Modified Porcine Patellar Tendon: An immunochemically modified and sterilized porcine ACL reconstruction device was developed with an understanding of the failure modes of prior ACL replacement products. Early on, investigations demonstrated the significance of the major antigen that causes rejection of pig cartilage and ligament xenografts in primates. Later investigations developed techniques to attenuate this rejection without sacrificing graft durability and without using high concentrations of glutaraldehyde, thus optimizing cellular infiltration and remodeling. The clinically used device consists of porcine bone-patellar tendon-bone treated with a-galactosidase to eliminate the Gal epitope, a low level of glutaraldehyde and sterilized with 17.8 kGy irradiation. Pilot clinical studies of ACL reconstruction have shown the utility of the technology with formal clinical studies in development toward commercialization. FUTURE DIRECTIONS FOR CARTILAGE XENOGRAFTS Xenogeneic cartilage as a scaffold represents an idealized biologic prosthesis. If intact cartilage can be transplanted and revitalized with human progenitor cells then true bio-resurfacing of the joint may be possible. The composite may lower the biomechanical barrier and present only the immunologic and cellular challenges to joint reconstitution. Stem cells and their progenitor variants may present the pathway to both immunologic suppression and anabolic stimulation for cartilage remodeling. Our work pushes forward in this direction at this time., Arthroscopic assessment provides important information on the outcome of cartilage repair procedures. There are currently two semiquantitative scoring systems available for systematic evaluation of the repair sites. Both the International Cartilage Repair Society (ICSR) cartilage repair assessment and the Oswestry Arthroscopy Score (OAS) have been validated (Smith et al. 2005, van den Borne et al. 2007). Mechanical properties of the repair tissue reflect the compositional and structural characteristics of the tissue and thus may describe the biological success of the repair process. Visual outcome measures Both the ICRS and OAS scoring systems were designed to evaluate the macroscopic outcome of cartilage repair and to focus the scoring system according to the clinical needs (Brittberg and Winalski 2003, Smith et al. 2005). The ICRS macroscopic evaluation grades the repair site in terms of the degree to which the defect is filled with repair tissue, integration to the border zone and macroscopic appearance of the surface. Each of these three estimates is classified into five levels (0–4 points) and the overall repair assessment has four grades: normal (12 points), nearly normal (8–11 points), abnormal (4–7 points), and severely abnormal (0–3 points). To evaluate the filling of the defect site after ACI or microfracture, the depth of the repair tissue is assessed. When estimating the outcome after osteochondral transfer, which immediately fills the defect, the survival of the initially grafted surface is evaluated. The OAS evaluation grades the repair tissue in terms of graft level with the surrounding cartilage, integration with the surrounding cartilage, appearance of the surface, color of the graft and mechanical stiffness to manual probing. Each estimate consists of three levels (0–2 points) and the points from the five estimates are added together to give an index with a range of 0 to 10 points. The ICRS macroscopic score and the OAS score showed a good inter- and intra observer reliability and the correlation between both scoring systems was excellent (van den Borne et al. 2007, Smith et al. 2005). Thus both scoring systems were considered valid research tools for cartilage repair surgery. The main difference between these scoring systems is that the OAS score takes into account hypertrophy of the repair tissue and tactile probing of the graft, which are not evaluated in the ICRS score. Although the arthroscopic evaluation of articular cartilage and repair tissue is the gold standard investigation to evaluate alterations in articular cartilage, it has also certain disadvantages. Quantitative measurements are seldom possible and evaluation of the changes is mostly qualitative. When trying to estimate, e.g., distances in the knee joint, disagreements of the arthroscopic measurements are usually greater than 34 % (Diaz and Albright 2008). The ICRS and the OAS scoring systems both improve the arthroscopic evaluation of the repair tissue by offering semiquantitative data of filling of the lesion, integration of repair tissue with adjacent cartilage and the macroscopic appearance of the repair site. The advantage of an arthroscopic scoring system is that it provides information of the whole repair area. A biopsy specimen for histology or biomechanical indentation usually provides more local information at a small part of the repair site. Mechanical outcome measures The goal of repair procedures is to produce functionally competent tissue that can withstand the high loads the joint is subject to. Therefore, knowledge on the biomechanical properties of the repair tissue would be important when evaluating new methods of cartilage repair. Manual probing of cartilage surfaces is a routine procedure during arthroscopies. Probing may reveal some information on the resiliency of cartilage and repair tissue, especially when the tissue is much softer or harder than normal cartilage. However, the procedure is a highly subjective and insensitive method, and thus this technique provides information with limited value, especially for research purposes. Laboratory studies have shown that the quantitative stiffness measurement by the indentation technique is a sensitive indicator of cartilage structural integrity (Jurvelin et al. 1989), possibly even more sensitive than the qualitative histological evaluation (Lane et al. 1979, Armstrong and Mow 1982). For example, after long-term immobilization, macroscopically healthy looking cartilage may show significantly impaired functional properties predisposing cartilage to further injury (Jurvelin et al. 1986, Arokoski et al. 2000). Further, studies with human cartilage have shown that indentation stiffness predicts histopathology of cartilage (Franz et al. 2001, Bae et al. 2003, Appleyard et al. 2007). Our group has previously presented and validated an arthroscopic indentation instrument that is capable of measuring compressive stiffness of articular cartilage in vivo (Lyyra et al. 1995). Later, few other indentation instruments have been introduced for in vivo use (Niedeauer et al. 2004, Appleyard et al. 2001). The studies have shown that instruments can detect functional changes in articular cartilage during tissue degeneration (Bae et al. 2003, Lyyra-Laitinen et al. 1999). The mechanical indentation instrument developed by our group consists of a handle and a measurement rod, equipped with a mechanical indenter and an integrated force gage at the tip (Lyyra-Laitinen et al. 1999). The force by which the tissue resists the constant indentation of articular cartilage is a measure for cartilage stiffness. The latest version of this instrument and measurement technique, enabling more controlled, rapid and reproducible measurements, has been recently introduced (Timonen et al. 2009.) Earlier studies have shown that different cartilage surfaces in healthy joints show significant variation in their mechanical properties (Laasanen et al. 2003, Kiviranta et al. 2006, Froimson et al. 1997). It has also been reported that the site-dependent variation exists within one cartilage surface (Samosky et al. 2005, Kiviranta et al. 2008). Due to the site-dependent variation, accurate site-matched reference values would be needed to assess cartilage integrity with this approach. As for now, there are no comprehensive normal values of normal cartilage stiffness. Therefore, values of the healthy adjacent cartilage are used for reference when measurements of cartilage repair tissue are conducted (normalized stiffness) (Vasara et al. 2005). A clinical follow-up study of thirty patients operated with autologous chondrocyte transplantation has shown that the indentation stiffness of the repair tissue improved to 62 % of the adjacent cartilage one year after the cartilage repair procedure (Vasara et al. 2005). The repair tissue stiffness in eight patients reached the same level as the adjacent cartilage stiffness, but there was great variation in the biomechanical properties of the grafts. The osteochondritis dissecans lesions generally had softer grafts suggesting that the deep lesions may need longer maturation time (Vasara et al. 2005). The delayed gadolinium-enhanced MRI of cartilage analysis of four patients showed that the proteoglycan (PG) concentration of the repair tissue had replenished during the one-year follow-up. However, the same grafts had very low stiffness values, indicating that PG concentration alone does not necessarily fully characterize biomechanical integrity of the graft (Vasara et al. 2005). Other studies have shown that after the mean follow-up of 1.8 or 4.5 years after autologous chondrocyte transplantation the normalized stiffness of the cartilage repair tissue was 104 % and 94 %, respectively (Henderson et al. 2007, Peterson et al. 2002). The same studies have revealed that grafts with histologically verified hyaline repairs exhibit indentation stiffness values comparable to surrounding cartilage and superior to those associated with fibrocartilage repairs. These data demonstrate that arthroscopic indentation can be used successfully to distinguish functionally and structurally appropriate tissue from unsatisfactory repairs. Thus, mechanical indentation of the repair tissue may be considered a biological outcome measure, supporting the use of arthroscopic indentation as a method for assessment of cartilage repair tissue., Introduction Many factors influence the evaluation of outcome following cartilage procedures. The outcome is influenced by the patient, the nature of the lesion, the procedure performed and the outcome measure utilized. All of these factors must be independently considered in great detail to appropriately evaluate any treatment or procedure for a cartilage lesion. Each of these factors will be considered separately to present the reader with an organized approach to evaluating the outcome for a patient who has undergone a cartilage regenerative or restorative procedure.1 The Patient Many factors must be considered when evaluating the patient. These factors will have a very large effect on the outcome of treatment and must be documented in detail. The age as well as the height and weight of the patient have an important effect on the outcome. The occupation will also affect outcome do to the relationship with activity level. Whether the patient is male or female should be documented. Perhaps one of the most important prognostic factors following cartilage surgery is the level of activity of the patient. This is a critical variable since with a decreased level of activity many patients can tolerate significant knee pathology. The measurement of patient activity is complex and can be difficult. The Tegner rating scale evaluates patients based on their participation in various sports2. While this scale has been used extensively in the past, it has limitations with respect to patients who do not participate in specific sports measured by the scale. Therefore individuals who are active but do not participate in one of the sports evaluated in this rating scale may be incorrectly rated as having a lower activity level. A rating scale that measures patients' activity independent of specific sports is desirable. One such scale has been published that was developed with patient input with respect to activities that are important and difficult for them to perform3. This rating scale asks patients four questions about the frequency with which they perform four activities. The activities are running, cutting (changing directions while running), decelerating (coming to a quick stop while running) and pivoting (turning the body with the foot planted…). This scale has been evaluated for reliability and validity in separate groups of patients3. The Lesion The characteristics of the cartilage lesion that is repaired have an important impact on the outcome after treatment. These characteristics should be documented in detail prior to surgery to allow an accurate evaluation of the results in light of what was actually treated. The size of the lesion and whether the lesion involves only cartilage or bone should also be determined since lesions involving subchondral bone generally require a more involved reconstruction. The diagnosis will also have an important effect on treatment in many cases. Avascular necrosis leading to a cartilage problem will affect underlying subchondral bone and may be related to systemic health problems. Osteochondritis Dissecans also involves the underlying subchondral bone and will often lead to large defects. The alignment of the lower extremity can also affect outcome depending on the location of the lesion. Alignment is ideally evaluated radiographically using three foot standing x-rays to determine the anatomical alignment and the mechanical axis. Other interarticular disease must also be evaluated, particularly the opposing articular surface. In general, if the opposing surface is degenerative, the patient would be diagnosed with arthritis and cartilage resurfacing may not be appropriate. Therefore, the articular surface opposing the cartilage injury site as well as the articular surfaces elsewhere in the knee must be evaluated. The Procedure When evaluating the results of surgery there are several factors that should be considered in addition to the actual type of operation performed. The indication for surgery should be documented. In general, the indication for surgery is pain. However, if the indication is not pain and the surgeon is performing the operation to avoid future problems in the knee, this should be explicitly indicated. If the patient has had a prior procedure this also should be documented. The type of procedure performed previously and the number of operations done should also be evaluated. The postoperative rehabilitation may have an important influence on the outcome. Factors such as use of continuous passive motion, weight bearing and strengthening exercises, as well as the timing of their incorporation can affect the result of the procedure. While it is obvious that the surgeon has a critically important effect on the outcome, this should be evaluated in studies comparing two treatments. In certain cases two procedures will be evaluated in a head to head comparison. If one surgeon performed all of the operations for the patients in one group while a different surgeon performed a different operation for the patients in a different group, the study is inherently biased. In this case, the study may be comparing the surgeons rather than comparing the procedure itself. In situations such as this, it is important to remember that the investigator must track not only what was done, but by whom. Evaluation of Outcome: Objective There are several “objective” measures of outcome such as physical examination, imaging and tissue biopsy. While these are generally very important to the surgeon, they may not be of any relevance to the patient. Patients are generally more concerned with their subjective complaints and function. Nevertheless, objective measures are important and often give critical information. Physical exam is a routine part of follow-up after surgery. For cartilage procedures about the knee physical exam includes an evaluation of gait, pain on palpation, effusion, range of motion and stability of the knee. Imaging is also an important part of the evaluation. Radiographs can demonstrate the progression of degenerative disease such as osteophytes, subchondral sclerosis, subchondral cysts and joint space narrowing. Change in alignment may also be related to degenerative osteoarthritis. An evaluation of the repair tissue itself is useful to determine the quality. Routine histology as well as immunohistochemical evaluation have been performed6,7. While the information gained by biopsy is valuable, many patients will not consent to this procedure. Despite the potential lack of patient interest in this approach, some authors have been able to evaluate patients with this methodology8. Evaluation of Outcome: Subjective There many factors that are termed “subjective” with respect to patient evaluation. They are termed as such because they are difficult to evaluate or measure quantitatively. Nevertheless, issues such as pain and function are of paramount importance to patients who are recovering from cartilage procedures. Symptoms and disabilities are generally evaluated using validated rating scales. There are many that have been published for use in this patient population9,10. The goal of using rating scales to measure patient outcome is to evaluate concepts that are critical to patients and to do so in a time efficient manner. Therefore, relatively shorter questionnaires are preferred to limit responder burden. Of the available knee rating scales, several will be discussed below with respect to their usefulness for this patient population. The modified Lysolm scale2 is an eight item questionnaire which was initially designed to evaluate patients after knee ligament surgery. It has 25 points attributed to knee stability, 25 to pain, 15 to locking, 10 each to swelling and stair climbing and 5 each to limp, use of support and squatting. It has been used extensively for clinical research studies mainly for the anterior cruciate ligament. However, it has recently been evaluated and found to be acceptable for chondral disorders of the knee11. The activities of daily living scale of the knee outcome survey is a useful instrument for cartilage patients and we have distributed this questionnaire to evaluate patients at our institution12. It was developed based on a review of relevant instruments with clinician input. It is designed for patients with disorders of the knee ranging from ACL injury to osteoarthritis. Therefore, it is generally applicable to most cartilage patients. The questions range from relatively simple basic functions to more advanced activity. It has been found to have excellent psychometric properties10. The international knee documentation committee (IKDC) developed a rating scale for “objective” parameters relating to knee function. These parameters include effusion, motion, ligament laxity, crepitus, harvest site pathology, radiographic findings and one-leg-hop tests. Patients were given a grade as normal, nearly normal, abnormal or severely abnormal for each. The lowest grade for a given group is the patient's final grade. The IKDC has subsequently developed a questionnaire relating to “subjective” factors16. While this questionnaire has not specifically been validated for patients with articular cartilage disorders, it is likely that it is a useful instrument. The knee injury and osteoarthritis outcome score (KOOS), was developed using input from patients who underwent meniscal surgery in the past14. Five separate scores are calculated for pain, symptoms, activities of daily living, sport and recreational function, and knee related quality of life. This scale is useful since the Western Ontario and McMaster University's osteoarthritis index (WOMAC) is incorporated into the KOOS15. The WOMAC involves 24 questions with 5 relating to pain, 2 to stiffness and 17 to difficulty with activities of daily living. It is mainly for patients with lower extremity osteoarthritis and therefore can be useful for patients with cartilage disease. The KOOS is a wide ranging scale since it applies to patients with degenerative disease, but also has questions about sport participation. This makes it an attractive alternative for evaluating outcome follow cartilage procedures., Microfracture is one of many methods available to treat articular cartilage lesions. The technique has been elaborated by Steadman et al. and applied chiefly in young athletes and in young patients generally1. There are some inherent advantages. The microfracture technique is minimally invasive and the costs are minimal since expensive cell cultures are not necessary. In comparison to an abrasion chondroplasty the subchondral bone plate is not completely destructed but partially preserved between the microfracture holes improving load-bearing characteristics following healing2. Unlike osteochondral or periosteal autograft procedures the problem of a harvest site morbidity is excluded. The intervention leads to a spontaneous repair response, which is based upon therapeutically induced bleeding from the opened subchondral bone spaces and subsequent blood-clot-formation3. However the technique has also some inherent disadvantages. Microfracture promotes resurfacing with predominantly fibrocartilaginous repair tissue of inferior quality. In this context a fibrous type of cartilage tissue was found in rabbits or canines treated with Pridie drilling or with microfracture4. Animal studies investigating the durability of fibrous tissue revealed a progressive failure of apparently well healed cartilage. Reason for the worse durability was the lack of physical and chemical bonding between the macromolecular components of the repair tissue and the residual adjacent cartilage following micromotion and macromotion between them. Furthermore the repair tissue over the re-established prominent tidemark was only half as thick as the surrounding original cartilage. These results were confirmed by clinical studies with MRI evaluation, that revealed osseous overgrowth between 25–50% of the patients and persistent gaps between the native and repair tissue in most of the microfracture repairs. Further MRI analysis showed an incomplete defect fill under the level of the intact adjacent hyaline cartilage5–7. While no validated treatment algorithm exists for treating articular cartilage lesions in the knee, the arthroscopic microfracture technique is commonly used as a first-line option and frequently serves as the standard technique against which other cartilage repair procedures are compared. A recent analysis of cartilage repair techniques has pointed out the methodological limitations of the available literature on articular cartilage repair8. This may be a reason for the heterogenity in outcome between worse and excellent results, reported in clinical studies with a follow up to more than 10 years after surgery. To provide a better understanding in the indications and limitations of the microfracture technique we performed a comprehensive analysis of the clinical literature on articular cartilage repair in the knee by this technique. For this purpose literature was searched for human studies reporting clinical, histological and MRI results after microfracturing chondral lesions. The used search engines were MEDLINE, EMBASE, CINAHL and Cochrane Central Register of Controlled Trials. The quality of the existing studies was analysed using modified Coleman Methodology scores. Clinical effectiveness of articular cartilage repair was evaluated by systematic analysis of short- and long-term functional outcome scores, macroscopic and microscopic repair cartilage quality, and findings of postoperative magnetic resonance imaging. Twenty-eight studies describing 3122 patients were included in the review. Average follow-up was 41 months with only five studies reporting follow-up of 5 years or more. Six studies were randomized-controlled trials and mean Coleman Methodology Score was 58 (range 22–97). During the first 24 months microfracture effectively improved knee function in all studies. Afterwards there are conflicting results regarding the long term durability. This heterogenity in long term outcome may be explained by several factors, influencing the final result. In a randomised trial comparing microfracture and ACI more fibrous as hyaline cartilage was found in the histological evaluation of biopsy specimens taken during a second-look arthroscopy after microfracture. The histological results were graded with 1 to 4 points. Most of the failures by 5 years occured with fibrocartilage and none of the patients with a failure had the best-quality cartilage9. The factors leading to more hyaline or to more fibrous cartilage are still unknown. However the quality of the repair tissue seams to play a major role in the long term durability of the repair tissue and subsequent good clinical outcome. The importance of the tissue quality was also addressed by another study comparing microfracture and ACI using characterized chondrocytes. After 12 months ACI revealed significant better histological results and after 36 months significant better clinical results could be detected with the ACI procedure10. We could find some other important factors, that may influence the clinical outcome or the histological result. In this context a good defect fill with improved clinical scores were identified primarily with defects on the femoral condyles. In contrast trochlear or retropatellar defects revealed in most cases a deterioration between 18 and 36 months after surgery7. Furthermore young patients under 40 years had the best clinical results6. The worse results of patients over 40 years compared to younger patients may be explained by the reduced regeneration capacity of the repair tissue. As stem cells age their mitochondrial function and synthetic activity decline. Therefore chondrocyte senescence decreases the efficacy of cartilage repair. Another important factor was the amount of defect fill on MRI, which was highly variable and correlated with functional outcome. Macroscopic repair cartilage quality positively affected long-term failure rate. Furthermore better clinical results could be detected in young sportive patients with a Tegner score over 4, a short duration of symptoms, a small lesion size, a low body mass index and the first surgical intervention. In all prospective clinical randomised controlled trials the lesion size was limited to 4cm2. Larger lesions are predominantly treated with autologous chondrocyte implantation. For these large lesions there are only few and not sufficient data with the microfracture procedure. In conclusion microfracture is a minimal invasive and cheap method, that provides effective short-term functional improvement of knee function especially in young active patients with small chondral defects. Only few data is available on its long-term results. Shortcomings of the technique include limited hyaline repair tissue, variable repair cartilage volume, and possible functional deterioration. Further well-designed prospective randomised controlled trials with an adequate randomisation procedure, power analysis, patient inclusion and exclusion criteria, validated outcome measures, independent investigators, clinical histological and MRI evaluation with a continuous follow up, a detailed rehabilitation protocol and a high Coleman Methodology Score are needed to determine the long-term effectiveness of microfracture and to define its specific clinical indications compared to other cartilage repair techniques., Repair processes often mimic or recapitulate developmental processes quite closely although not exactly. Thus, an understanding of developmental processes is essential if we wish to derive rational strategies in attempting to biologically augment articular cartilage repair. Interestingly, whilst we know a great deal about the signalling molecules that regulate the transition of phenotypic states within the epiphyseal growth plate, we know very little about those that regulate the growth and development of articular cartilage. However, first we must compare the similarities and differences between these two tissues. It is often considered that both the epiphyseal growth plate and articular cartilage are remnants of the embryonic cartilaginous rudiment. Whilst this still holds for the growth plate, it is now known that articular cartilage derives from a sub-set of cells that reside at the periphery of the embryonic epiphysis adjacent to the interzone (Hyde et al., Dev Biol) In terms of the mechanisms of growth, then there are certainly similarities in that they both grow by apposition with stem/ progenitor at the ‘top’ of the tissue, differentiating then proliferating and eventually terminally differentiating to be replaced by bone. Seminal work by Vortkamp elucidated the BMP/IHH/PTHrP pathway that regulates the transition of cell state in the growth plate and she continues to refine these pathways (refs. Science and more recnt) In contrast to the growth plate, little if anything is known of the signalling pathways that regulate the transition of cells in articular cartilage. Current evidence suggests that it is not the same as the growth plate but this is an area that requires urgent attention. In terms of the regulation of the proliferation of stem cells that gives rise to appositional growth, we know that the Delta/Notch pathway plays an important role in articular cartilage as about 70% of the cells at the articular surface are positive for Notch1 and inhibition of Notch signalling abolishes clonality of the progenitor population (Dowthwaite et al., 2004). At the same time, Notch1 is also expressed by the cells immediately above the terminally differentiating cells and is similarly placed above pre-hypertrophic cells in the growth plate where Notch1 has an inhibitory effect of progression to terminal differentiation. Lastly, I will speculate further on other aspects of the development of articular cartilage that may be useful in better understanding repair and pathological processes., Articular cartilages provide within a diarthrodial joint, together with the lubricants lubricin and hyaluronan secreted also by the synovium, an almost frictionless articulating surface capable of handling the stresses, strains and dissipation of loading that is required over many decades of use. It is a truly unique tissue the properties of which are determined by its extensive and very specialized extracellular matrix. What is interesting is how the properties and responses of this tissue to the environment, be it physiological or pathological, can vary according to the site within a joint and between joints. A variety of information collected over many years is now giving us better insights into the variability in cartilage form, function and response to injury. Some of this will be reviewed focussing on the ankle, knee and hip joints. The overall determining factor in cartilage thickness would appear to be the biomechanical loading that the joint experiences. From studies of animals thickness appears to be directly related to the to body size and is in a linear logarithmic relationship to body weight1. In the case of the human lower limb the mass of the donor is significantly correlated with the mean cartilage thickness for ankle2, knee2,3 and hip joints2. Height is also correlated with thickness in the hip and knee joints whereas body mass index and cartilage thickness are correlated in the ankle joint2. The latter studies have also indicated an inverse relationship between cartilage thickness and its compressive modulus. The knee has thicker cartilage than the ankle and hip while hip cartilage is usually thicker than the ankle2. This thickness may be determined by the congruence of the joint as thicker cartilage is found in incongruent joints4. Thus it has been suggested that congruent joints with thin cartilage only deform a small amount yet the area of contact is large enough to distribute load and maintain an acceptable level of stress. In incongruent joints deformation of the thicker cartilage increases the contact area between joint surfaces to decrease the stress. Talar (ankle) cartilages have a higher proteoglycan content and lower water content, consistent with the higher equilibrium modulus and dynamic stiffness found in these cartilages5. Anatomical, structural and biomechanical differences in cartilages are also seen within a joint. The surface layer is very different to the deeper layers with its high tensile stiffness and fracture stress. This decreases progressively with increased depth as proteoglycan aggrecan content increases and cell density decreases6. Even the matrix around cells varies in structure with the distance from the cell6. The fattened cells of the cell surface layer express distinct proteins such as lubricin7. The superficial cells of the ankle and knee cartilages are also quite different in their arrangement. In the knee chondrocytes are present as single cells or doublets. Whereas in the ankle they are arranged in planar clusters containing multiple cells within chondrons8. Adjacent to focal ankle lesions the clusters increase in size. This differential response to degenerative focal lesion development is of special interest. This is because biochemical studies of knees versus ankles have revealed that in knees the reponse of articular cartilage in an early lesion emphasizes degradation of type II collagen. In contrast, in the ankle the emphasis is on increased synthesis of this collagen and increased turnover of the proteoglycan aggrecan with no increase in matrix degradation9. Moreover the changes that occur in the ankle are not restricted to the lesion site as in the knee10. They are seen throughout the joint11. Together these observations demonstrate that there is a strong reparative response to focal damage throughout the ankle joint, something not seen in the knee where matrix destruction is emphasized. Chondrocytes in the knee are more sensitive than those in the ankle to the harmful effects of IL-1, a cytokine considered to be important in cartilage pathology in osteoarthritis12 the superficial cells being most sensitive with a greater number of high affinity receptors for IL-113. These structural, metabolic and mechanical differences could account for the much higher incidence of osteoarthritis in the knee compared to the ankle. Regional differences within joints also exist. In the case of the adult bovine knee joint significant differences exist in femoral condyles for proteoglycan content, this being higher in the lateral femoral condyle than the medial and femoral condylar groove14. Collagen content is higher in the groove than in condylar cartilage as is pyridinoline cross-linking. This is reflected in higher tensile strength in the groove although failure strains are similar in these different joint sites. All these differences no doubt reflect the different mechanical environments acting upon chondrocytes and determine how they may respond in different joint locations to create a matrix that is best suited to that environment. The marked differences between the ankle and knee articular cartilages provide insights that can better help us understand the development of osteoarthritis., Introduction Focal cartilage damage in the knee is most commonly caused by trauma and osteochondritis dissecans. The choice of treatment of such a cartilage lesion depends on a variety of factors. Operative cartilage repair techniques usually consisted of debridement and microfracturing the defect (1,9). Autologous chondrocyte implantation (ACI) and osteochondral autograft transplantation (OAT) are also advocated (1–4). These different techniques all claim substantial clinical improvement and the available literature does not provide evidence to declare one technique superior to another. On the contrary, the few randomized controlled studies available comparing these three techniques conclude with conflicting results (1,4,9). We focus on the options and limitations of osteochondral autograft transplantation in an attempt to facilitate appropriate patient selection for this specific treatment option. Biomechanical Aspects The technique of osteochondral autograft transplantation is based on the concept of transplanting vital hyaline articular cartilage from a minor weight bearing area of the (knee) joint to a chondral defect in the weight bearing area. Thus, the clinical problem of a large articular defect in an important area of the joint is addressed with a solution where a larger number of relatively small defects in a less symptomatic part of the joint is accepted. Since the fixation options of hyaline articular cartilage to the subchondral bone remain insufficient, the cartilage has to be transplanted as an osteochondral plug. There are several biomechanical dilemmas' to address with this technique. For example, the osteochondral plug has to be long enough to allow stable fixation in the recipient hole. Without a stable press fit fixation the plug will not incorporate in the host bone with eventual failure of the transplant. In addition, the articular surface has to be reconstructed as anatomically as possible. Various cylindrical plugs are used to resurface the joint (mosaicplasty). Careful positioning of the transplanted osteochondral plugs is mandatory to achieve a congruently reconstructed joint surface. Protruding plugs may introduce peek stresses on the graft and thus lead to early degeneration. On the other hand, it is equally important not to place the plugs below the recipient joint surface, since this will lead to peak stresses on the border. Surgical technique is thus critical to achieve an optimal reconstruction of the lesion with intact hyaline cartilage and a congruent joint surface to allow an adequate load distribution. We performed cadaver studies on the optimal surgical technique for osteochondral autologous transplantation in human knees. One study focussed on the optimal length of the osteochondral plug to achieve adequate press fit stability (6). In addition, it was hypothesized that transplanted plugs would have more intrinsic stability when the length of the graft was matched exactly with the depth of the recipient hole. This way the plug was ‘bottomed’ in the defect and would therefore be less susceptible for subsidence. Different lengths of (un)bottomed osteochondral plugs were tested using compressive forces from a loading apparatus. It appeared indeed that longer plugs needed higher forces to begin displacement. At flush level, bottomed plugs needed significantly higher forces than unbottomed plugs to become displaced below flush level (mean forces of 404 N and 131 N, respectively), especially when short plugs were used. In clinical practice we therefore recommend to use short bottomed plugs. If, however, unbottomed plugs are still chosen, the longer the plug the higher the resulting stability will be because of higher frictional forces. In a second biomechanical cadaver study surface congruency after osteochondral transplantation was evaluated (7). Restoration of surface congruency and stability of the reconstruction may be jeopardized by early mobilization. To investigate the biomechanical effectiveness of osteochondral transplantation, we performed a standardized osteochondral transplantation in eight intact human cadaver knees, using three (un)bottomed cylindrical plugs. Surface pressure measurements with Tekscan pressure transducers were performed after five conditions. In the presence of a defect the border contact pressure of the articular cartilage defect significantly increased to 192% as compared to the initially intact joint surface. This was partially restored with osteochondral transplantation (mosaicplasty), as the rim stress subsequently decreased to 135% of the preoperative value. Following weight bearing motion two out of eight unbottomed mosaicplasties showed subsidence of the plugs according to Tekscan measurements. This study demonstrates that a three-plug mosaicplasty is effective in restoring the increased border contact pressure of a cartilage defect, which may postpone the development of early osteoarthritis. Unbottomed mosaicplasties may be more susceptible for subsidence below flush level after (unintended) weight bearing motion. Histological aspects Various studies have shown good clinical results from mosaicplasty of the weight-bearing part of the femoral condyle (1–4). After 10 years of follow-up, good-to-excellent results have been described in 92% of 597 treatments with mosaicplasties (2). Despite these enthusiastic reports on mosaicplasty, there is still concern about the fate of transplanted cartilage and the repair potential of the donor site defect (11). Most studies have reported sufficient repair of both the original cartilage defect and the donor site defects on (occasionally performed) repeated arthroscopy (2). Unfortunately, these studies have been based mainly on data from subjective clinical scores. Histology is of course difficult to obtain for ethical reasons. Occasionally, limited histology from a needle biopsy of the graft has been described, with promising results. We report on a good quality full-thickness histological specimen from an entire osteochondral transplantation (5). The sample could be obtained for histological evaluation because of a total knee arthroplasty performed 3 years after the mosaicplasty. Histology of the recipient site showed vital grafts with excellent incorporation in the subchondral bone. The transplanted cartilage had retained its hyaline structure and seemed to provide a good resurfacing of the joint. The donorsite defect, however, showed only limited repair with fibrous tissue filling of a persistent subchondral defect. In clinical practice the donor site defects are commonly left empty, and spontaneous repair with fibrocartilagenous tissue is assumed. From earlier animal experiments on donor site defects, this potential for spontaneous repair appears to be very limited (11). In an attempt to stimulate the repair of the donor site defect an animal experiment was repeated in the goat. A standardized osteochondral donor defect was created in the knee. Defects were addressed by transplanting an extra-articular osteo-periosteal plug from the proximal tibia to the donor site defect. Empty defects and defects with a plug without covering peristeum served as controls. Incorporation of this graft into the subchondral bone was observed; however, no chondrogenesis from the covering periosteum could be detected. In this animal model no substantial benefit could be detected from this approach, except perhaps some structural support to the adjacent subchondral bone, preventing early collapse. We believe that the repair potential of the donor site defect, as well as the value of an additional osteoperiosteal plug from the proximal tibia, must not be overestimated. A second animal model in the goat focussed on the vitality of transplanted osteochondral plugs and the susceptibility of the transplanted plugs to subsidence. A standardised two-plug (ø 6 mm) osteochondral transplantation was performed in the goat knee. Histological evaluation is currently performed on fuorochrome vitality staining of the transplanted articular cartilage cylinder, trochlear donor site defect repair and subsidence of bottomed versus unbottomed plugs. The overlying articular cartilage clearly survives the transplantation procedure of the osteochondral plug, as appears from the diffuse green staining of the chondrocytes. No red staining, representing cell death, was observed at the (cutting) edges or at the surface. Some subsidence of the plug could be observed in a number of unbottomed plugs (personal data). Clinical aspects Osteochondral lesions in the knee remain a clinical challenge and do not not heal spontaneously in adults. There is not yet an optimal treatment intervention defined due to the limited amount of prospective research available. Various studies have reported on good results in a larger number of patients using different techniques (1–4). The main limitation of these studies is the heterogeneity of the treated cartilage defects. We treated a small subgroup of patients with homogenuous OD lesions with OCT and also reported on a significant improval (p⇠0.003) on various clinical scoring systems (ICRS package) (10). Prospective follow-up MRI was also performed using a semi-quantitative scoring system. Magnetic resonance imaging evaluation at 1-year follow-up showed good surface congruency, no edema or protuberance of the cylinders, good similarity of cartilage thickness and a non-complete osseous integration. Despite these promising clinical results, the potential for donor site morbidity remains a concern with OCT. Prospective bone scintigraphic evaluation both for the donor and the recipient site was performed in patients who were treated with OCT (8). In a group of 13 patients with a symptomatic articular cartilage defect bone scintigraphies were obtained pre-operatively, one year after osteochondral transplantation and finally at an average follow-up of 4 years (31–65 mnths). We learned that elevated bone scintigraphic activity from an osteochondral lesion in the knee could be restored with OCT. However, increased scintigraphic activity was introduced at the donorsite, which activity reduced again with longer follow-up. The use of fairly large osteochondral plugs appeared to correlate with retropatellar crepitus and increased scintigraphic activity and therefore is not recommended. Conclusions We conclude that osteochondral transplantation remains a valid treatment option in selected cartilage defects. A subgroup of osteochondral defects on the lateral border of the medial femoral condyle with a limited size improves significantly following osteochondral transplantation. Donor site morbidity remains a concern and clear limitation with this technique. Donor site problems can be controlled using a limited amount of plugs (maximum 3–4) with a diameter of no more then 8 mm. Future research should probably focus on identifying the appropriate choice of operative treatment (OATS, chondrocyte transplantation or microfracture) for well defined subtypes of articular cartilage lesions, instead of searching for one superior technique for all., Cartilage is widely distributed throughout the human body and is comprised of a combination of connective (or skeletal) tissue cells and extracellular matrix [1]. The specific organization of the various cartilaginous tissues is related directly to the temporal and spatial functional demands on the tissue, both static and dynamic. Generally, these functional demands pertain to the following: (1) the protection and support of related non-skeletal tissues and organs, (2) the articulations between skeletal elements, and (3) the dynamic processes related to skeletal growth [1]. Articular cartilage is an extremely important mechanical entity in articulating joint function where it provides a wear-resistant surface for one diarthrodial element to slide over the other [2]. Other types of cartilage tissues fulfill mechanical function as well, although different from that of the articulating surfaces of joints. Cartilage of the intervertebral disc acts as a load transmitter and shock absorber between bony vertebral bodies [3]. The functional roles of cartilage in the trachea, nose, ribs, ears, and pharynx involve maintaining form and resisting deformation, while providing some degree of flexibility [4]. Because of these differences among the cartilage tissues, the extracellular matrix, which possesses a defined biochemical composition and confers specific biomechanical properties, is composed differently. Whereas a large body of work has focused on repair and regeneration of articulating hyaline cartilage, a growing body of research on repair and regeneration of craniofacial and other cartilages offers other paradigms for cartilage repair with some similarities as and differences to be discussed. Cartilage of all types is a relatively simple, but highly specialized, connective tissue consisting of chondrocytes embedded in an extracellular matrix composed primarily of proteoglycans, collagen, and water[1]. Since cartilage has no internal vascular network and, therefore, possesses limited innate ability for repair and regeneration [5–7], injury to cartilage often results in scar formation and permanent loss of structure and function. Nutrition by diffusion rather than through a vascular network, however, allows cartilage to be easily transferred to repair sites and to be used in a multitude of ways. For example, autologous cartilage can be sculpted into delicate structures like an ear, or fill defects and restore contour in areas throughout the face [8–11]. One possible solution for providing quality structural tissue could be to engineer cartilage tissues to meet the anatomic requirements for the repair. As such, the material properties of synthetic or natural compounds could be manipulated to allow the delivery of an aggregate of dissociated cells into a host in a manner that will result in the formation of new functional tissue [12]. Using a sufficient quantity of cells combined or composited with a polymer(s) and transplanted into the defect site could restore normal function. To achieve the desired result, however, one must consider both the properties of the tissue native to the site and the properties of the polymer(s) being used to generate cartilage repair tissue. Although cartilage is a relatively simple tissue containing only one cell type-chondrocytes—the cartilage from different anatomic areas have different structural and functional demands. Entrapped within the extracellular matrix, the chondrocytes continuously produce various macromolecules such as collagen and sulfated glycosaminoglycans to replenish the extracellular matrix according to these anatomical differences. Cartilage can be divided into categories according to the composition of the matrix, and its biological role in the body. Hyaline cartilage, which is rich in type II collagen, can be found in the ribs, trachea, and covering the articulating surfaces of bones where it functions as a gliding surface and shock absorber for skeletal elements[13]. Elastic cartilage, which contains elastin, occurs in tissues such as the external ear, the epiglottis, and portions of the larynx [6]. Fibrocartilage, which is rich in type I collagen fibers, can be found in tissues that are subjected to tensile forces like the outer portion of intervertebral discs, the knee menisci, and in certain ligament and tendon attachments to bone [14]. Specialized cartilage tissues, like those in the epiphyseal growth centers of long bones, contain highly specialized chondrocyte elements that precisely control elongation and mineralization of growing bones [13]. Therefore, the structural and normal biological function of each of the diverse cartilages should be considered when attempting to engineer replacement tissue. The biochemical composition is closely related to the mechanical factors to which the specific cartilage structures are subjected. Hyaline cartilage in the diarthrodial joints are under constant shear and compression forces, while simultaneously providing low friction across the interface [5,7,13]. The extracellular matrix is designed to absorb compression and return cartilage to a normal state once the force is removed. By contrast, the elastic cartilage in the ear has internal structural support and different extracellular matrix molecules to give the ear an external shape and flexibility [14]. In healing and repair of cartilage, the tensile forces can be a measure of integration of the engineered cartilage with the native cartilage at the defect site [15–17]. Anderson and Athanasiou have reported on generating cartilage for replacement of the temporomandibular joint, which in itself has specific mechanical demands [18]. Consideration of the mechanical demands affecting the tissue will help guide approaches to engineer new cartilage specific to the defect. Placing chondrocytes in three-dimensional matrices, similar to their natural environment, can permit the cells to retain their native phenotype and produce their extracellular components. Using polymers that undergo a controllable bulk erosion process in vivo, the polymer can be made to resorb at a rate proportional to the rate at which cartilaginous extracellular matrix is being deposited into the intercellular spaces. A critical element for engineering cartilage is finding or developing suitable scaffold materials that permit or accelerate the formation of new extracellular matrix according to the cartilage tissue desired. Using polymers, both natural and synthetic, that undergo controllable bulk erosion or resorption can be favorable for engineering cartilage tissues in vitro or in vivo. For example, polymers that degrade at a rate proportional to which cartilaginous extracellular matrix is being deposited into the intercellular spaces could be employed to generate cartilage in situ. Several scaffolds, both natural and synthetic, have been tested in animal models for engineering cartilage. Xu et al. have reported on the formation of cartilage from different anatomical sources and differences in mechanical properties of the neocartilage [19]. Investigators have demonstrated numerous techniques for improving the biological and biomechanical properties of tissue-engineered cartilage including techniques to: 1) to improve the bioproperties of extracellular cartilaginous matrix, 2) provide internal support to tissue-engineered cartilage, and 3) add external (pseudoperichondrium) support to tissue-engineered cartilage. One objective is to improve the flexibility of tissue-engineered cartilage framework, particularly in tissues other than joint cartilage. Some possibilities to enhance the neocartilage matrix properties by incorporating non-resorbable materials to meet the needs for reconstruction will be discussed. Cao et al. engineered cartilage in the shape of human auricles in nude mice using articular chondrocytes and a biodegradable internal PGA/PLLA scaffold to attain the desired shape of an ear [20] demonstrating that tissue-engineered cartilage could be generated with a resorbable endoskeletal scaffold. There have been no reports of successfully generating complex three-dimensional cartilage structures using PGA or PLLA in immune-competent animals, however, possibly due to intense inflammatory responses. Recent work by Kusuhara et al. has improved on the generation of three-dimensional human ear shapes using a co-polymer of poly(L-lactic acid) and poly(e-caprolactone) and costal, articular and nasal septal chondrocytes from bovine sources [21]. Arevalo-Silva and colleagues [22] investigated the use of nonbiodegradable endoskeletal scaffolds made from the following materials: 1) high-density polyethylene, 2) soft acrylic, 3) polymethylmethacrylate, 4) extrapurified silastic and 5) conventional silastic. They concluded that using a permanent biocompatible endoskeleton demonstrated success in limiting the inflammatory response to the scaffold, especially the high–density polyethylene, acrylic, and extrapurified silastic. Our laboratory examined the mechanical function of perichondrium in ear cartilage. We found that intact perichondrium prevented fracture of ear cartilage tested [14]. From these studies we concluded that providing a perichondrial layer was important for confer flexibility to engineered cartilage tissue intended for craniofacial reconstruction. To simulate a perichondrial layer, we investigated expanded polytetrafuoroethylene and lyophilized perichondrium as structural components for supporting the engineered cartilage. Recent work by our group has evaluated the use of rigid nondegradable materials, such as porous polyethylene, enhance the structural stability of ear cartilage. Similar hybrid approaches could be employed for generating tissues like the meniscus or vertebral discs. In summary, the primary goal of engineering cartilage as a therapeutic approach is to restore the physiological conditions of an affected or defective tissue in the body and the neotissue should possess the organization related to the specific structural and mechanical demands of a particular anatomical region., Introduction Blood has been considered an armful factor for the articular cartilage. Previous studies have demonstrated that recurrent intra-articular bleedings represent a negative factor for articular cartilage, inducing a deterioration of the tissue. This could ultimately lead to degenerative osteoarthritis, even though the mechanism is not yet entirely understood (1–3). Other authors have investigated the effect of peripheral blood on articular cartilage both in vitro and in vivo: Roosendal and Hooiveld have shown that a short-term exposure of human articular cartilage to whole blood in vitro induced an irreversible dose-dependent inhibition of proteoglycan synthesis and it was accompanied by cell apoptosis (4,5). However, when a short-term exposure was performed in vivo after injection of autologous blood into the canine knee, the initially adverse changes in cartilage proteoglycan synthesis turned into normalization after 10 weeks (6). Recently, the same group has tested the threshold of blood exposure time and concentration that lead to irreversible joint damage (7). However, many current surgical procedures for articular cartilage repair, like subchondral bone drilling (8), abrasion artrhoplasty (9) and microfracture (10), are based on the capacity of bone marrow cells to produce a fibrocartilaginous tissue when migrated in a joint environment (11). In contrast, in the performance of the techniques based on the transplantation of autologous chondrocytes, the presence of blood has been considered a disturbing factor for the development of the new cartilage tissue (12). The more recent techniques for cartilage repair and reconstruction utilize autologous chondrocytes seeded onto a biocompatible scaffold, in which they can duplicate, mature and produce new cartilage matrix in vitro and in vivo after surgical implantation. Also in this approach, it is recommended care in protecting the reparative cells from the contact with blood, which could derive from the subchondral bone or any other part of the joint injured during the surgical implantation (13), both in open and arthroscopic approach. However, the nature of engineered cartilage differs from that of cartilage explants examined in the studies mentioned above. Therefore, we believe that the influence of the contact of peripheral blood to the engineered cartilage represents an important but still unclear issue that needs to be investigated. The engineered cartilage, however, is structurally and biologically different from native articular cartilage, as it is supposed to complete the maturation in vivo. Therefore, it is probably more susceptible to the adverse effects of an articular bleeding, as indicated by studies of other authors, who investigated the effect of blood on immature joint (14). The effect of blood on engineered cartilage was only recently investigated (15). We have developed an in vitro model to investigate the effect of blood contact on the tissue-engineered implant and demonstrated that a 3-day exposure of cartilage to 50% (volume/ volume) blood results in a temporary and reversible effect on engineered cartilage tissue obtained from chondrocytes seeded onto collagen scaffold. However, some important issues remain to be clarified: could the different blood concentration negatively affect the chondrocytes' vitality and synthetic properties? Is the negative effect of blood on the engineered cartilage due to the toxic effect of the peripheral blood or to the lack of nutrients occurring during the exposure to blood? The aim of this study was to investigate the effect of different concentrations of blood and of the lack of nutrients on the morphological, biochemical and biomechanical properties of engineered cartilage, synthesized by articular chondrocytes seeded onto a biological scaffold. Additionally, we have analyzed the effect of the main pro-inflammatory chemokine IL-1p on engineered cartilage based on human articular chondrocytes cultured for two different culture times. Experimental data Tissue engineered cartilage was developed combining expanded chondrocytes with a collagen membrane scaffold. Two sets of experiments were performed: one testing swine chondrocytes and allogeneic blood as inflammatory factor (“Blood study”); the other testing human adult chondrocytes and IL-1β (0.05 ng/ml) (“IL-1 study”). In the “Blood study”, articular chondrocytes were isolated from swine joints, expanded in monolayer culture, seeded onto collagen membranes and cultured for 2 weeks. During this period, an immature extracellular matrix, produced by cells, stabilized the chondrocytes to the biological membrane. This method generally duplicates the protocol for membrane seeding used in current clinical practice. After 2 weeks (t0), some samples were retrieved for analysis; others were exposed for 3 days to the contact with swine peripheral blood diluted with culture medium at 2 different concentrations (B50% group = 50% blood / 50% medium; B80% group = 80% blood / 20% medium); others were exposed for 3 days to the contact with a PBS solution. Following these 3 days (t3), some samples were retrieved for analysis, others were returned to standard culture conditions for 21 additional days (t3+21), in order to investigate the “long term effect” of the blood contact. For all the listed experimental times, some samples belonging to the control group were left in standard culture conditions without having any contact with blood or PBS. All groups of seeded membranes were analyzed grossly, by optic microscopy (OM), biochemically, histologically, and by biomechanical test. In particular, for morphological analysis, samples were analyzed macroscopically and by OM at all experimental times. Samples were weighed and sized with a calliper (products of axis) upon retrieve from culture. The edge of membranes was evaluated by OM analysis. For biochemical analysis, rate of cellular proliferation was evaluated by mitochondrial redox reaction to the tetrazolium salt (MTT) at all experimental times. For histological analysis, samples were processed and stained with safranin-o. Few sections (only at the time t3+21) were processed for immunohistochemical analysis and stained for type II collagen. For biomechanical analysis, samples were tested under unconfined geometry for compression using an electromagnetic testing machine. In the IL-1 study, human articular chondrocytes were harvested post-morted, expanded in monolayer, seeded onto collagen I/III scaffolds and cultured for 2 and 4 weeks. IL-1 was added during the last 3 days of culture. Samples were analyzed with histology, immunohistochemistry for collagen II, and biochemistry. In the “Blood study”, all seeded samples showed an increase in the weight and an evident cartilage-like matrix production. The evaluation of the samples with an optic microscope showed similar results for all study groups. It demonstrated the presence of spherically-shaped cells homogeneously distributed around the edge of the samples and firmly attached to the membranes. A specific concentration-dependent reduction of the mitochondrial activity due to blood contact was evident at the earlier culture time, followed by a partial recover at the longer culture time. An initial reduction of the biomechanical properties of the membranes, followed by a late stabilization, was recorded, regardless the presence of blood. In the “Il-1 study”, all samples exposed to IL-1 demonstrated a reduction in the intensity of Safranin-o and collagen II staining and in the quality of the biochemical composition. This reduction was more marked for the samples cultured for 2 weeks only, while samples cultured for 4 weeks had a better response to the pro-inflammatory stimulus. Conclusion The results from this study demonstrated that isolated chondrocytes could be seeded onto a biological scaffold, producing cartilage-like matrix with tissue specific morphology, composition, and biomechanical integrity. The blood contact seemed to produce a delay in the weight increase of the samples with respect to the control group. The analysis of the mitochondrial activity seemed to indicate a negative effect of the blood contact on the seeded membranes. In fact, samples exposed to a medium diluted with 80% and 50% blood recorded a depression of the MTT values with respect to group C. The same negative effect was recorded for PBS group where the lack of medium nutrients was probably the cause of the reduction of the chondrocytes' vitality. However, the negative effect of the blood contact was specific, because the samples exposed to a medium diluted with different quantity of blood showed different depression of the MTT values. At the longest time period (t3+21), the cellular activity of the blood groups increased almost reaching the values achieved before the blood contact. This indicates that the toxic effect on the chondrocytes was temporary and reversible. The analysis of the biomechanical data did not support these evidences, because the biomechanical results were not affected by blood contact. It could be hypothized that exposure time was not long enough to produce differences in the biomechanical properties of the constructs. We can conclude that the negative effect of the blood on the engineered cartilage is evident at the cellular level. However, it does not seem to be perceptible at the (engineered) tissue level, with the model utilized here. Three days of exposure did not entirely devitalize the tissue cells and did not seem to influence the synthetic properties of the chondrocytes and the biomechanical integrity of the immature cartilage at the longest time point. In the “IL-1 study” the negative effect of IL-1 seemed to be more marked than that of blood in the “Blood study”, although a direct comparison of the 2 model results very speculative. Moreover, this negative effect was clearly dependent on the level of maturation of the construct. A further in vivo study is probably needed to investigate the potential facilitation of the biological environment in reversing the negative effects of bleeding on the cells of the engineered cartilage as shown by other authors for native articular cartilage (14). Future studies are also desirable to test the effect of the different level of maturation of the engineered cartilage on its capacity of surviving and integrating in a joint environment exposed to bleeding., Lesions of the meniscus are frequently observed in orthopedic practice. Injury or loss of meniscal tissue potentially leads to pain, knee dysfunction, and osteoarthritis at long term. [4, 16] In cases of extensive destruction and complete loss of the meniscus, only two methods are available in clinical practice today for meniscal substitution: allograft transplantation and collagen meniscus implantation. However, their long-term success, durability, safety, and chondroprotective effects are still uncertain.[10] Several materials have been tested as partial meniscus substitutes in animal models. Total meniscus substitution remains difficult and its chondroprotective effect has been poorly described in the literature. A polyvinyl alcohol-hydrogel meniscus in rabbits showed promising results in terms of chondroprotection, but problems persisted, such as durability of the polymer, the fixation method, and complete tissue regeneration in a material that does not adhere to tissue.[3, 5] Welsing and van Tienen et al.[11, 15] published a 2-year follow-up study of a degradeable poycaprolacton-polyurethane meniscus implant in dogs. Although they showed promising results in respect of tissue formation a final remodelling into a neomeniscus was not possible because the polymer was still present after 24 months. Cartilage degeneration was not prevented. Tissue engineering with application of cells has recently been proposed as a possible solution for meniscal regeneration aiming at a more stable construct, thus preventing cartilage degeneration more effectively.[8, 9, 12–14] Walsh et al.[13] used a collagenous sponge loaded with mesenchymal stem cells to heal a partial meniscus defect in rabbits and reported that the presence of cells augmented the repair process but did not prevent degenerative osteoarthritis. Martinek et al.[8] reported better macroscopic and histological results in CMI implants seeded with meniscal fibrochondrocytes in comparison to cell-free implants in sheep. However, the tissue-engineered meniscus was biomechanically unstable and the implant size reduced during the 3-months observation period. Different cell sources have also been analyzed in vitro to find the most suitable source for cell augmentation of tissue-engineered meniscus.[2, 7] The surgical technique used in animal models for total meniscal replacement has also not been highly investigated and consistent, as authors use different animal models (rabbit, dog, and sheep) and diverse surgical techniques. We tried to develop a novel approach towards meniscus substitution and developed a new implant made from polycaprolactone and hyaluronan. In a pilot study, the implant was tested for total and partial meniscal substitution in sheep for 6 weeks. Tissue integration between the joint capsule and the implant was observed with tissue formation, cellular infiltration, and vascularization. At this early timepoint no cartilage protection was observed.[1] Our hypothesis was that the application of a tissue engineering approach, using cells seeded onto this scaffold, would offer some benefits in patients submitted to total meniscectomy by increasing the biological response and remodeling processes. Therefore, the aim of the following study was to investigate the feasibility of using this novel material for meniscal tissue engineering and to evaluate the tissue regeneration after the augmentation of the implant with autologous articular chondrocytes expanded ex vivo. The secondary aim was to evaluate two different surgical scaffold implantation techniques in an animal model: suture to the capsule and to the meniscal ligament, with or without transtibial fixation of the horns. The animals were sacrificed after 3 and 12 months. All implants showed excellent capsular ingrowth at the periphery. Macroscopically, no difference was observed between cell-seeded and cell-free groups. Better implant appearance and integrity was observed in the group without transosseous horns fixation. Using the latter implantation technique, lower joint degeneration was observed in the cell-seeded group with respect to cell-free implants on a macroscopic level. The histological analysis indicated cellular infiltration and vascularization throughout the implanted constructs. Cartilaginous tissue formation was significantly more frequent in the cell-seeded constructs. Mankin Scores were not significantly different between cell-seeded and non-cell-seeded implants and they were not better in treated sheep than those of control sheep.[6] To date there is no evidence that meniscus substitution is capable of preventing osteoarthritis. However, the studies show promising results and it became obvious, where developments need go. Enhancement of biomechanical stability, proper selection of cell sources and cell augmentation techniques as well as special surgical techniques will have to be the goal of future research., Articular cartilage has been notorious for its limited healing potential for centuries. In order to enhance the cartilage repair the tissues engineering (TE) concept was introduced into the orthopedic surgery 15 years ago. The classical technique of autologous chondrocyte implantation (ACI) and its 2nd and 3rd generation upgrades proved to be successful in the treatment of small to mid-sized traumatic or osteochondritis dissecans lesions. The outcome of TE methods in degenerative cartilage lesions was inferior, therefore only small and contained degenerative lesions may be treated at present1, 2. As the number of young and active patients with debilitating joint disease is increasing steadily, the currently available cartilage regeneration or substitution techniques need further development3. The possible future applications will be reviewed in the lecture. When addressing a degenerative cartilage lesion the surgeon is dealing with a disease of all joint structures (cartilage, subchondral bone, menisci, ligaments, synovia, and capsule) due to genetic and environmental factors4. A successful cartilage regeneration or substitution must be focused also to the inflammation and mechanical issues together with the joint resurfacing3. Persistent high levels of synovial fluid markers after cartilage repair in non-degenerative lesions lesions, which may indicate graft remodeling or early degeneration, suggest that the resurfacing alone cannot entirely stop the disease progression5. At the final point the cartilage regeneration or substitution needs to promote the anabolic events over the catabolic degenerative mechanisms. Certain conservative and joint reconstruction strategies for the treatment of degenerative joint surfaces have been used for decades. Thank to these strategies the degenerative joint process may be transiently slowed down. They need also to be applied prior or together with the next cartilage resurfacing by regeneration or substitution to provide as stimulating environment as possible3. First, the treated joint has to be systemically prevented from the repetitive injuries or overuse by weight balance, activity modification, and regular exercise programs. Second, the joint reconstruction includes ligament repair/reconstructions, meniscus reconstruction, and unloading procedures6. Since the degeneration is an ongoing process, the treatment requires a proper timing. Any cartilage TE requires a cell source. Today the mature chondrocytes from outside the injured area are used as gold standard. If the same technology is transferred into the degenerative milieu, the source cells will also be diseased. The chondrocytes in OA are lower in number, they have different capacity to proliferate in vitro, their response to growth factors is different, and they express significantly higher levels of matrix degrading enzymes3. In spite of these drawbacks Tallheden et al. demonstrated that OA chondrocytes retain their differentiation potential upon isolation and proliferation in vitro which could make them suitable for cartilage TE7. Mesenchymal stem cells (MSC) were able to differentiate into different tissues in animal studies, but the processes could not be exactly controlled. The clinical evidence of MSC usage in cartilage is limited and no specific applications for degenerative lesions are available. Scaffolds represent another key component in cartilage regeneration or substitution. They are of different origins and can be divided into four chemical classes: protein-based polymers, carbohydrate-based polymers, artificial materials, and combinations. A scaffold needs to be sterile, biocompatible, biodegradable, and mechanically stable to protect seeded cells during the implantation and early postoperative rehabilitation. Numerous scaffolds were successfully used in clinical settings for focal lesions showing comparable results to classical ACI, but enabling quicker and less invasive operative procedures8. Unfortunatelly, no comparative studies between scaffolds are available and none of the scaffold has been specifically designed for the degenerative joint lesions. Growth factors are not routinely used in the cartilage repair surgery today, but the evidence on their positive influence is growing. They will be unavoidable in the reconstruction of a degenerated joint. BMP-7 has been most extensively studied and it has the highest clinical potential at the moment. It demonstrated a strong pro-anabolic and an anti-catabolic activity and a good safety profile in animal studies9. Other mixtures of growth factors will be combined in the future. They could be delivered as recombinant proteins with cells/on the scaffold or as genes in genetically modified cell. To summarize, the degenerative lesions are not an isolated problem, but they constitute a piece in the spectrum of a whole joint disease. There are no successful cartilage regeneration or substitution methods available at present for degenerative lesions. It seems that many old and new strategies will be combined for a successful and stable outcome. However, any planned strategy for the degenerative knee lesions has to meet patient's expectations and agreement on the post-operative protocol., Introduction When confronted with a young patient who has an old knee several parameters have to be assessed: the alignment of the limb, the stability of the knee joint, the status of the menisci and the extent of articular cartilage damage. For each deranged parameter, a validated surgical approach exists. Malalignment can be corrected with osteotomy, stability can be restored by ligament-reconstruction, and meniscal transplantation or cartilage repair surgery can reestablish the load distribution and shock absorption of the knee joint. When all these interventions fail their goals, unicondylar or total knee arthroplasty is the ultimate alternative. Alignment In the young active patient, high tibial osteotomy (HTO) can protract the implantation of a total knee arthroplasty. The frequency of HTO has diminished due to the rise in total knee arthroplasties. Few data are available about the long-term follow-up of HTO. Gstottner et al presented their results of 134 lateral closing wedge osteotomies with survival rates of 94%, 79.9%, 65.5% and 54.1% at respectively 5,10,15 and 18 years. Age was the only parameter with significant influence on survival contrary to gender and mechanical axis. 10 The long-term results in an older population (mean age 69 years) of 76 HTO showed the best survival of 90% at 10 years (with arthroplasty as an end point) when X-ray valgus angle at 1 year postop was between 8–16°. The overall survival at 10 years was 74%. 15 A survival analysis on 67 knees showed a cumulative survival probability of 89.5% at 5y, 74.4% at 10 years and 66.9% at 15 and 20 years. More than 90% of patients had an improvement in pain score and would have the operation again.16 Another well-established indication for HTO is medial, unicompartmental cartilage degeneration in patients with varus malalignment. Studies have led to 8–10° of valgus alignment as widely accepted. A recent laboratory study indeed showed a decrease in contact pressures and contact area when the alignment was shifted from varus to valgus. All contact pressure is shifted to the lateral compartment between 6–10° of valgus. This is regardless of condylar width, baselinge alignment, body weight or chondral defect size. 11 Based on these findings, one could say that HTO could be combined with autologous chondroctye implantation (ACI) for patients with varus malalignment and chondral defects. A small cohort study of 8 patients who underwent ACI and medial opening wedge HTO, showed favorable results at 28 months.7 Stability Anterior cruciate ligament (ACL) reconstruction has been performed for more than 20 years. It is a widely accepted procedure to restore the stability of the knee joint. Some authors hypothesized that a concomitant meniscal allograft transplantation (MAT) could improve the results in patients with an injured medial meniscus and ACL. Short-term results of ACL-reconstruction + MAT were promising and not different from MAT alone in a stable knee. 14, 18 Long-term results of small patient numbers showed a significantly improvement of knee function in symptomatic meniscus-deficient knees. The addition of an ACL-reconstruction probably improved these results.9 ACL-reconstruction combined with ACI results in sustained improvement in pain and function at short-term. ACL-reconstruction + ACI gives better results than ACI after a successful ACL-reconstruction.1 Shock absorption To restore the impaired function of damaged menisci or articular cartilage, both meniscal allograft transplantation (MAT) and autologous chondrocyte implantation (ACI) are procedures with good to excellent short to long-term follow-up. MAT for articular cartilage treatment is not comparable to ACI and gives worse results on long-term. 3 Recently some reports have evaluated the results of a combined MAT+ACI procedure. Short-term results for the combined intervention showed statistically significant improvements in outcome scores, but not always better results compared to the procedures in isolation. 6, 13 This is readily explained by the indication to perform a combined procedure, which allows to operate on patients with worse defects that would otherwise be contra-indications for one of the procedures in isolation. Ultimate alternative In some young patients, even combined procedures are not an option or fail prematurely. Total knee arthroplasty is then the only way out. The unicondylar knee arthroplasty (UKA) can be used in selected patients with a well functioning stability system of the m uscles and ligaments and of course an isolated medial gonarthrosis. It has been shown that a deficient ACL results in higher failure rates after UKA. 5 Combined ACL-reconstruction and unicondylar knee arthroplasty, restoring the knee stability and keeping the advantages of uni versus total knee arthroplasty, yield encouraging short-term results.17 Long-term results of TKA show reliable and durable results at 18 year follow-up with an estimated survival of 100% at 15 years and 93.7% at 20 years in one study on patients less than 55 years with rheumatoid arthritis. All patients received a cemented condylar prosthesis. 2 Another study showed survival rates of 96% at 10 years and 85% at 15 years in 52 patients with OA, all 55 years or younger. A press-fit condylar knee system was used. 4 A series of 80 knees in 63 patients who received a mobile bearing knee prosthesis showed a cumulative survival rate of 96% at a 12-year maximum follow-up. 12 Finally a prospective follow-up of 1047 patients of 55 years or younger in a community registry showed the best survival rate for cemented TKA with 85% survival at 14 years. Cementless designs and unicondylar knee arthroplasty independently increased the revision risk.8 Conclusions HTO has the ability to protract the implantation of a TKA and to improve the patient's quality of life with high satisfaction rates. Combined HTO + ACI seems to be a viable option for medial chondral defects. MAT+ACL-reconstruction may probably improve stability and even provide protection for the cartilage. The anterior cruciate ligament and the medial meniscal allograft seem to protect each other both ways. ACL-reconstruction + ACI gives better results than ACI after a successful ACL-reconstruction Combined MAT+ACI could neutralize some contraindications for MAT or ACI in isolation and offers a safe alternative with acceptable results on short-term. Patients in which none of the interventions above are optional, knee arthroplasty remains a final solution. UKA should only be done in stable knees or in association with an ACL-reconstruction. The long-term results of TKA in young patients are good to excellent., The predominant functions of articular cartilage are mechanical in nature, providing both protection for the subchondral bone and, in association with the synovial fluid, an highly efficient low friction articulation during joint activities. Consequently researchers have attempted to provide a complete mechanical characterization of articular cartilage in heath and disease, with seminal studies having been reported over 60 years ago. This research has been enlivened by the research interest in providing a design template for mechanical parameters critical for the evaluation of tissue engineered repair strategies for articular cartilage, which will provide long-term functionality post implantation. Early laboratory-based tests revealed that the nature of the testing certainly influenced the mechanical responses. As an example, to ensure simulation of physiological situations, cartilage had to be tested in an hydrated state. In addition, the heterogeneous structure of the tissue contributes to the variations in mechanical properties with cartilage depth, as evidenced by tensile testing of thin cartilage specimens, sectioned parallel to the articular surface. These depth-dependent variations also had a major influence on the boundary conditions associated with compression testing. This has led to two separate approaches involving osteochondral constructs, full-depth cartilage constructs with thin section of subchondral bone, being tested in either confined or unconfined compression. The former approach involves fluid movement restricted to one direction through the porous loading platen. Nonetheless it lends itself to modeling using an assumption of isotropic biphasic behaviour, which yields material parameters, namely Young's modulus, aggregate modulii and hydraulic permeability. By contrast, unconfined compression permits lateral fluid movement through the sides of the cylindrical constructs and barreling between the impermeable loading platens. Similar values of Young's modulus have been estimated, approximately 0.80 MPa, from cartilage explants from the bovine humerus (Korhonen et al., 2002). The use of isolated constructs also permits the examination of structure-function relationships, often with the use of proteolytic enzymes to selectively degrade the ECM components of articular cartilage. To minimize the changes associated with sample excision from its natural state, an alternative in vitro approach involves examining the response of articular cartilage to indentation. This testing mode has also proved problematic, particularly in ensuring that the axis of indentation was perpendicular to the articular surface. Two geometries of indenter have generally been employed, using either a fat ended or an hemispherical probe. An analysis of each was provided in a seminal paper, which assumed that the loading was transmitted to an infinite isotropic elastic material on a rigid substrate (Hayes et al. 1972). Nonetheless, the complex nature of the internal strain field within the heterogeneous tissue under an indenter necessitates the determination of structural parameters associated with force-deformation behaviour. To covert to material properties both the local thickness of the cartilage and the exact nature of the cross-sectional area of the tissue under the indenter need to be determined. Another important consideration in mechanical testing involves the inherent viscoelastic behaviour of articular cartilage. Accordingly both stress relaxation and creep tests have been performed. However, the long tem creep experiments to determine the equilibrium or aggregate modulus do not simulate the normal dynamic loading patterns in joints. This could by overcome using an underdamped mechanical loading system, where the short-term loading response of unconfined cartilage constructs could yield both the elastic and viscous parameters separately (Bader et al. 1994, 2000). This approach has demonstrated that the elastic response of the loaded cartilage is controlled by the collagen network. It also revealed values of initial compressive modulus of approximately 10 MPa. With the emergence of arthroscopic examination of joint structures, it soon became clear that any testing device that could be used arthroscopically, would enable the in vivo assessment of early tissue changes in localized areas. This was addressed by the seminal work from Jurvelin's group in Kuopio who designed an arthroscopic-based indenter (Lyyra-Laitinnen et al., 1999), which was developed into a commercial product (Artscan Oy, Helsinki, Finland). It consists of a handheld tool, equipped with a mechanical indenter and an integrated force gage at the tip. The force by which the tissue resists the constant indentation is a measure of the cartilage stiffness. Since then a few groups have presented and validated instruments involving indentation during arthroscopy that are capable of measuring compressive stiffness of articular cartilage in vivo (Appleyard et al. 2001; Bae et al. 2003; Niederauer et al., 2004). These instruments have been shown to be sensitive in detecting superficial changes in degenerated tissue. A later development by the Finnish group involved the integration of a miniature ultrasound (US) transducer into the probe tip of the indenter. This enabled measurements of original cartilage thickness, thus permitting strain measurements and enabling a realistic estimation of compressive modulus (Laasanen et al., 2002). With this instrument, cartilage thickness, dynamic modulus and US reflection coefficient of cartilage surface (RUS) can be determined during short-term, clinically applicable measurements. These parameters are sensitive indices to early cartilage degeneration. US reflection at cartilage surface has been shown to be a sensitive and specific measure of the quality of superficial cartilage tissue (Kiviranta et al. 2007). A recent study demonstrated, that RUS was able to discern degeneration of the samples with high sensitivity (0.77) and specificity (0.98) (Kirivanta et al. 2008). US reflection measurement shows potential for diagnostics of early OA, obviating the need for site-matched reference values. In addition, the high linear correlations between indentation and reference measurements suggest that these arthroscopic indentation instruments can be used for quantitative evaluation of cartilage repair post-surgery. The use of such arthroscopic-based techniques, however, is limited by the invasive nature of the procedure, and restricted to joint areas accessible by the probe. However in the last decade the emergence of quantitative magnetic resonance imaging (MRI) techniques probing macromolecular composition and structure has provided the means to indirectly assess the mechanical properties of normal, early degenerated, and surgically manipulated tissue in vivo. Quantitative MRI techniques have successfully been developed to provide measures of the macromolecular environment within cartilage, made possible via the interaction between interstitial water and the macromolecular constituents of cartilage that affect the nuclear magnetic relaxation properties. Indeed the anisotropic behavior of T2 relaxation time of MRI has been correlated with the 3D arrangement of the collagen network and thus might serve as a parameter for assessing the integrity of the collagen network (Nieminen et al. 2000). Indeed, an increase in T2 relaxation time was observed in early symptomatic degeneration of human articular cartilage in vivo (Mosher et al. 2000). An alternative MRI approach has been recently demonstrated to be sensitive to cartilage proteoglycans. This involves a delayed Gadolinium-enhanced MRI technique (dGEMRIC), which yields a value of the relaxation parameter, T1, which can be used in conjunction with T2 to describe up to 87% of the variations in specific biomechanical properties in cartilage (Nieminen et al., 2004). It is evident that this combined approach can provide important information on the mechanical properties of articular cartilage (Kiviranta et al., 2007). The results are encouraging with respect to functional imaging of cartilage, although in vivo applicability may be limited by the inferior resolution of clinical MRI instruments and, of course cost and limited availability., Introduction Limb skeleton develops through the process called endochondral bone formation. In this process, mesenchymal cells condense and differentiate into proliferative chondrocytes that then differentiate to hypertrophic chondrocytes. Hypertrophic cartilage is gradually replaced by bone. Bone morophogenetic proteins (BMPs) have been thought to play important roles in this process. BMP signals are transduced intracellularly by Smad proteins and non-Smad proteins. Along these pathways, several regulatory mechanisms work. Antagonists such as noggin antagonize BMPs extracellularly, and inhibitory Smads such as Smads 6 and 7 inhibit activation of Smad proteins. It was recently reported that articular cartilage expresses inhibitory Smads in arthritic conditions. Methods and Materials To clarify roles of BMPs and their inhibitory factors in cartilage formation, we generated transgenic mice that overexpress BMPs or related molecules in cartilage under the control of the Col11a2 promoter/enhancer. Results Overexpression of BMP4 or GDF5/CDMP1 expanded cartilage and accelerated chondrocyte hypertrophy. Noggin overexpression severely inhibited cartilage formation, suggesting that BMP signals are essential for cartilage formation. Smad6 overexpression delayed chondrocyte hypertrophy but did not affect cartilage formation, suggesting possibilities that non-Smad pathways transduce BMP-mediated cartilage formation. To examine BMP signals in various steps during endochondral bone formation, we generated conditional transgenic mice for Smad7 using Cre / loxP system. Smad7 overexpression inhibited both initial cartilage formation from mesenchyme and later chondrocyte differentiation to hypertrophy. Our in vivo and in vitro results suggested that Smad7 inhibit cartilage formation, at least in part, by down-regulating BMP-activated MAP kinase pathways. Conclusions BMP signals regulate cartilage formation and differentiation at multiple steps., Scaffolds for cartilage tissue engineering serve as a template to guide cells and extracellular matrices to organize into new tissues, provide the tissues with initial mechanical strength, control tissue shape and size, and promote their integration with adjacent tissues. The scaffolds should be biocompatible and biodegradable, allow cell attachment, proliferation and differentiation, facilitate gas exchange, nutrient diffusion and waste metabolism, and be processible into designed shapes. A number of three-dimensional porous scaffolds fabricated from various kinds of biodegradable materials have been developed and used for cartilage tissue engineering. Especially, polymer materials have received increasing attention and been widely used for cartilage tissue engineering. The polymers used to prepare scaffolds for cartilage tissue engineering include biodegradable naturally derived polymers such as collagen, hyaluronic acid, fibrin and alginate, and biodegradable synthetic polymers such as poly(glycolic acid) (PGA), poly(L-lactic acid) (PLLA), poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone. Despite various scaffolds have been developed, significant challenges remain in the development of functional scaffolds. Especially, improvements on mechanical properties, cell seeding efficiency and interconnectivity of scaffolds are strongly desired. Two kinds of collagen porous scaffolds with controlled pore structures and interconnectivity were prepared by template method. One is collagen mesh that was prepared by using a PLGA knitted mesh as a template. At first, a hybrid mesh of PLGA and collagen was prepared by forming web-like collagen microsponges in the openings of a PLGA knitted mesh. And then, the hybrid mesh was immersed in an alkaline aqueous solution and incubated at 37 ° C for 24 h to selectively dissolve the PLGA knitted mesh to obtain the collagen mesh. The interconnected porous structure of the collagen mesh was confirmed by SEM observation. Another collagen scaffold is collagen sponge with open surface structure that was prepared by using embossing ice particulates as a template. Ice particulates were formed on a plate surface and collagen aqueous solution was poured onto them. Collagen sponge was prepared after freeze-drying. SEM observation demonstrated that the collagen sponge had a surface structure of open large pores and a bulk porous structure of small pores. The surface large pores and bulk small pores were interconnected. The open surface and interconnected porous structures facilitated cell seeding and homogeneous cell distribution. The synthetic and naturally derived polymers have their respective advantages and drawbacks. The biodegradable synthetic polymers can be easily formed into designed shapes with relatively high mechnical strength. However, the scaffold surface of these polymers is relatively hydrophobic, which is not good for cell seeding. On the other hand, naturally derived polymers have specific cell interaction peptides, and their scaffolds have hydrophilic surfaces, which are beneficial to cell seeding and cell attachment. However, naturally derived polymers are mechanically too weak. Hybridization of naturally derived polymers with synthetic polymers has been developed to to combine their advantages and to avoid their drawbacks by forming sponge or microsponges of a naturally derived polymer in the openings of a synthetic polymer skeleton. The synthetic polymer skeleton enabled easy formation into the desired shapes and provided the appropriate mechanical strength, while the enclosed collagen sponge or microsponges facilitated cell seeding and cell attachment. Several kinds of such hybrid scaffolds were developed. One example is a hybrid sponge prepared by introducing collagen microsponges in the pores of a PLGA sponge. The PLGA-collagen hybrid sponge was prepared by immersing a PLGA sponge in a bovine collagen type I acidic solution under negative pressure, freezing at – 80 Ë š C, and freeze-drying. The hybrid sponge was further crosslinked by treating with glutaraldehyde vapor and washing with glycine aqueous solution and water. Collagen microsponges were formed in the pores of the PLGA sponge. The ultimate tensile strength, the modulus of elasticity, and the static stiffness of the PLGA-collagen hybrid sponge were higher than those of PLGA and collagen sponges, in both dry and wet states. The second example is a PLGA-collagen hybrid mesh. This was prepared by forming collagen microsponges in the openings of a knitted PLGA mesh. SEM observation confirmed that web-like collagen microsponges were formed in the openings of the PLGA mesh. The hybrid mesh exhibited a significantly higher tensile strength than did the collagen sponge alone, and was similar to the PLGA mesh. The hybrid mesh was used to culture chondrocytes for cartilage tissue engineering. Cartilage with high mechanical strength was regenerated and the size of the engineered cartilage could be controlled. Histological examination of these specimens using hematoxylin and eosin stains indicates a uniform spatial cell distribution throughout all the implants both radially and longitudinally. The chondrocytes showed a natural round morphology in all the implants. The bright safranin-O-positive stain indicated that glycosaminoglycans (GAG) were abundant and homogeneously distributed throughout the implants. Toluidine blue staining demonstrated the typical metachromasia of articular cartilage. Immunohistological staining with an antibody to type II collagen showed a homogeneous extracellular staining for type II collagen. The third example is a cylinder-type PLLA-collagen hybrid sponge prepared by enclosing collagen sponge in a PLLA porous cylinder. The PLLA sponge cylinder was prepared using a method of porogen leaching using a Teflon mold. SEM observation showed that the pores were distributed evenly in the wall and bottom sections of the PLLA sponge cylinder. The inner and outer surfaces had smaller pores than did the cross-section. This might be due to a contact effect of the NaCl particulates and wall of the Teflon mold. The porosity and pore size of the PLLA sponge cylinder were 86.8 ± 0.8% and 84.3 ± 5.5 μ m, respectively. The PLLA sponge cylinder was then filled with aqueous collagen solution by introducing collagen solution into the pores of the cylinder and the central space. The collagen solution-filled PLLA sponge cylinder was freeze-dried and cross-linked to form a PLLA-collagen hybrid sponge. The PLLA-collagen hybrid sponge had the same shape as that of PLLA sponge cylinder. SEM observation demonstrated that collagen sponge was formed in the center of the PLLA sponge cylinder and collagen microsponges were formed in the pores of the wall of the PLLA cylinder sponge. The collagen sponge in the center space was connected with the collagen microsponges in the pores of cylinder wall by collagen fibers that passed through the interstices of the PLLA sponge). The interconnection protected the shrinkage of the central collagen sponge. Micrographs of horizontal and vertical cross-sections of the central collagen sponge indicated its tubelike structure in the vertical direction. The porosity of the PLLA-collagen hybrid sponge was 91.7 ± 3.6%, significantly higher than that of the PLLA sponge cylinder (86.8 ± 0.8%). Introduction of collagen sponge in the central space of the PLLA sponge cylinder increased the total porosity of the hybrid sponge. The PLLA-collagen hybrid sponge showed significantly higher Young's modulus than did that of the PLLA sponge cylinder and collagen sponge alone. These results indicate that the PLLA sponge cylinder served as a mechanical skeleton and reinforced the hybrid sponge. The hybrid sponge was used for the cell culture of bovine articular chondrocytes. Most of the seeded cells were trapped within the hybrid sponge. The seeding efficiency was 96.1 ± 2.1%. The special porous structure with central collagen sponge surrounded with a PLLA-collagen sponge cylinder is thought to be advantageous for cell seeding. The outer layer of the PLLA-collagen hybrid sponge cylinder might protect against cell leakage during cell seeding. The chondrocytes adhered on both the central collagen sponge and the collagen microsponges of the PLLA-collagen hybrid sponge cylinder. The collagen sponge facilitated cell adhesion in the hybrid sponge. Biochemical analysis indicated that the DNA and sulfated glycosaminoglycans (GAG) contents increased with culture time. HE staining indicated that the chondrocytes showed round morphology. Safranin O-positive staining indicated the existence of glycosaminoglycans (GAG), which was abundant and homogeneously distributed around the cells. Toluidine blue staining demonstrated the typical metachromasia of cartilage. Immunohistological staining with antibodies to type II collagen and cartilage aggrecan showed that the type II collagen, and cartilage aggrecan were positively stained. These matrices surrounded the cells. Gene expression analysis by real-time PCR indicated that theã € € chondrocytes expressed type II collagens and aggrecan. The hybrid sponge promoted the formation of cartilaginous tissue when bovine chondrocytes were cultured. To summarize, a few kinds of porous scaffolds of collagen with controlled pore structures and PLGA-collagen hybrid porous scaffolds with high mechanical strength and cell seeding efficiency were developed by template and hybridization methods. These porous scaffolds facilitated cell seeding, cell adhesion, distribution and proliferation, and promoted cartilage tissue formation. These scaffolds will be useful for cartilage tissue engineering., Philosophy Scaffold-free TEC is feasible to cartilage and meniscal repair with advantages in various aspects such as safety issues, cost effectiveness, minimal surgical invasion and quick surgical time, with comparable repaired tissue quality with other cell-based therapies in cartilage repair. Background Immune-tolerance of MSCs Safety issues regarding the implantation of animal-derived or chemical materials in clinical settings High medical cost with the use of scaffold Trends in promoting minimally invasive surgery Risk of complications by long surgery In vitro characterization of the TEC -human study- The purpose of this study was to characterize a tissue engineered construct (TEC) generated with human synovial mesenchymal stem cells (MSCs). MSCs were cultured in medium with ascorbic acid 2-phosphate (Asc-2P) and were subsequently detached from the substratum. The detached cell/matrix complex spontaneously contracted to develop a basic TEC. The volume of the TEC assessed by varying initial cell density showed it was proportional to initial cell densities up to 4×105 cells / cm2. Assessment of the mechanical properties of TEC using a custom device showed the load at failure and stiffness of the constructs significantly increased with time of culture in the presence of Asc-2P, while in the absence of Asc-2P the constructs were mechanically weak. Thus, the basic TEC possesses sufficiently self-supporting mechanical properties in spite of not containing artificial scaffolding. TEC further cultured in chondrogenic media exhibited positive alcian blue staining with elevated expression of chondrogenic marker genes. Based on these findings, such human TEC may be a promising method to promote cartilage repair for future clinical application. Large animal studies on cartilage repair The objective was to in vitro generate a mesenchymal stem cell (MSC)-based tissue-engineered construct (TEC) to facilitate in vivo repair in a porcine chondral defect model. Porcine synovial MSCs were cultured in monolayer at high density and were subsequently detached from the substratum. The cell/matrix complex spontaneously contracted to develop a basic TEC. Immunohistochemical analysis showed that the basic TEC contained collagen I and III, fibronectin, and vitronectin. The basic TEC exhibited stable adhesion to the surface of a porcine cartilage matrix in an explant culture system. The TEC cultured in chondrogenic media exhibited elevated expression of glycosaminoglycan and chondrogenic marker genes. The TEC were implanted in vivo into chondral defects in the medial femoral condyle of 4-month-old pigs, followed by sacrifice after 6 months. Implantation of a TEC into chondral defects initiated repair with a chondrogenic-like tissue, as well as secure biological integration to the adjacent cartilage. Histologically, the repair tissue stained positively with Safranin O and for collagen II. Biomechanical evaluation revealed that repair tissue exhibited similar properties similar to those of normal porcine cartilage in static compression test but the TEC-repaired tissue had lower micro-friction properties than normal articular cartilage. We also conducted the same surgical model study using mature (12m-old-) pigs and there was no significant difference in the modified ICRS histological scoring and biomechanical properties except for lubrication paroperties. This technology could potentially be a unique and promising method for stem cell-based cartilage repair. Large animal study on meniscal repair The meniscus is not entirely vascularized and has limited healing capacity. When the lesion involves the avascular region, complete reparative process cannot be expected. Recent studies revealed that the mesenchymal stem cells (MSCs) from synovial membrane have the potential to differentiate into a variety of connective tissue cells. With the synovial MSCs, we have developed a novel scaffold-free tissue engineered construct (TEC). Although scaffold-free, in vitro study demonstrated that the TEC possesses sufficiently self-supporting mechanical properties to withstand surgical handling. Moreover, the TEC is adhesive to the cartilage matrix and have chondrogenic differentiating capacity. Further, we have reported the feasibility of the TEC to repair partial thickness cartilage defect in a porcine model. Likewise, the TEC might also be applicable to incurable meniscal legions. The purpose of this study is to test the feasibility of the TEC to meniscal repair. The synovial MSCs were isolated from the synovial membrane of the skeletally matured Crown miniature pig's knee, and cultured to be confluent with 0.2mM ascorbic acid-2 phosphate (Asc-2P). The monolayer cell-matrix complex was detached from the substratum at confluent condition as reported. The detached complex actively contracted to develop a thick, three-dimensional tissue (the TEC), which was implanted to 4mm diameter cylindrical defect at the anterior horn of medial meniscus. All animals were put a cast on bilateral legs for 1 week and then back to the free-caged activity. The implamted menisci were evaluated grossly and microscopically The meniscal defects treated with the TEC were securely filled with fibrous tissue with good tissue integration. In contrast, there were none, or only partial, repair observed in the untreated defects. Histomorphometrical evaluation revealed that the meniscal defects was significantly better filled when treated with TEC (79% vs 33%) and integration ratio to adjacent meniscal tissue was also significantly better in the treated defects with the TEC (83% vs 51%). Furthermore, SO staining was decreased in 70% of the untreated menisci at the central portion, while SO staining was preserved in the TEC-treated menisci. Notably, focal articular erosions as mirror changes were observed at both the femoral and tibial articular surfaces in the 30% of the untreated knees (n=3), while no mirror lesion was developed in any TEC-treated knee. The present study demonstrated that the TEC implantation resulted in secure filling and repairing the incurable meniscal defect with good integration to the adjacent meniscal tissue. Moreover repair of the defect resulted in the prevention of body degeneration of meniscus as well as of chondral damage facing the meniscal lesion. Taking into account that the TEC does not require any extrinsic (biological or chemical) material for scaffold, this unique implant could be a promising therapeutic tool for the repair of incurable meniscal lesions. Clinical Experiences Clinical Trial has been registered and started in Spring 2009 at Osaka University. https://center.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&recptno=R000000960&type=summary&language=E, Transplantation of Tissue-engineered cartilage We have been performing transplantation of tissue-engineered cartilage made ex vivo for the treatment of osteochondral defects of the joints (108 cases) as a second generation of chondrocyte transplantation since 1996. Sixty knees who had received transplantation of tissue-engineered cartilage for cartilage defects were followed up for at least 5 years. Although the clinical results were satisfactory, we need the surgical approaches to treat large cartilage defects with less invasive technique. Articulated Distraction Arthroplasty Bone marrow stimulating procedure has been a well-accepted procedure for a large osteochondral defect. However, there are two potential weak points to induce hyaline cartilage. One is compressive overload on the drilled or micro fractured area at the early stage. In order to reduce the overload, we have developed external fixators, which allow almost full ROM with joint distraction for animals. Although joint distraction has been used to treat osteoarthritis, there has been little basic research on articulated joint distraction for the repair of osteochondral defects. We examined the effects of joint distraction with range of motion after drilling on a fresh osteochondral defect at the weight bearing area of the rabbit knee joint. A full thickness osteochondral defect was experimentally created at the weight bearing area of both medial femoral condyles of an adult Japanese white rabbit. After drilling to the defect, the experimental knee joint was distracted for 1.5 mm using a pair of external fixators to decrease compression force. The contralateral knee joint was used as a control with no apparatus. Gross findings and histological evaluation were assessed to study the morphology of the repaired cartilage. A partial repair with cartilage-like tissue was observed in the joints of the experimental group at 4 weeks. While cartilage-like tissue stained with Safranin O was found in the experimental group at 8 and 12 weeks, there were destructive changes in the control joints. Morphological changes were evaluated using the histological grading scale. There was no significant difference between experimental and control groups at 4 weeks. However, the mean scores of the experimental groups at 8 and 12 weeks were significantly better than those of the control groups at the same time points. Between the experimental groups, the scores at 8 and 12 weeks were both significantly better than those at 4 weeks. In conclusion, a combination of subchondral drilling, joint motion and distraction by an articulated external fixator promoted repair of a fresh osteochondral defect at the weight bearing area. Although distraction for 4 weeks was not a long enough period to repair the defect, distraction for 8 and 12 weeks resulted in a good outcome (Kajiwara and Ochi et al. JOR in 2005)1. Then we developed a new articulated device which permits smooth exercising of the joint during fixation for human. We evaluated the clinical results of a new distraction arthroplasty device after arthroscopic bone marrow-stimulating technique for the treatment of osteoarthritis of the knee. We developed a new distraction arthroplasty device that allows active ROM and compared preoperative and postoperative findings for 6 knees. The age ranged from 42 to 58 years. The fixation period for the distraction device ranged from 7 to 13 weeks, and the follow-up period was over 1 year. The Japanese Orthopaedic Association knee score, range of motion, and joint space values were significantly improved in all six cases at the latest follow-up (P ⇠.05). Scores on a visual analog pain scale were also significantly improved (P ⇠.05). We conclude that treatment using this new arthroplasty device in combination with a bone marrow-stimulating method was effective for osteoarthritic knees in middle-aged patient (Deie and Ochi et al. Arthroscopy in 2007)2. Another weak point is a small number of mesenchymal stem cells obtained from drilled holes, a new approach is injection of cultured MSCs in the joint after drilling. This injection was demonstrated to be effective for a cartilage defect, although this study was an animal study (Nishimori and Ochi et al. Br JBJS in 2006)3. This approach should be a new one for a large defect. Future direction for cartilage repair with minimally invasive tissue-engineering technique The most optimal procedure to repair cartilage defects is just injection of cytokines or growth factors and cells. Our completely novel approach was to use cell delivery system using an external magnetic field (Kobayashi and Ochi et al. Arthroscopy in 2008)4. We investigated whether it is possible to successfully accumulate magnetically labeled mesenchymal stem cells (MSCs), under the direction of an external magnetic force, to the desired portion of osteochondral defects of the patellae after intra-articular injection of the MSCs. MSCs were cultured from bone marrow and were labeled magnetically. Osteochondral defects were made at the center of rabbit and pig patellae, and magnetically labeled MSCs were injected into the knee joints either under the direction of an external magnetic force or with no magnetic force applied. In the rabbit model we evaluated the patellae macroscopically and histologically, and in the pig model we observed the patellae arthroscopically. Accumulation of magnetically labeled MSCs to the osteochondral defect was demonstrated macroscopically and histologically in the rabbit model and was demonstrated by arthroscopic observation to be attached to the chondral defect in the pig model. Thus, we could show the ability to deliver magnetically labeled MSCs to a desired place in the knee joint. In the clinical setting, our novel approach is applicable for human cartilage defects and may open a new era of repairing cartilage defects caused by osteoarthritis or trauma with a less invasive technique., Cartilage defects are a major clinical challenge because of the very limited regenerative capacity of articular cartilage. The solution to this might be regenerative medicine, a newly emerging area that aims to restore tissue function by applying principles of engineering and life sciences to develop biological substitutes. Cartilage was proposed as an ideal tissue for tissue-engineering treatments, being a “simple” tissue with one cell type (chondrocytes), a homogeneous matrix, and devoid of blood vessels and nerves. However, cartilage has proved difficult to engineer, and despite the significant efforts (∼1600 PubMed articles) over the past 15 years, the translation to the clinic has been limited. In fact, it may be the (oversimplified view of cartilage where the problem lies. Articular cartilage is a highly organized tissue with distinct structural patterns depending on the scale at which the tissue is examined. Over the range of nanometers to micrometers, the cartilage extracellular matrix is arranged as a network of collagen fibers and proteoglycans that allow for cell adhesion and mechanical support, and transduction of chemical and mechanical signals from the surrounding tissue to enter the cell [1]. Over the range of micrometers to millimeters, there are topographical differences in cartilage thickness and matrix content across the surface of the joint, which are associated with the level of loading [2, 3]. Further, the properties of articular cartilage change with depth from the articular surface, resulting in a “zonal” structure that is typically divided into three levels: superficial (surface to 10–20% of thickness), middle (20%-70%), and deep (70%-100%). Cells of each of the different zones of articular cartilage are organized distinctly and express zone-specific markers. In the superficial zone, cell density is high and cells are relatively fattened and clustered in a horizontal fashion.[4, 5] Here, the cells secrete proteoglycan 4 (PRG4) [6, 7] and clusterin [8], a molecule that is overexpressed in osteoarthritis [9]. In mature bovines, Notch1 is expressed solely in the superficial zone of articular cartilage [10], however this distribution appears to be species-, development-, and disease-specific [11, 12]. In the middle zone, cells are more spherical and are randomly oriented. Cartilage intermediate layer protein is a potential marker for the middle-deep zones[13], with apparent autoimmune activity and positive correlation with expression and progression of osteoarthritis[14] and chondrocalcinosis[15]. In the deep zone, chondrocytes are larger and organized in vertical columns. Several markers exist in adult cartilage including members of the Notch-Delta signalling pathway, which are expressed throughout cartilage during development, but are localized to the deeper layers in mature mouse tissue[12], in particular Jagged 1 [16]. Additionally, collagen type X, a marker for chondrocyte hypertrophy and indicator of endochondral ossification during development, is expressed in the deepest layers of articular cartilage [17]. To artificially mimic this zonal organization, an increasing number of investigations is currently directed at the development of a zonal tissue-engineered cartilage implants [18]. To achieve this, we are employing organ printing technology or “bioprinting” which combines the deposition of specific cell populations with the simultaneous deposition of biomaterials [19]. This allows the development of zonal cartilaginous grafts and by using hydrogels, a more physiological environment can be created. Cell suspensions can be mixed into in situ cross-linkable hydrogels (e.g., gelatin, agarose, alginate or PEG) in a cartridge and subsequently printed following a programmed 3D pattern[20–22]. Also, these water-based “bioinks” can contain biologically active components, such as proteins, peptides, DNA, hormones, extracellular matrix molecules and natural or synthetic polymers[23] to further enhance and direct the behavior of the cells. We characterized the use of bioprinting technologies to design and build heterogeneous cell-laden 3D structures. More specifically, we tested the design of various heterogeneous scaffolds, and tailored the porosity and elastic modulus of the grafts by modulating the distance between the strands and their configuration. Further, we are currently evaluating this technology for the printing of layered cartilaginous constructs using cells from the different zones of the articular cartilage. To this end we first investigated if zonal differences between chondrocytes isolated from the deep, middle and superficial zones of the cartilage are maintained during in vitro expansion and redifferentiation. For this study we elected to use equine chondrocytes, since this animal model is becoming increasingly important in the preclinical evaluation of orthopaedic treatments. Chondrocytes were isolated from cartilage of the patella-femoral groove of fresh equine cadavers (7–14 yrs). The separation into the different zones was confirmed using histology. Significantly more cells per gram tissue were isolated from the superficial zone compared to the deeper zones. During the expansion, the proliferation rate did not differ significantly between the cells of the different zones, whilst the expression of collagen types I, II and VI was lost in all zonal populations. After expansion in monolayers, cells were redifferentiated in alginate bead and pellet culture for up to 4 weeks. During redifferentiation staining for these markers re-appeared and was most intense after 4 weeks of culture. Interestingly, collagen type IX staining was also lost during expansion, but only re-appeared gradually in the middle and deep zone cultures. Similarly, COMP could not be immunolocalized after expansion and staining did only re-appear in the middle and deep zone cultures. Finally, we investigated the presence of clusterin [8]. In healthy articular cartilage clusterin is present within the superficial zone of the cartilage. Indeed, after isolation of the cells from the tissue, staining for clusterin was only found in association with cells from the superficial zone and it disappeared after expansion. During (re)differentiation in alginate, clusterin was present in cultures of all zones, but more pronounced within the cultures derived from the superficial zones of the cartilage. This indicates that clusterin would be a useful additional zonal marker, besides e.g. PRG-4, collagen type X and cartilage intermediate layer protein, (CILP), rather than an exclusive one, to further characterize tissue-engineered cartilage constructs with biomimetic zones. Thus, we have observed differences between zonal cell populations during in vitro culture, suggesting that the use of cells from the different zones could yield a tissue-engineered construct that better mimics the zonal native tissue. We have recently combined chondrocytes and osteogenic progenitors in printed constructs, yielding macroscopically heterogeneous grafts with distinct tissue formation. Printed zonal cartilage cells remained viable in alginate, in which they expressed cartilaginous markers including collagen types II and VI, as well as proteoglycans. However current culture experiments will demonstrate if printing cells of the different zones of the cartilage yields a construct with distinct zonal differences. Clearly, appreciation of zonal differences in the cartilage tissue could lead to important advances in cartilage tissue engineering., Despite satisfying clinical results autologous chondrocyte transplantation shows some technical disadvantages: insufficient initial mechanical stabilityuncertain cell distribution within the defectFixation of the periosteal flap with sutures penetrating healthy cartilageNecessity of intact cartilage shoulder surrounding the defectChance of periostal hypertrophy Various research groups are in the process of developing three-dimensional cell-carriers to improve techniques for cartilage repair. Specially designed scaffolds are one of the key components in tissue engineering. Research is focused on developing bioresorbable scaffolds that exhibit optimal physical properties coupled with excellent biocompatibility. Scaffolds act as shape and guidance templates for in vitro and in vivo tissue development. For cartilage and bone tissues, a suitable scaffold provides initial mechanical stability and supports even cell distribution. Various bioresorbable materials are currently used in various forms and shapes: Collagens of animal origin (mostly collagen type I and III)HyaluronanPolymers (PLA, PGLA)and others These matrices may be fixed by auto-adhesion, with fibrin glue, sutured or anchored transosseously. Biomecanical and preclinical studies showed that the stability of fixation varies tremendously with obvious clinical implications. Arthroscopic implantation of autologous chondrocytes on bioresorbable cell-carriers is feasible. Using these scaffolds, more even cell distribution within the defect may be achieved with operative handling being improved at the same time. Various techniques are currently used for the implantation of matrices (availability may vary from country to country): After debriding the defect, a size-matching scaffold is sutured or glued into the defect. Different materials may simply be attached by adhesion forces.Examples Hyaluronan Matrix (Fleece) of animal origin / Hyalograft® (Fidia)Collagen-Gel / CaRES® (ArthroKinetics AG)Collagen matrix / MACT® (CellTec)Collagen matrix / MACI® (Genzyme AG)After exact determination of the defect size a matching implant is prepared. The implant will then be pre-armed with resorbable threads (eg. Vicryl) which are to be knotted using a special technique. Anchoring holes will be placed anterogradely or tibially using a guide instrument. After insertion of pulling threads the pre-armed matrix is anchored within the defect by pulling the knots into the holes. Polyglactin/poly-p-dioxanon Fleece/Bioseed-C® (Biotissue-Technologies)Stable matrices enable a fast and stable but more costly fixation with intraosseous pins (Smart Nail®) Poly-glycolic-acid (PGA) Fleece/Chondrotissue® (Biotissue-Technologies) One of the first studies to examine the stability of implants for cartilage repair was done by Driesang in 2000. He applied autologous chondrocyte transplantation with a periosteal flap in goats and discovered that all sutured flaps (n = 6 animals) became detached from nonimmobilized joints during the recovery period. The purpose of this study was further to ascertain whether postoperative restriction of joint movement could prevent the delamination of flaps. Partial-thickness defects were created in the knee joint cartilage of 27 goats. These defects were then filled with a fibrin matrix and covered with periosteal (n = 6) or fascial (n = 21) flaps, which were sutured with simple, interrupted stitches to the surrounding tissue. The joints were immobilized by means of a modified Robert Jones bandage for periods of 2–6 weeks, after which time they were inspected for the absence or presence of flaps. In four animals, joint immobilization for 3 weeks was followed by free movement for a similar period. Four of the six periosteal flaps and two of the 21 fascial ones became delaminated after the period of immobilization. In the four goats permitted 3 weeks of free joint movement following a similar period of joint immobilization, all flaps (which had been retained up to the end of the immobilization period) became detached. These findings indicated that joint immobilization hinders the delamination of flaps but that this restriction of movement must be sustained for an undefined period of time. The nature of the tissue used for flaps also influenced the rate of retention by immobilized joints. Drobnic published a paper in 2006 about the comparison of four techniques for the fixation of a collagen scaffold in human cadaveric knee. Four fixation techniques for a fibrinogen and thrombin coated collagen fleece, used as a scaffold in the cartilage repair, were compared simulating the initial postoperative period in the cadaveric knee joints. Full-thickness chondral lesions were made on the medial femoral condyles of seven human cadaveric inferior extremities. Four scaffolds without seeded chondrocytes were implanted into each lesion using four fixation techniques consecutively: self-adhesion without additional material (SA), fibrin sealant (FS), bone sutures (BS), and periosteal cover (PC). After each implantation 150 cycles of continuous passive motion (CPM) were performed. Two cases were additionally exposed to 50 cycles of 10 and 20 kg loading each after the completion of CPM. The scaffolds were evaluated after every 30 cycles, and the fixation strength was tested after the motion was completed. All the SA scaffolds were detached prior to reaching 60 cycles. The other scaffolds remained stable throughout the testing with only minor disruptions. The endpoint fixation strength was higher for BS and PC than for the FS scaffolds. The FS scaffolds were detached as a result of additional load cycles, while the BS and PC scaffolds showed substantial deformations. SA of tested scaffold did not provide sufficient fixation. The FS fixation was easy to perform and assured satisfactory scaffold stability. BS and PC provided excellent scaffold stability, but the techniques were difficult and caused additional injuries. Regardless of the fixation technique used, the tested collagen scaffold may not be exposed to loading in the initial postoperative period. Hunziker found that surgical suturing of articular cartilage induces osteoarthritis-like changes. In clinical tissue-engineering-based approaches to articular cartilage repair, various types of scaffolds are used to retain an implanted construct within the defect, and they are usually affixed by suturing. The authors established a large, partial-thickness defect model in the femoral groove of adult goats. The defects were filled with bovine fibrinogen to support a devitalized flap of autologous synovial tissue, which was sutured to the surrounding articular cartilage with single, interrupted stitches. The perisutural and control regions were analyzed histologically, histochemically and histomorphometrically shortly after surgery and 3 weeks later. Compared to control regions, chondrocytes were lost from the perisutural area even during the first few hours of surgery. During the ensuing 3 weeks, the numerical density of cells in the perisutural area decreased significantly. The cell losses were associated with a loss of proteoglycans from the extracellular matrix. Shortly after surgery, fssures were observed within the walls of the suture channels. By the third week, their surface density had increased significantly and they were filled with avascular mesenchymal tissue. The authors concluded that the suturing of articular cartilage induces severe local damage, which is progressive and reminiscent of that associated with the early stages of osteoarthritis. This damage could be most readily circumvented by adopting an alternative mode of scaffold fixation. Knecht et al. performed an in vitro biomechanical testing of fixation techniques for scaffold-based tissue-engineered grafts. In this study, the authors have mechanically tested the fixation stability of four commonly used biomaterials for ACI attached by four different fixation techniques (unfixed, fibrin glue, chondral suture, and transosseous suture) in situ. Scaffolds based on polyglycolic acid (PGA) and polyglycolic acid and poly-L-lactic acid (PGLA), collagen membranes, and a gel-like matrix material were fixed within rectangular full-thickness cartilage defects of 10 × 15 mm(2) and loaded in tension until failure. Fibrin glue fixation of PGLA-scaffolds withstood a load of 2.18 6 +/- 0.47 N, chondral sutured PGA-scaffolds of 26.29 6 +/- 1.55 N, and transosseous fixed PGA-scaffolds of 38.18 6 +/- 9.53 N. The PGA-scaffold could be loaded highest until failure for all fixation techniques compared to the PGLA-scaffold and collagen membrane. The findings might serve as basis for selecting the most suitable fixation technique for scaffold-based tissue-engineered grafts according to the expected in vivo loads. Marlovits et al. examined the early postoperative adherence of matrix-induced autologous chondrocyte implantation for the treatment of full-thickness cartilage defects of the femoral condyle with MRI. In this clinical pilot study, a Matrix-induced Autologous Chondrocyte Implantation (MACI) technique with a three-dimensional collagen type I-I II membrane was used for the treatment of full-thickness, weight-bearing chondral defects of the femoral condyle in 16 patients. The cell-scaffold construct was implanted in the debrided cartilage defect and fixed only with fibrin glue, with no periosteal cover or further surgical fixation. All patients were followed prospectively and the early postoperative attachment rate, 34.7 days (range: 22–47) after the scaffold implantation, was determined. With the use of high-resolution magnetic resonance imaging (MRI), the transplant was graded as completely attached, partially attached, or detached. In 14 of 16 patients (87.5%), a completely-attached graft was found, and the cartilage defect site was totally covered by the implanted scaffold and repair tissue. In one patient (6.25%), a partial attachment occurred with partial filling of the chondral defect. A complete detachment of the graft was found in one patient (6.25%), which resulted in an empty defect site with exposure of the subchondral bone. Interobserver variability for the MRI grading of the transplants showed substantial agreement (kappa=0.775) and perfect agreement (kappa(w)=0.99). The authors concluded that implantation and fixation of a cell-scaffold construct in a deep cartilage defect of the femoral condyle with fibrin glue and with no further surgical fixation leads to a high attachment rate 34.7 days after the implantation, as determined with high resolution MRI. Petersen and his group described arthroscopic techniques for the fixation of a three-dimensional scaffold for autologous chondrocyte transplantation and examined the structural properties in an in-vitro model. The aim of the study was to evaluate the structural properties of matrix-associated autologous chondrocyte implantation (Bioceed-C®/ BioTissue Technologies) with multiple fixation techniques implanted in fresh porcine knees after they had undergone load to failure. The ultimate failure load, yield load, and stiffness of 3 different techniques for the fixation of a 2-mm thick polymer fleece was evaluated: (1) fixation with biodegradable polylevolactide pins (Smart Nail®), (2) a transosseous anchoring technique, and (3) conventional suture fixation. Techniques 1 (pin) and 2 (transosseous anchoring) can be used arthroscopically. Maximum load and yield load were significantly higher in the group 1 (pin fixation) and group 2 (transosseous anchoring) compared to group 3 (conventional suture). Stiffness was significantly higher in group 1 than in groups 2 or 3. This biomechanical dataset showed that two fixation techniques (pin fixation and transosseous anchoring) have a higher ultimate load, yield load, and stiffness than the conventional suture technique at time point zero. The described techniques provide an outstanding fixation strength with arthroscopic techniques for autologous chondrocyte transplantation There is still uncertainty about the importance and the best technique for the fixation of scaffolds in cartilage repair. Extensive research will have to be done to establish new techniques with new scaffolds to ensure biologic performance AND biomechanical stability., The use of biomaterials for cartilage repair has strongly increased in the last decade, due to promising results obtained with the development of new therapeutic options for the treatment of articular cartilage lesions. Actually in clinical practice there are two main concepts for biomaterials application for cartilage repair: cartilage regeneration promoted by cultured autologous chondrocytes, supported by the 3D scaffold (so-called second generation autologous chondrocyte transplantation) or implant of various biomaterials for “ in situ” cartilage repair which exploits bone marrow stem cess differentiation induced by the scaffold properties. The use of classic ACI (first generation) has been associated with several limitations related to the complexity and the morbidity of the surgical procedure. To address these problems-called second generation ACI techniques have been developed. Essentially, the concept is based on the use of biodegradable polymers as temporary scaffolds for the in vitro growth of living cells and their subsequent transplantation onto the defect site. Essential properties of these scaffolds include biocompatibility and biodegradability through safe biochemical pathways at suitable time intervals. It is known that chondrocytes in two-dimensional cell cultures alter their phenotype and dedifferentiate to fibroblast cells that no longer posses the capacity to produce collagen type II and proteglycans. The use of three-dimensional scaffolds has been shown to favor the maintenance of a chondrocyte differentiated phenotype1,2. The clinical application of this second generation tissue engineered approach is well documented for different types of scaffold with an evaluation of the clinical outcome at short and medium-term follow up3,4,5,6. Autologous chondrocyte transplantation on a three-dimensional matrix was introduced in clinical practice in Europe from 1998–1999, so it is very difficult to obtain long-term clinical findings. Matrixes mainly used in clinical practice in Europe are collagen or hyaluronic acid based. In the USA there still no FDA approval for matrix-assisted chondrocyte transplantation in human application. Considering that from a surgical and commercial standpoint, an ideal graft for chondral or osteochondral defect repair would be an off-the-shelf product; thus, some new biomaterials were recently proposed to induce “ in situ” cartilage regeneration after direct transplantation onto the defect site. The possibility to create a cell-free implant to be sufficiently “ intelligent” to bring into the joint the appropriate cues to induce orderly and durable tissue regeneration is still under investigation in numerous animal studies7,8,9,10,11, and/but only few of these have been introduced into the clinical practice12,13. Scaffolds composed of synthetic or natural materials in a variety of physical forms (fibers, meshes, gels) have been used for cartilage regeneration. Solid scaffolds provide a substrate upon which cells may adhere, while gel scaffolds function to physically entrap the cells. Commonly used synthetic materials are the polylactides, like polylactic (PLA) and polyglicolic (PGA) acids. The mechanical properties and the degradation of synthetic biomaterials are more easily modified that for the natural polymers, but their degradation products may cause damage to native tissue or implanted cells. However new chemistry of these materials has improved their biocharacteristic and biocompatibility. Natural materials used to produce scaffolds include agarose, alginate, hyaluronic acid, gelatin, fibrin glue, collagen derivatives and acellular collagen matrix. They have impeccable biocompatibility, can be processed in a reliable and reproducible way and may enhance cell performance. The materials mostly used actually in clinical practice are protein-based (collagen or gelatine), but the use of polysaccharides is in rapid growth. There are several studies pointing to the critical role of saccharide moieties in cell signalling schemes and their importance in cartilage regeneration14,15. One of the more important properties of polysaccharides in general is their ability to form hydrogels. Hydrogel formation can occur by a number of mechanisms and is strongly influenced by the types of monosaccharide involved: for example thermal gellation is tipical for agarose and pH-dependent gellation for chitosan. Chitosan (partially de-acetylated derivative of chitin, found in arthropod exoskeletons) seems to have an important potential in stimulating chondrogenesis15 and there are some chitosan-based gels and scaffolds under clinical investigation for “ in situ” cartilage regeneration after direct transplantation onto the defect site. The treatment of osteochondral lesions is biologically challenging since two different tissues are involved (bone and articular cartilage) with a distinctly different intrinsic healing capacity. There some composite materials in pre-clinical and clinical experimentation for tissue engineered and “ in situ” regeneration approach for cartilage repair (Martin), but only one is widely commercialized for this application. This is bilayer porous PLGA-calcium-solfate biopolymer (TruFit) proposed for direct application into the defect site. Although pre-clinical experimentation is promising12 there still no clinical results available in the literature of the use of this material. RIZZOLI EXPERIENCE We utilized osteochondral nanostructured biomimetic scaffold (Fin-Ceramica S.p.A., Faenza - Italy) with a porous 3-D tri-layer composite structure, mimicking the whole osteochondral anatomy. The cartilaginous layer, consisting of Type I collagen, has a smooth surface to favour the joint flow. The intermediate layer (tide-marklike) consists of a combination of Type I collagen (60%) and HA (40%), whereas the lower layer consists of a mineralized blend of Type I collagen (30%) and HA (70%) reproducing the sub-chondral bone layer. In vitro and animal studies showed good results in terms of both cartilage and bone tissue formation. We observed same macroscopic, histological and radiographic results when implanting scaffold loaded with autologous chondrocytes or scaffold alone. The scaffold was able to induce an in situ regeneration through stem cells coming from the surrounding bone marrow16,17,18. We have performed clinical pilot study on 30 patients where the newly developed scaffold was used for the treatment of chondral and osteochondral lesions of the knee joint in order to evaluate the safety and the reproducibility of the surgical procedure and in order to test the intrinsic potential of the device. The clinical outcome of all patients was analyzed prospectively, at 6 moths and 1 year and an high resolution MRI. 29 of 30 patients (mean age of 29.3 years) were prospectively evaluated at 6 and 12 months follow up(one patient lost at follow up). In 29 patients 35 lesions were treated, average size of the defects was 2.8 cm2 (range: 1.5– 5.9 cm2). Statistical analysis demonstrated a significant improvement (Non Parametric paired Wilcoxon test, p⇠0.0005) from pre-operative to 12 months follow up. IKDC objective score showed preoperatively 46.1% of normal or nearly normal knees and 79.3% of normal or nearly normal knees at 12 months. Statistical analysis showed a significant improvement in the IKDC subjective score from preoperative (37,5± 14,6) to 12 months follow up (82,4± 11,9) (Non Parametric paired Wilcoxon test, p⇠0.0005). MRI evaluation showed complete filling of cartilage defect was noted in 86.2% of the patients and the congruency of the articular surface was seen in same patients. Subchondral bone changes (edema or sclerosis) were noted in 53.3% of patients. This scaffold, composed of Type-I collagen and nanostructured hydroxyapatite was designed for the treatment of cartilaginous and osteocartilaginous defects and have demonstrated to stimulate in situ bone and cartilage regeneration. Obviously, this short follow-up does not allow us to draw conclusions about the clinical effectiveness and histological quality of cartilage repair tissue in the long-term follow-up of this procedure, but clinical and MRI analysis allowed us to study and better understand the potential of this novel developed scaffold. The ability of the scaffold to induce orderly osteochondral tissue repair without necessarily including autologous cells makes it attractive (i) from a practical standpoint, since it could be used as an off-the-shelf graft in a one-step surgical procedure, (ii) from a surgical standpoint, since due to its flexibility it could inserted under minimally invasive conditions. Previous animal study highlighted the good potential of the graded biomimetic osteochondral scaffold in promoting by itself bone and cartilage tissue restoration, probably by inducing selective bone marrow stem cell differentiation in osteogenic and chondrogenic lineages18. Further systematic evaluation is necessary to determine the clinical and morphological outcome, especially compared to other treatment options such as bone-marrow stimulation techniques, mosaicplasty, and autologous chondrocyte transplantation., Purpose Focal articular cartilage lesions can be treated by surgical techniques that puncture holes in the base of the defect to induce bleeding and to generate conduits for stem cell migration into the defect 1–4. An optimal hyaline cartilage repair tissue contains type II collagen and high levels of GAG 5, however “marrow stimulation” techniques frequently elicit fibrocartilage or fibrous repair tissue that contains type I collagen and low levels of glycosaminoglycan (GAG), which is mechanically unstable and fails under load-bearing 3,6,7. Since marrow stimulation is a relatively simple and inexpensive out-patient-based surgery, a genuine interest has developed around finding methods that stimulate a more hyaline, marrow-derived repair. We previously showed in preclinical animal models that more hyaline repair cartilage is formed after microfracture, when the defect is covered with a hybrid polymer-blood clot implant 8,9. Hybrid clots are formed by mixing liquid autologous whole blood with a cytocompatible solution of chitosan, a polysaccharide biomaterial, which then coagulates to form a more adhesive and stable clot 9,10. Hybrid clots were shown to promote hyaline repair by attracting neutrophils and by stimulating transient subchondral angiogenesis and bone repair 9,11, however the step-wise cellular events linking these processes remain unclear. Blood clots 12,13, and chitosan particles 14,15 can attract innate immune cells to skin wounds. When macrophages become activated, they release angiogenic factors 16,17. However “classically” activated macrophages also release cytokines that can trigger cartilage breakdown (TNF-a, IL-1b), whereas “alternatively” activated macrophages express arginase-1 and release wound-healing factors such as IGF-1 18. In this study, we investigated the role of chitosan and blood clot in eliciting neutrophils and activating macrophages both in vitro and in vivo. Materials and Methods Experiments involving healthy, non-fasting human subjects and animals were carried out with institutionally-approved protocols. Hybrid clots were formed by aseptically mixing 3 volumes human or rabbit whole blood with 1 volume sterile liquid chitosan-glycerol phosphate (chitosan-GP: 1.6% w/v chitosan, 100 mM GP pH 6.6, Bio Syntech Inc, QC, Canada), or 1.5% w/v alginate (FMC, QC, Canada), hydroxyethyl cellulose (Spectrum, QC, Canada) or hyaluronic acid (Sigma, ON, Canada) in phosphate-buffered saline. Chitosan was biodegradable (80% degree of deacetylation, DDA, ie 80% glucosamine and 20% N-acetyl glucosamine, medium viscosity: “80M”) or non-biodegradable (95% DDA, ie 95% glucosamine and 5% N-acetyl glucosamine, “95M”). To track scaffold fate, some chitosan-GP samples contained fuorescent rhodamine B isothiocyanate–chitosan (RITC-chitosan) tracer with identical DDA and molecular mass 19. Clots were solidified in sterile glass tubes and cultured for up to 6 hours at 37°C, and exuded serum was analyzed for platelet and inflammatory cytokines, and ability to attract purified human neutrophils using a trans-well migration assay 20. In vivo implants were generated in rabbit dorsal subcutaneous sites (using autologous rabbit blood), and recovered after 1 day (N=7) or 7 days (N=6). In a separate experiment, implants were delivered to knee trochlea full-thickness articular cartilage defects with four, 0.9 mm microdrill holes (N=16 rabbits, analyzed at 1, 2, or 8 weeks of repair), using thrombin to accelerate in situ solidification 10. Control defects were treated with thrombin alone. Implants and decalcified femur ends were sectioned and stained with Safranin O-Fast green-iron hematoxylin, arginase-1, or collagen type I, and analyzed by stereology, or by histomorphometry (Empix, ON, Canada). Neutrophils were identified by nuclear morphology and CD14 expression, and macrophages by RAM-11, CD68, and arginase-1 expression. ANOVA was used to test for significant differences (p⇢0.05) induced by chitosan or time. Results Biodegradable chitosan elicits neutrophils and alternatively activated macrophages in vivo. Neutrophils strongly accumulated around subcutaneous implants containing 80M chitosan while few neutrophils were attracted to 95M chitosan, blood clot, LPS/blood, and hybrid clots incorporated with other polysaccharides (p⇠0.0005, Fig. 1A). After 1 week in vivo, neutrophils further accumulated and degraded 80M chitosan particles as shown by a depletion of RITC-chitosan (Fig. 1B & C). Neutrophils more easily infiltrated chitosan-GP/blood implants (Fig. 1C) compared to chitosan-GP implants (Fig. 1B). Altogether, these data demonstrated that the combination of blood and biodegradable 80M chitosan particles in hybrid clot implants promoted neutrophil chemotaxis, invasion of the implant, and cell-mediated chitosan particle clearance. After 1 day in vivo, macrophages were scarcely detected around subcutaneous implants. However after 1 week in vivo, macrophages expressing phagocyte markers RAM-11 and CD68 collected specifically at the edges of implants with neutrophils. As macrophages migrated into the neutrophil-dense implant, they phagocytosed chitosan, lost the RAM-11 epitope, and expressed arginase-1+ (Fig. 1D) which indicated that macrophages elicited by chitosan were alternatively activated. These data are important because they demonstrated that biodegradable chitosan particles could induce macrophages to adopt an angiogenic, wound-repair phenotype in vivo, through a phase requiring strong neutrophil attraction. Mechanisms of neutrophil attraction. IL-8 is highly chemotactic for neutrophils in vitro 21, and proteomic analysis of serum revealed that out of 25 inflammatory chemokines, IL-8 and MCP-1 were the dominant factors released from leukocytes in whole blood and in chitosan-GP/blood, while IL-6 and IL-1b were the dominant cytokines released from LPS/blood clots. These data showed that chitosan structurally stabilized blood clots without stimulating blood leukocytes to secrete catabolic cytokines. By transwell migration assay, chitosan-GP/blood clots released more potent neutrophil chemotactic factors to serum than blood clot alone. These data suggested that blood leukocytes in contact with biodegradable chitosan generated de novo neutrophil chemotactic factors distinct from IL-8 that recruited neutrophils to 80M chitosan. Chitosan attracts alternative macrophages to granulation tissues in drilled subchondral bone. Chitosan-GP/blood implants solidified over microdrilled cartilage defects doubled the level of arginase-1+ macrophages in granulation tissues formed in the trabecular drill hole at 1 week (p⇠0.05, Fig. 2A). After 2 weeks of repair, arginase-1+ cells persisted in neovascularized granulation tissues below treated defects, while arginase-1+ macrophages diminished in control repair tissues (Fig. 2A). After 8 weeks of repair, RITC-chitosan particles were completely cleared, arginase-1+ macrophages were no longer present, and drill holes below treated defects were repaired with more trabecular bone compared to control defects (p⇠0.05, Fig. 2B). Conclusions Biodegradable chitosan stimulated the release of neutrophil chemotactic factors by blood leukocytes and strongly attracted neutrophils in vivo. Neutrophils attracted to chitosan particles elicited and alternatively activated more macrophages than blood clot alone. When applied to marrow-stimulated articular cartilage defects, chitosan-GP/blood implants recruited significantly more arginase-1+ macrophages than blood clot alone. Increased density of arginase-1+ macrophages was associated with more vascularized granulation tissues. After 8 weeks of repair, chitosan particles, neutrophils, and arginase-1+ macrophages were no longer present, and trabecular bone was more completely restored, indicating that the therapeutic and transient effect was directly related to timely clearance of the biodegradable biomaterial by innate immune cells. Our novel findings generate a new paradigm for using biodegradable biomaterials to attract and activate macrophages in order to stimulate revascularization and repair of damaged subchondral bone., Introduction Osteochondral Defect (OD) has a poor spontaneous regenerative capacity and they can present with a variety of complaints from blister cartilage to full-thickness articular cartilage lesions with subchondral bone exposition. Since articular cartilage defects can progress to osteoarthritis (OA) in some cases1,2, surgical measures should be considered. Large ostheochondral defects (ICRS 4 ⇢ 4–10cm2) are difficult to treat, but several treatment options are available. In our country surgical options include drilling, microfracturing, and transplantation of osteochondral plugs but are often insufficient for the treatment of large defects1. From the last 7 years members of our laboratory has been working to find new alternatives to treat this kind of lesions using Biological Therapies. The works had been principally in the harvest process, expanding and differentiating mesenchymal stem cells (MSCs) into cartilage and bone. As these cells are easy to isolate, culture, and manipulate in vitro and have great plasticity3 they have become an important tool in cell replacement therapy and are considered as candidates for clinical applications4, we has been trying to development techniques to use in clinical setting. THE BEGINNING: ANIMAL MODEL STUDIES. The first work with MSCs were performed in New Zealand rabbits. The aim of this work were to obtain, culture and differentiate rabbit Bone Marrow derived MSCs in vitro to chondral lineage in the latinamerican reality. An iliac puncture was performed percutaneously over liac crests to obtain the bone marrow samples. By differential centrifugation the mononuclear cell level was obtained. After the expansion of MSCs we exposed the cells in micropellet for 21 days to TGF-b1 as described by Johnson et al5. We evaluated the quality of differentiation by histomorphometric analysis performed in the Mayo Clinic. In our hands, the MSCs expanded easily in vitro: at day 3 the cells in all cultures were adherent, at day 6 in all cultures was visible cells with spindle-shaped morphology (fibroblast-like morphology), characteristic of MSCs and in average at day 18 (15–19 day) the cells achieved 70% of confluence. After 21 days of culture with TGF-b1 the MSCs achieved an optimal differentiation quality (Figure 1). With this work we demonstrated that was possible achieve the isolation, culture and chondrogenic differentiation of rabbit bone marrow MSCs in the latinamerican reality. These results were submitted for publications and were presented in congress6. Figure 1: Chondrogenic Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells. A: Aliquots of rabbit BMMSCs forming a spherical pellet in differentiation medium containing TGF-β1. B: Safranin-O/Fast Green stain showing chondrogenic differentiation of rabbit BMMSCs in a 21 day pellet culture with TGF-β1 (10 ng/ml). C: Quality of chondrogenic differentiation of rabbit BMMSCs. 10 pellet (83%) showed score 3, 1 pellet (8.5%) score 2 and 1 pellet score 1. In our next work with animals we compared the results of repair full-thickness cartilage defects in a rabbit model with a collagen I/ III matrix seeded with autologous chondrocytes or MSCs. After the harvest process, the cells were cultured in vitro and then the cells were seeded over the matrix at concentration of 20.000 cells/cm2. After 5 days, the collagen I/III matrix seeded with cells were implanted over a full-thickness defects produced in the femoral condyle of New Zealand rabbits. After 6 weeks the rabbits were euthanasied and the quality of repair was evaluated by gross and histologic examination with hematoxylin-eosin and safranin/O staining. The quality of tissue reparation was significantly better in defects treated with collagen I/III matrix seeded with chondrocytes or MSCs. In defects treated with MSCs the tissue appeared to have better integration and the histology showed hyaline-like tissue than did repaired by chondrocytes. These results were presented in congress and are being written to be sent for publication. MOLECULAR APPROACH In the next years our laboratory started studies performed only in vitro to evaluate our capacity to harvest human MSCs from different tissue sources and the quality of MSCs differentiation by techniques of molecular biology. In this context, we harvest human MSCs from bone marrow, umbilical cord blood and adipose tissue. Because the adipose tissue is a promising source of MSCs and they can be obtained by a minimal invasive method and in large quantities we tried to reproduce the two principal published methodology to harvest MSCs from this tissue but we fail, so we propose a new method based in mononuclear cells from adipose tissue. This new methodology to isolate human adipose tissue MSCs is easier than those published and the cells are expanded faster with similar differentiation capacity to that reported. This methodology is currently being patented. With the idea to use clinically the MSCs we evaluated the ability to obtain human bone marrow derived MSCs in a surgical room by density gradient with the same methodology described for the animal studies. In a surgical time an iliac punctures were performed and bone marrow aspirated were collected (15 ml, 3 patients). The density gradients were performed and the mononuclear cell levels were obtained. To determine the presence of MSCs in the mononuclear cell levels, this portion was expanded and differentiated to osteoblastic and chondrogenic lineage in our laboratory. The osteogenic differentiation was evaluated by alkaline phosphatase histochemistry and the chondrogenioc differentiation was evaluated by Western blot for collagen II (Figure 2). We demonstrated that was possible to obtain MSCs by density gradient in a surgical room and these MSCs had osteogenic and chondrogenic capacity7. Figure 2: Osteoblastic and Chondrogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells Isolated in a Surgical Room Osteogenic differentiation evaluated by alkaline phosphatase histochemistry and chondrogenic differentiation evaluated by Western blot for collagen II. CLINICAL PILOT STUDIES In the last 3 years and as a result of presentations of researches performed in our laboratory in different meetings has consulted several patients with large osteochondral defects (around 10cm2) that had no other chance of treatment. The first 3 patients with the informed consent were operated in 2004 and all they were carrying osteochondral defects ICRS 4 (2 in talus and 1 in femoral head). In the 3 patients we concentrate bone marrow cells harvested in the same surgical time by centrifugation. The defects were debrided and the size of defect was measured after debridement. Then a periosteal flap was extracted according to the defect size. From the iliac crest autologous cancellous bone was gained to restore the osseous part of the defect and then the periosteal flap was sutured to the cartilage rim of the chondral defects. The concentrate bone marrow cells were injected into the defects and then the flaps were sealed with fibrin glues. After 2 years of follow-up the 3 patients are satisfied with the results regarding pain during full weight bearing. The 2 patients with talus lesions improved the articular functionality according with AOFAS score in 37 points without evidences of progression of the osteochondral defects. These results were presented in congress and are being written to be sent for publication8,9. 4 years ago a 30 years old patient was diagnosed with an autoimmune disease and underwent cronic corticosteroid therapy. After 2 years of continuos corticosteroid medication the patient started complaining of bilateral knee pain, more severe on the left knee. Weightbearing plain radiographs showed a radiolucent area in the femoral lateral condyle and magnetic resonance imaging revealed an extense area of bone necrosis (⇢10 cm2) and subchondral edema in the weight bearing area of the lateral femoral condyle. At this point the Lyshom score was 15 points. For these reasons a treatment of the osteochondral defect with MSCs was decided with the informed consent of the patient. Briefy, an iliac puncture was performed and bone marrow aspirated was collected (15 ml). Five hundred ml of the patient's blood were collected and 230 ml of autologous serum were obtained. MSCs were obtained from bone marrow aspirated by a density gradient and they were plated at concentration of 106 cells/ml and incubated at 37° C/5% CO2 in expansive medium with 10% of autologous serum. When MSCs achieved 80% of confluence, they were tripsinizated and replated over a collagen type I/III matrix (Chondro-gide®) at 37° C/5% CO2 in expansive medium for 14 days. To induce chondrogenic commitment, 3 days before the surgical procedure the expansive medium was supplemented with TG F-b1 and ITS+Premix. The surgical procedure consisted on open debridement. Subchondral bone defect was filled with autologous cancellous graft and the MSCs in a Chondro-gide® matrix were positioned over the top of the lesion and sutured to the cartilage rim of the chondral defect. Clinical and radiological follow-up was performed every 3 months and 2 years after the surgical procedure a second-look arthroscopy was performed and biopsy specimens for histology and molecular analysis were obtained from the osteochondral defect area. At two-years follow-up the patients have recovered articular functionality. The Lysholm score improved 80 points (15 to 95) and in the radiological studies was observed complete scaffold integration. The cartilage was normal at second-look arthroscopy (Figure 3). The biopsy histology (H-E and safranin-O) was normal. Western blot and real time PCR for SOX-9 and collagen II from biopsy were similar to normal cartilage. The patient is currently asymptomatic of the left knee. These results were presented in congress and are being written to be sent for publication10,11. Figure 3: Treatment of osteochondral defect ICRS 4 ⇢ 10 cm2 with Mesenchymal Stem Cells induced to chondrogenic lineage. Osteochondral defect ICRS 4 ⇢ 10 cm2 and MSCs in a collagen type I/III matrix (Chondro-gide®) sutured to the cartilage rim of the chondral defect. CONCLUSIONES Since Wakitani et al12. demonstrated that bone marrow MSCs had chondrogenic potential and because MSCs-based cell therapy is clearly promising, several clinical trials have been developed. In countries with few economic resources staggered studies appear to be a good option. Although clinical applications of MSC are promising, the clinical use should be carefully evaluated., Chondrocytes and fibrochondrocytes adapt to changes in their biomechanical environment. In this presentation, the mechanoresponsiveness of condrocytes and fibrochondrocytes under normal and inflammatory conditions in vitro is presented. The effects of Continuos passive motion (CPM) on arthritic cartilage and arthritic meniscus fibrocartilage is also demonstrated in vivo (rabbit). Fibrochondrocytes and condrocytes from rat meniscus were exposed to Cyclic Tensile Strain (CTS) in vitro at various magnitudes and frequencies. The mRNA and protein analyses revealed that CTS at magnitudes of 5% to 20% did not induce proinflammatory gene expression. IL-1b induced a rapid increase in the iNOS mRNA. CTS strongly repressed IL-1b-dependent iNOS induction in a magnitude-dependent manner. Exposure to CTS resulted in 90% suppression of IL-1b-induced mRNA within 4 h and this suppression was sustained for the ensuing 20 h. The mechanosensitivity of fibrochondrocytes was also frequency dependent and maximal suppression of iNOS mRNA expression was observed at rapid frequencies of CTS compared with lower frequencies. Like iNOS, CTS also inhibited IL-1b-induced expression of proinflammatory mediators involved in joint inflammation. The examination of temporal effects of CTS revealed that 4- or 8-h exposure of CTS was sufficient for its sustained anti-inflammatory effects during the next 20 or 16 h, respectively. Fibrochondrocytes constitutively also expressed low levels of RANKL and RANK but marked levels of OPG. IL-1b upregulated expression and synthesis of RANKL and RANK significantly, whereas expression of OPG was unaffected following 4 and 24 h. When fibrochondrocytes were simultaneously subjected to CTS and IL-1b, expression of RANKL and RANK was significantly downregulated as compared to that of IL-1b-stimulated unstretched cells. The inhibitory effect of CTS on the IL-1b-induced upregulation of RANKL and RANK was sustained as well as magnitude and frequency dependent. The arthritic menisci and cartilage from rabbits subjected to CPM or immobilization were investigated for glycosaminoglycans (GAG), interleukin-l b (IL-1 b), matrix metalloproteinase-1 (MMP-I), cyclooxygenase-2 (COX-2), and interleukin-10 (IL-10) were determined by histochemical analysis. Within 24 h, immobilized knees exhibited marked GAG degradation. The expression of proinflammatory mediators MMP-I, COX-2, and IL-I p was notably increased within 24 h and continued to increase during the next 24 h in immobilized knees. Knees subjected to CPM revealed a rapid and sustained decrease in GAG degradation and the expression of all proinflammatory mediators during the entire period of CPM treatment. More importantly, CPM induced synthesis of the anti-inflammatory cytokine IL-10. These in vitro and in vivo studies explain the molecular basis of the beneficial effects of mechanical stimulus observed on fibrocartilage and articular cartilage and suggest that mechanical stimulus suppresses the inflammatory process of arthritis., Cartilage tissue has very low capabilities to respond an injury and in the best scenario it recreates a repair tissue far away from the original mechanical resistance, therefore, cartilage has being seen as a great challenge and numerous approaches has being attempted in order to leave as much of this precious tissue in place, or even regenerate it (1)-When performing an arthroscopic surgery, it is commonly foundcartilage injuries that present as fibrillated surface and loose fragments or unstable pieces hanging from the subchondral bone by atiny area of contact, degenerative changes derived by trauma, overuse or systemic diseases.(2,3). Traditionally, when we see such an scenario, cleaning the house cometo mind as the first step of operative procedures. Mechanical shavingand lavage with removal of fragment and fibrous synovial tissue arethe golden standard(4). As technology evolution, other tools likeRadiofrequency appear to solve same problems with apparent advantages over mechanical shaving in chondroplasty procedures (5). Radiofrequency has being launched as a resource to help the surgeoncut, coagulate, marketed as something rather recent from the 90'sdecade, however, RF is as common as the electrical scalpel that we use in every operating room, or at least, the principles are the same. Developed by Dr. Harvey Cushing and a Harvard Physicist William Bovie back in 1920. A glimpse in basic science behind RF When charged particvles such as electrons are accelerated, anelecrtomagnetic field is creatd. Electricity is a flow of electronstraveling through a conductor and a force must push this flow to theentire length and this force is called Voltage which in turn iscomposed of units called volts. The larger number of electrons thelarger the voltage traveling through the conducting material. Resistance to the passage of the current by the medium is calledimpedance which is reflected as Ohm unit. If one volt is sent throughone Ohm of resistance the resultant is an Ampere (Amp.). The electriccurrent also depends on its alternance switching from one pole to theother, therefore when one cycle is ccompleted in one second isreferred as one Hertz (Htz). Radiofrequency starts from 10,000 Htz to 30 Mega Hertz. RF is basically a thermal energy when applied to tissues. When in monopolar mode, the current flows from the dispersive electrode know as the skin patch through the body and leaves the active elecrode known as the one leaves the energy in the tissues to affect. Bipolar meantime does not drive the energy across the tissues since both electrodes are in the immediate neighborhood, ti does close the circuit by mean of the saline conduction properties and those tissues nearby get the thermal influence (6). Tissue response to RF Thermal modulation on collagena, has being studied by Arnockzky(7) The temperatures required to alter the molecular bonding of collagen and thus cause tissue shrinkage (65 degrees C to 70 degrees C) are also known to destroy cellular viability. Therefore, thermally modifiedtissues are devitalized and must undergo a biologic remodelingprocess. Meniscal tissue is capable of full recovery after exposicionto RF thermal energy pulses and has being shown to help heal someavascular inuries in Rabbits and human After the exposicion themeniscal tissue goes through a known curve of cell death, followed bycell repopulation and full histologic recovery after three months dueto vascular neoformation and fibroblast production of collagen (8,9,10). The expression of heat-shock proteins in human chondrocytes and ratfemoral head cartilage following heat shock was analyzed by Westernblotting, and red-blood-cell-induced chondrocyte death was assessed by cell viability and apoptosis by flow cytometry. Heat-shock inducedexpression of heat-shock protein 70 (HSP70) (rat and human) and HSP32 (human). Blood and blood products reduced rat cartilage proteoglycan synthesis and human chondrocyte viability, and induced human chondrocyte apoptosis at concentrations considerably lower than those reported previously. The induction of HSP70 in rat cartilage was ineffective in reducing chondrocyte death in the absence or presence of red blood cells or red cell products. Heat shock to humanchondrocytes reduced low levels of apoptosis (⇠20%) and cell deathinduced by low levels of blood products, but not higher levels(11) Radiofrequency in Chondroplasty Cartilage injuries are quite frequent findings, Curl et als, showed an incidence of 63% in a serie of 31,516 arthroscopies (12). A recent serie on 25,124 concur with the former one showing a prevalence of 60% as for total cartilage injuries (13). When a chondral injury is spoted, several aspects must taken in account such as depth, extension and stability. Chondroplasty goal is to regularize the borders, eliminate unstable fragments and smooth the surface. Traditionally mechanically shaving has being for a long time the method of choice due to it's availability and ease of use (14) One of the main concerns is the prevention or diminishing in reactivesynovitis caused by loose cartilage tissue in the joint, that in turnmay cause the appearance of the feared Matrix Metalloproteinases, MMP, that digest cartilage matrix (15). At the down of the first studies of thermal energy, the initialconcerns were either to use monopolar o bipolar and the depth oftissue penetration. After in vitro study of fresh bovine cartilageexposed to three RF systems two bipolar and one monopolar, the results were t smoothing of the surface and chondrocyte death in all three and that bipolar Rf device got 78% to 92% deeper than monopolar(16).A in vitro study with cartilage confirmed the chondrocyte death and the deeper penetration of the BPRF system(17). Therefore, the general feeling among surgeons was that these systemswhere not suitable for a safe chondroplasty. Furhter studies, on despite were carried on and as better understanding of the RF principles, interactions with the tissue and time of application, rendered more promising results. In a prospective study of mechanical vs BPRF after two year of FU in patella chondral injuries, the group with BPRF has better score Fulkerson-Shea (18) Gambardella in a study in sheeps compared the Cartialge injuries treated with RF, Mechanical shaving and microfracture. He found identical histological findings inthe RF and Microfracture group regarding the filling of the defect, and in the mechanical treated group, there was no filling. Kaplan et alshowed an interesting study on chndorcyte recover after severalthermal expositions of 45,50 and 55. He found that chondrocyte would recover up to 50 degree exposure but will not on 55 degrees Celcius(19) thus a new parameter emerged, the temperature. As time went by and more studies were published a new concern was born, Osteonecrosis apparently related to RF (20). However, there seemed to be a bias and a coincidence because Avascular Osteonecrosismay be also related to repeated trauma to subchondral bone due to alack of meniscus, and a meniscetomy was performed in some cases thatEventually developed thid complication due to tissue overload not tothermal damage(21,22,23). In a clinical study comparing Monopolar RFand shaver in chondroplasty in grade III femoral condyle cartilagelesion. They found no avascular necrosis, and the endpoint for bothgroups was an equal outcome in pain control. Another clinical studycarried out by Voloshin (24) of arthroscopic evaluation ofradiofrequency chondroplasty of the knee in humans Only 3 of 25lesions demonstrated progression. More than 50% showed partial orcomplete filling of the defect. Bipolar radiofrequency chondroplastyis an effective way to treat partial-thickness cartilage lesions;however, long-term effects of this treatment on cartilage remainunknown. A study with basic science evidence compared Monopolar chondroplasty vs mechanical debridement in a human patella. Pre and post treatment samples were incubated with viability stain and examined for smooth surface. The tissue depth affectation for Mechanical debridement was 385 micron meanwhile the RF group was 286 microns. Also the surface was visually smoother (25). In summary RF is a thermal source that produce some tissue response. Before theknowledge of how RF interact with tissue, large areas of chondrocytedeath were the common byproduct of thermal application for Chondroplasty. Modern and actual science stress the new tip probessurface contact, speed of probe passage over the defect or affectedtissue, water flow, temperatures on the range of 50 degreesCelsius. There is a decrease of stiffness of 71% of the tissue comparedwith non treated healthy cartilage. Depth of damage of mechanicalshaver about 386 microns compared with MRF of 286 microns. Someclinical studies shows second look with total and partial filling of the partial thickness defect whereas mechanical debridementarthroplasty has a null response. Some studies show in a short termbetter scores for the RF chondropasty than for the Mechanicalones. Osteonecrosis does not seems to be closely related to RF forthere are several factors that may be of more importance likesubchondral and meniscal trauma, providing the RF was properly applied. As we go further in research, we will have more proof of the total knowledge on safety for long term bases. The actual evidence warrant exciting horizons and promising clinical evidence goes to encourage it's use with careful trust. We must remember that a knowledgeable surgeon must fully understand the RF principles, the biological bases of thermal application like temperature, duration and probe passage over the cartilage tissue as well as power settings which are quite different from Monopolar toBipolar. We are not yet sure wether a RF treated chondromalacicArticular Cartilage will sustain at long term an initial stabilizationjust because we see and want instant gratification, there is the needto think ahead in time, the tissues will not be the same over themonths or years to come and they may have some degree of recovery or destruction. Chondral smoothening by radiofrequency has beingremarked, but it is really important to the clinic, how much does italters the friction coefficient.?, I look forward to see some studies. The field of the Heat Shock proteins may have an important role in Chondral protection, a subject to research. “Not all the new things are good, but the good things were once new”., PRP System: evidence for its use in tissue regeneration Over the last decade, a different approach has emerged to repair demaged cartilage or to fully regenerate it. Currently, the possible tecniques are: cells (bone marrow stem cells or differentiated cells)biomaterial mimicking extracellular matrix (scaffold)growth factors (GF) as regulatory signals. The therapeutic effect of these three individual components is enhanced when they are used together. Scaffolds are cell carriers playing an important role in maintaining cells in defect sites. However, physiological slowdown of cell proliferation may occur according to the scaffold constitution. GF help recreating a microenvironment favourable to cell proliferation and differentiation. In 1998, Marx demonstrated the effectiveness of platelet rich plasma (PRP) as a natural cocktail of growth factors acting in concert to accelerate healing and restoration of damaged tissues. Many in vitro studies show mitogenic effects of PRP on various type of cells (osteoblasts, chondrocytes, endhotelial cells, fibroblasts, marrow derived stem cells). PRP supplies of platelet-derived growth factor (PDGF), transforming growth factor (TGF) - β, insulin-like growth factor (IGF), vascular endhotelial growth factor (VEGF) and basic fibroblast growth factor (b FGF) which influence cell proliferation and differentiation. The tissue healing properties of PRP-gel are linked to some fundamental preparation features: the number of platelets in PRP must not be lower than 1 ×109 mLplatelets in PRP shouldn't be activated, since platelet stress induces increases in the release of GF in the supernatant platelet poor plasma (PPP) that is discarded after platelet concentration. PRP must be activated to form a gel. There are several reports about the use of platelet gel or PRP in dental implant, bone lesions or musculoskeletal injuries. One of the goals of PRP-gel is to improve cohesion of organic fragments (bone, biomaterial and cells) thus creating biologically active growth-factor enriched 3D structure. Platelet gel production follows a two-step procedure. First, hyper-concentrated platelets are prepared through platelet sedimentation (differential centrifugation of whole blood); and second, the gel is formed by adding fibrinogen cleaving agents (Ca ++, thrombin, or batroxobin) to PRP, leading to fibrin formation and polymerization, platelet activation and growth factor release. PRP can be prepared either in a blood bank environment, using good laboratory or manufacturing practice (GLP/GMP), or in the clinical point-of-care, using platelet sedimentation devices (tubes & centrifuge). Various devices are commercially available to prepare PRP in the clinic and the gel can be prepared in proximity to the patient. Some devices are also on the market for PRP gelification. Among these there are a few which devices can perform both phases (platelet sedimentation & PRP gelification). Which kind of PRP should we opt for? The answer is very difficult because each method has its own intrinsic property: DevicesProcessed blood (mL)PRP volume (mL)Platelet recovery(%)ActivatorPRGF Kit209.5 ± 4.1(%) 35 ± 16Autologous thrombinPRP (Landesberg)6010.6 ± 2.430 ± 10Ca++, thrombinAG Curasan157.6 ± 1.633 ± 10Ca++, thrombinPCCS508.5 ± 3.568 ± 9Ca++, thrombinHarvest5010.0 ± 067 ± 10Ca++, Autologus thrombinVivostat1205.0 ± 017 ± 6Neutralization of acidified batroxobinRegen Kit105.0 ± 0.590 ± 5Autologous thrombinFibrinet84.4 ± 0.265 ± 10Ca++, high speed centrifugationPlateltex85.0 ± 0.479 ± 7Ca++, batroxobinMazzucco HM608 ± 495 ± 5Ca++, Autologous thrombin Certainly the home made (HM) preparation of PRP gel using good laboratory or manufacturing practice is the best, because quality concentration (4 – 6 folds) and purity of preparation (no red cells) are manteined. Moreover this allow us to prepare thombin without haemolysis and cryoprecwipitate (if higher gel texture is required). The drawbacks are the time of preparation that is longer and the standardization of the processing because there is a “variety” of protocols of preparation and each laboratory uses its own parameters. Currently the clinical protocols about the use of PRP and PRP gel are based on the concept that platelet growth factor lead the healing processes; autologus use of these hemocomponent has made it safe to apply and combine different cell types, starting from the assumption that there are no adverse reactions and that it is not necessary to make quantitative checks of GF. We have evaluated three commercially available devices and one manual procedure (HM) with respect to resulting platelet concentration, growth factor (PDGF-BB, TGF-β1, b-FGF, VEGF, EGF, IGF-I) content and the kinetics of growth factor release from gel. Our studies showed (currently in print), the amount of GF made bioavailable to tissues depends on the amount of the factors that are contained in the platelets, in the plasma moiety, absorbed in the gel, and released in time both from platelets and gel. The results have shown several phenomena: a lack of correlation between platelet count and growth factors content in the PRP; procedure-dependent and procedure-independent features in growth factors release; process-dependent differences in the kinetics of the bioavailability of growth factors to tissues. Since lesions of almost all tissues are said to benefit from topical application of platelet-derived factors and since different tissues are formed by particular cells and by particular extracellular matrix composition. It is likely that different kinetics of growth-factors bioavailability might be more or less appropriate to treat different kind of lesions or different kind of tissues., Members of the Stem Cell Research Center (SCRC) have isolated various populations of myogenic cells from the postnatal skeletal muscle of normal mice by means of the cells' adhesion characteristics, proliferation behavior, and myogenic and stem cell marker expression profiles. Although most of these cell populations have displayed characteristics similar to those of skeletal muscle satellite cells, we also have identified a unique population of muscle-derived stem cells (MDSCs). The MDSCs exhibit long-term proliferation abilities, elevated self-renewal rates, increased resistance to stress, and they are multipotent and can differentiate toward a variety of tissue types including: muscle (skeletal and cardiac), neural, endothelial, osteogenic, and chondrogenic lineages, both in vitro and in vivo. In contrast to other myogenic cell types, MDSCs show very efficient engraftment and regeneration of various musculoskeletal tissues due to their ability to highly survive post-implantation through the high anti-oxydant expression by MDSC. Interestingly, it has been observed that female MDSCs (F-MDSCs) can more efficiently regenerate the dystrophic skeletal muscle of mdx mice (a mouse model of Duchenne muscular dystrophy) than their male MDSCs (M-MDSCs) counterparts. MDSCs are influenced by environmental cues released within dystrophic or injured skeletal muscle which has been shown to negatively impact MDSCs and cause the cells to differentiate toward a fibrotic cell lineage and hence produce scar tissue rather than healthy skeletal m uscle fibers. Potential strategies are being explored to prevent the formation of scar tissue within injured skeletal muscle by blocking the action of TGF-b1. Finally, blood vessels contain several cell types, including myo-endothelial cells and pericytes, and are likely the place of origin of the murine MDSCs discussed above. The results outlined above open new avenues by which researchers could use muscle stem cell-based gene therapy and tissue engineering to improve tissue regeneration., Many focal cartilage defects can be effectively treated with autologous chondrocyte implantation, a two-step procedure requiring cartilage harvest at the index surgery, cell expansion, and subsequent reimplantation. In an effort to provide easier surgical delivery of autologous chondrocytes to repair chondral defects, minced cartilage may be an option. In this single-stage option, cartilage tissue, either processed intraoperatively (autologous) and loaded onto a scaffold or processed in advance (allogeneic) and available “on the shelf,” can treat chondral defects. In the lab, cartilage was harvested from both human and bovine trochleas, minced into small fragments (∼1mm3), and loaded onto PGA/PLA nonwoven felt or PGA/PCL foam reinforced with PDS, and cultured. The minced cartilage with scaffold samples were then implanted in mice for 4 weeks to assess chondrocyte migration and growth. The study showed that there is an inverse relationship between cartilage fragment size and amount of outgrowth (smaller size, more chondral growth) and the highest level of cellular activity is localized at the edge of the minced cartilage. Further, we tested the effectiveness of the minced cartilage on goat specimens. A 7mm trochlear defect was created and randomized to one of three treatment options: no treatment (empty), scaffold alone, and scaffold with minced autologous cartilage fragments. All treatments generated tissue within the defect. However, the scaffold with minced fragments produced the best results: whiter tissue with better congruency, more intense staining for proteoglycans, zonal architecture, and had a higher collagen type II to type I ratio. A Phase I FDA clinical study icompleted enrollment in 2007 and are now enrolling patients as part of a prospective comparison of this Cartilage Autograft Implantation System® (Mitek, Inc, Rayham, MA) in a Phase III multi-center study. The purpose of this study is to determine the safety and efficacy of CAIS compared to microfracture at 12 months post-treatment, with the primary efficacy assessment based on an analysis of non-inferiority of CAIS to microfracture for reduction in knee pain., CCI to stage autologous cartilage regeneration ACI has been shown to be an effective method of restoring articular cartilage and joint function in recent years several publications have shown the capability to regenerate a tissue which to some extent histologically resembles functional articular cartilage long-term results of clinical patient outcome are good if the hurdles in initial restoration as for example in the first two postoperative years are passed without complications. However there is a clear need for a better understanding of how cellular technology can be optimized to improve the reliability of patient outcome. Factors which come into play are aspects such as quality of the initial biopsy the number of passages and expansion volume of cells to be used. Also preoperative intra-articular joint homeostasis will be of influence to cartilage culture quality and the outcome of integration and tissue restoration after the transplantation surgery has been completed. Characterized chondrocytes are an autologous cartilage cell population consisting of phenotypically stable cells. As expansion of autologous chondrocytes leads to dedifferentiation and loss of chondrogenic capacity manufacturing methods needed to be developed with the aim of preserving as much as possible the phenotypic characteristics of the expanded cell population. ChondroCelect (TiGenix N.V., Haasrode, Belgium) is an autologous cell therapy product introducing the patented concept of a chondrogenic potential score (patent number WO2008061804) whereby a gene marker profile is used in the identification of in vivo cartilage forming ability. A controlled and consistent manufacturing process optimizing a number of parameters including enzymatic release, seeding density, time and procedure at harvest and shipping conditions is based on ectopic cartilage formation. The correlation between the marker profile and the ectopic cartilage forming activity shows an R2 value of 0.608 The total amount of cells administered is dependent on the size (surface in cm2) of the cartilage defect. The dose used is 0.8 to 1 million cells/cm2, corresponding with 80 to 100 microliter of product/cmÂ2 of defect. Characterized chondrocyte site implantation is the next generation of ACI it builds upon the established principle of culturing autologous chondrocytes but provides the surgeon and patient with a panel of molecular profiling which predicts the stability of cartilage tissue formed. While the method still involves a biopsy surgery and second stage procedure for implantation arthrotomy it has the additional benefit of a reliable cellular products being implanted. We have previously shown that after one year patients that have been treated by CCI have a histological tissue regenerate which is superior to that of a microfracture repair. The clinical outcome has been shown to be beneficial with a statistical superior results for relevant parameters of patient described clinical outcome using KOOS. However this study also indicates further challenges for clinical application of this regenerative technique. These lie in the need to better understand which patients are optimal candidates for such biological reconstruction. How come we predefined the success or failure of the biological regeneration process, which patients are eligible for reconstruction and which joints have passed the point of no return towards osteoarthritic degeneration for which cellular therapy at this moment has not been shown to be a viable option. CCI makes a difference in quality of tissue regeneration it's provides better clinical outcome three years post surgery however improvements are needed for matrix-based implantation patient profiling arthroscopic implantation and eventually maybe even one stage surgical interventions., Articular cartilage lesions, with their inherent limited healing potential, remain a challenging problem for orthopaedic surgeons. Various techniques, both palliative and reparative have been used to treat this pathology with variable success rates. In recent years regenerative techniques, such as ACI, have emerged as a potential therapeutic option. Recent studies [1,2] suggest the durability of this treatment, especially at long-term follow-up, due to its ability to produce hyaline-like cartilage that is mechanically and functionally stable, and also allows integration with the adjacent articular surface. However, despite the favourable clinical results obtained by many authors, the use of classic ACI (first generation) has been associated with several limitations related to the complexity and the morbidity of the surgical procedure, as well as the frequent occurrence of periosteal hypertrophy. Also some recent randomised studies [3,4] report controversial results regarding the better performance of the first generation ACI technique compared to other procedures used for cartilage repair. To address these problems Second generation ACI has been developed and biodegradable polymers as temporary scaffolds for the in vitro growth of living cells and their subsequent transplantation onto the defect have become widely used. Second generation ACI represents a modern and viable technique for cartilage full thickness chondral lesion repair [5,22,23]. However second generation ACI is a two step procedure which includes an arthroscopic biopsy for cell culture and implantation. Aside from the risk of harvest site morbidity and two surgical procedures, the total cost of the operation, scaffold and chondrocytes cultivation is still very high. Future directions in cartilage repair are moving towards the possibility to performing one step surgery. These could include the use of stem cells and growth factors. The use of autologous mesenchymal stem cells (MSC) and growth factors represents an improvement on the currently available techniques as this avoids the primary surgery for cartilage biopsy and subsequent chondrocytes cultivation and seeding on a scaffold. Many authors have recognized that nucleated cells found in bone marrow are a useful source of cells for restoration of damaged tissue [6,7]. Once MSC are cultured in the appropriate microenvironment, they can differentiate to chondrocytes and form cartilage. The onset of chondrogenesis requires a chemically defined serum free medium supplemented with dexamethasone, ascorbic acid and growth factors such as TGF-B [8]. In conjunction with appropriate scaffolds, these has been demonstrated that cells can be used to regenerate cartilage in a variety of applications [6]. However, some animal and laboratory studies have shown the chondrogenic potential of MSC but only few clinical human studies have been published [9,10]. Wakitani et al. [11] used autologous culture of expanded bone marrow for repair of cartilage defects in osteoarthritic knees; they chose 24 knees of 24 patients with knee OA who underwent a high tibial osteotomy; patients were divided into cell transplanted group and cell free group. After 16 months follow-up, they concluded that MSC were capable of regenerating a repair tissue for large chondral defects. Ochi et al. [12] observed that in a rat model the injection of cultured MSC combined with bone marrow stimulation can accelerate the regeneration of articular cartilage; they noted that this cell therapy was a less invasive treatment for cartilage injury. In their animal study [13] they introduced a MSC delivery system with the help of an electromagnetic field, enhancing the proliferation of cartilage inside the chondral defect after intra-articular injection, decreasing ectopic cartilage formation. Fortier et al. [14] concluded in animal studies that development of patient-side configuration techniques for intra-operative stem cell isolation and purification for immediate grafting have significant advantages in time savings and immediate application of an autogenous cell for cartilage repair. In order to find a more effective and simpler technique for cartilage repair we have attempted to use high concentration MSC combined with biologic scaffolds for osteochondral defect repair. Our technique for this operative procedure, consists firstly in preparation of the osteochondral defect with debridement by arthroscopy, and extraction of bone marrow from iliac crest. High concentration MSC are obtained by harvesting 40 −60 mL of bone marrow aspirate from the iliac crest with aspiration kit and a centrifugations system (Harvest Smart PreP2 System - Harvest Technologies, Plymouth, MA, USA). Implantation of the MSC are performed after adding batroxobin obtaining a sticky clot material that is implanted into osteo-chondral defect. Bilayer porcine collagen type I/ III matrix (Chondro-Gide; Geistlich Pharma AG, Wolhusen, Switzerland) is then applied and sutured or fixed with fibrin glue to the surrounding cartilage tissue to cover the entire defect and the BMC gel. In our first group, we prospectively followed grade 3 and 4 cartilage knee lesions implanted with concentrated MSC from the iliac crest with or without the use of a scaffold. All patients followed the same specific rehabilitation program after MSC implantation. With a mean follow-up of 12 months, patients showed improvements in all scores. No adverse reaction or post–op complication were noted in all patients to this date. Simplicity and low cost are the two major advantages of one step M.C.I.. This technique does not require cartilage harvesting, transportation to a G.M.P. laboratory and subsequent cells cultivation, seeding on the scaffold and re-implantation. Though more patients and longer follow up is needed to confirm our results. This one-step procedure permits a significant reduction of operating time and the related costs. And could very well be the future procedure of choice in cartilage repair., Articular cartilage in adults has a limited capacity for self-repair after a substantial injury. In addition to bone marrow stimulating procedures such as microfracturing surgical therapeutic efforts to treat cartilage have focused on delivering new cells capable of chondrogenesis into the lesions. In the classic autologous chondrocyte transplanation (ACT) technique chondrocytes are isolated from small slices harvested from a minor weight-bearing area of the injured knee. The extracted cells are then cultured and once a sufficient number of cells has been obtained, the chondrocytes are implanted into the cartilage defect using a periosteal patch over the defect as a method of cell containment. Further improvements in tissue engineering have contributed to the next generation of ACT techniques, where cells are combined with resorbable biomaterials, as in matrix associated autologous chondrocyte transplantation (MACT). These materials secure the cells in the defect area and enhance their proliferation and differentiation. MR imaging as a non-invasive technique is the method of choice in the preoperative evaluation and follow-up of patients with these different surgical cartilage repair techniques. MR imaging of the morphology of cartilage and cartilage repair tissue has significantly improved in recent years due to the development of clinical high-field MR systems operating at 3 Tesla. The improved performance has also been achieved as a result of the higher gradient strengths and the application of dedicated coils with modern configuration such as phased array coils. MR should be performed with cartilage sensitive sequences such as fat-suppressed PD/T2-FSE or three-dimensional (3D) GRE sequences, which provide a good signal to noise ratio (SNR) and contrast to noise ratio (CNR). High spatial resolution is mandatory and can be best achieved with dedicated coils at 3T. High resolution imaging is necessary for a better visualization of graft morphology, in particular for the evaluation of transplant integration to the adjacent hyaline cartilage and bone. MR imaging also helps to evaluate the filling of the defect by repair tissue, the surface and structure of repair tissue, the signal intensity of repair tissue with respect to the time interval to surgery and the status of the subchondral bone. Complications such as periosteal hypertrophy, incomplete and complete delamination, arthrofibrosis and adhesions, incongruencies of the cartilage surface at the repair site, graft failure and reactive changes of the joint such as effusions and synovitis can be visualized. The evaluation of the success of cartilage repair procedures requires particular grading systems, one of which is “Magnetic resonance Observation of CARtilage repair Tissue” or MOCART. The MOCART scoring introduced by Marlovits and Trattnig is postulated to allow subtle and suitable assessment of the articular cartilage repair tissue. Indeed in many recent original articles, review articles and book chapters, the MOCART score is used and discussed in the follow-up after different cartilage repair procedures. In contrast, new isovoxel sequences (GRE and well as FSE) have the potential for high-resolution isotropic imaging with a voxel size down to 0.4mm3, and can thus be reformatted in arbitrary planes without any loss of spatial resolution. Using this possibility of multi-planar reconstruction (MPR), the cartilage repair tissue could be visualized three-dimensional in every plane and its classification and grading by an MR-based scoring system might benefit. Recently, an improved MOCART scoring system using the possibilities of 3D MPR in the post-operative evaluation of cartilage repair tissue after MACT was developed (3D MOCART). In addition to morphological MR imaging of cartilage repair tissue, an advanced method to non-destructively and quantitatively monitor parameters reflecting the biochemical status of cartilage repair tissue is a necessity for studies which seek to elucidate the natural maturation of ACT and MACT grafts and the efficacy of the technique. For example glycosaminoglycans (GAG) are known to be responsible for stiffness properties of cartilage, which gains even more importance with cartilage implants and the content and organization of the collagen network reflects further mechanical properties of cartilage. Therefore, several MR techniques were developed, which allow detection of biochemical changes that precede the morphological degeneration in cartilage. To date, the most promising technique for visualizing the loss of GAG seems to be the delayed Gadolinium-Enhanced MRI of Cartilage (dGEMRIC). It is based on the fact that GAG molecules contain negatively charged side chains which lead to an inverse proportionality in the distribution of the negatively charged contrast agent molecules with respect to the concentration of GAG. Consequently, T1 which is determined by the Gd-DTPA2- concentration becomes a specific measure of tissue GAG concentration. Earlier clinical studies of early cartilage degeneration showed that the differences of the pre-contrast T1 values between degenerative cartilage and normal cartilage were so small that they could be neglected; however, this is not true for cartilage repair tissue. For a correct evaluation of glycosaminoglycan concentration in cartilage repair tissue the pre-contrast T1 values have to be calculated, too. If a quantitative T1 analysis is also performed prior to contrast administration, it is possible to calculate the concentration of Gd-DTPA in cartilage repair tissue. The concentration is represented by ï„ R1, that is the difference in relaxation rate (R1=1/T1) between T1precontrast and T1postcontrast. This places time limitation problems on the patient evaluation since both pre-contrast MR imaging and delayed post-contrast MR imaging must be performed in cartilage repair patients. Furthermore, standard Inversion Recovery sequence for T1 mapping is time consuming. To overcome these problems a fast T1 determination by using different excitation flip angle values in gradient echo based sequences was optimized for dGEMRIC technique. For the follow-up of cartilage implants quantitative T1 mapping based on dual flip angle excitation pulse GRE technique allows in plane resolution of 0.3 × 0.3 mm with a slice thickness of 3mm and a scan time of about 4 minutes and was validated in vitro and in vivo. This dGEMRIC technique can be applied to patients following cartilage repair surgery as a way to obtain information related to the long-term development and maturation of grafts. While GAG content reflects stiffness properties of repair tissue, the organisation of the collagen matrix in repair tissue over time is important too, as failure within the collageneous fibre network is considered to entail further cartilage breakdown. The extracellular matrix of native articular cartilage is shaped by a highly organized collagen network, which is the basis of the histologic zones. Under ideal circumstances cartilage repair tissue produced following ACI and MACT, or other repair techniques, should, over time, develop a collagen network with a similar shape and collagen concentration to normal hyaline cartilage. Quantitative T2 mapping has been reported to be sensitive to collagen content and organization Using quantitative T2 mapping of patients at different post operative intervals after MACT surgery significantly higher T2 values in cartilage repair tissue in the early stage (3–6 months) compared to native hyaline cartilage were found with a decrease in repair tissue T2 values over time with the T2 values becoming similar to native healthy cartilage by approximately 10 to 13 months. With high resolution MR T2 mapping it is possible to assess zonal variations within the cartilage layer and use the measurement of the organization of articular cartilage as an additional tool to differentiate between cartilage repair tissues. This is based on the fact that the collagen fibers in the deep portion of cartilage are running perpendicular to cortical bone with consecutive dipolar effect and less mobility of protons resulting in a decrease of T2 values. Since the collagen fibers are randomly oriented in the superficial cartilage zone T2 values are longer. No differences between deep and superficial aspects within cartilage repair tissue and in total shorter T2 values compared to healthy cartilage after microfracture therapy was found indicating disorganized and more fibrous tissue. After MACT, zonal variation with T2 mapping could be measured, however compared to healthy cartilage sites the increase from deep to superficial zones was less pronounced. These findings may indicate that after MACT, cartilage repair tissue is, in terms of organization, more hyaline like. Quantitative T2 mapping may therefore help to better differentiate between normal maturation and development of abnormality in ACI and MACT. This technique may further help in the non-invasive, non destructive follow-up of patients operated on new generations of matrix-associated ACT using new scaffold or carriers. Better knowledge on the macromolecular organization in the implants may help in the planning of rehabilitative procedures after cartilage repair surgery. One encouraging alternative to these above mentioned sequence modalities for the evaluation of cartilage microstructure is the use of diffusion weighted sequences. Diffusion Weighted Imaging (DWI) is based on molecular motion that is influenced by intra- and extra-cellular barriers. Consequently, it is possible, by measuring of the molecular movement, to reflect biochemical structure and architecture of the tissue. To avoid long scan times and susceptibility changesassociated with spin-echo and echoplanar imaging sequences diffusion imaging can be based on steady state free precession sequences (SSFP) which realize a diffusion weighting in relatively short echo times. For the assessment of diffusion weighted images, a three-dimensional steady state diffusion technique, called PSIF (which is a time reversed FISP (Fast Imaging by Steady State Precession) sequence), has been used For evaluation, the quotient image (non-diffusion weighted / diffusion-weighted image) was calculated on a pixel-by-pixel basis. Recently, a quantitative SSFP based diffusion-weighted sequence was developed, but has to be validated in clinical studies. Most recent developments in MR imaging of cartilage repair comprise Magnetization Transfer imaging (MT) and its special variant: Chemical Exchange Saturation Transfer (CEST), the use of ultrahigh MR operating at 7T in vivo and the biomechanical MR imaging of cartilage repair tissue using unloading technique., Introduction Articular cartilage lesions are a common pathology of the knee joint and many patients could benefit from cartilage repair. Untreated, however, cartilage defects may lead to osteoarthritis (OA). Thus, surgical treatment options may offer a possibility for patients with cartilage defects to avoid OA or to delay the progression of OA. Therefore, cartilage repair techniques require sophisticated follow-up, if possible non-invasively. Although clinical findings are the primary criteria, a more objective outcome measure would possibly have the ability to provide predictive values. Advanced magnetic resonance imaging (MRI) is able to depict the morphological and biochemical condition of the cartilage repair tissue and the surrounding structures [1–4]. Histological samples are still the gold standard to gain information on the ultrastructure of cartilage repair tissue, however their impact is limited due to the required reoperation and the fact that a small biopsy may not provide information on the whole cartilage transplants, its borders and the relationship to the adjacent native cartilage. A sophisticated MR protocol in the follow-up after cartilage repair procedures nevertheless is able to visualize the morphological and the biochemical properties of the whole cartilage transplant and its adjacent cartilage non-invasively as a “virtual biopsy”. An ideal MRI protocol for articular cartilage and cartilage repair should provide accurate assessment of cartilage thickness and volume, reveal information about signal changes within cartilage, and demonstrate clear delineation of the cartilage surface, the cartilage and bone interface, as well as the subchondral bone and also provide information about the biochemical composition of articular cartilage and cartilage repair tissue. To evaluate such MRI protocols, cartilage repair procedures, such as arthroscopic or open surgical approaches as well as marrow-stimulation techniques, osteochondral grafting, and chondrocyte implantation/transplantation could be used. The uniqueness of cartilage repair in the use of advanced morphological and especially biochemical MR methodologies is bases in the fact that these procedures include defined areas of repair cartilage in addition to an often-intact surrounding cartilage in mostly young patients. Thus, modified and healthy cartilage can be compared within one subject. Nevertheless, the aim of an ideal MRI protocol must be its ease of implementation in scientific studies as well as day-to-day clinical routine. The combination of high-resolution morphological MR evaluation with biochemical assessment in a clinically applicable scan time needs to exploit high-field MRI together with advanced imaging techniques and sophisticated coil technology. Aim of this manuscript is to demonstrate advanced MRI techniques to depict the morphological and especially the biochemical constitution of cartilage repair tissue as a multiparametric approach in the follow-up of cartilage repair procedures. Methods and Discussion Histology -what do we expect? Native cartilage Native articular cartilage is a complex connective tissue, basically composed of water(∼75%), collagen(∼20%), and proteoglycan aggregates(∼5%). Water either freely moves throughout the matrix or is bound to macromolecules. Collagen in hyaline cartilage is largely type II, creating a stable network throughout the cartilage. The negatively charged proteoglycan is composed of a central core protein to which glycosaminoglycans (GAG) are bound. Hyaline articular cartilage is stratified primarily according to the orientation of collagen within a three-dimensional network [5, 6]. This complex network is stratified and arranged in zones. Cartilage repair In available studies a after MFX, OAT, ACI/ACT, and MACI/MACT histological evaluation show varying results; nevertheless, some trends are visible and may help to characterize the constitution of cartilage tissue. Gudas et al. [7], in a comparison of MFX and OAT, described all biopsies after OAT as hyaline cartilage, whereas after MFX, fibrocartilage (57%) and soft fibroelastic tissue(43%) was found. Bentley et al. [8], who compared OAT with ACI, reported biopsies obtained after ACI as hyaline-like(37%), mixed hyaline-like/fibrocartilage(37%), and fibrocartilage(28%), whereas after OAT hyaline cartilage is usually present within the different plugs, however problems are described in between the plugs. Horas et al. [9], on the other hand, reports histological samples after ACI as hyaline-like near the base of repair tissue, but with no tidemark and rather fibrous tissue in the central and superficial layers. After OAT, biopsies demonstrated the original tidemark and did not differ from surrounding cartilage. Bartlett et al. [10] compared ACI and MACI with nearly identical biopsies of hyaline-like cartilage(∼30%), mixed hyaline-like and fibrocartilage(∼10%), and fibrocartilage(∼60%). Other studies have reported much higher numbers(up to 75%) of biopsies with hyaline-like cartilage after ACI or MACI. When looking at one of the most widely recognized studies in this field, by Knutsen et al. [11], who compared ACT and MFX, two very important points become clear: i) Biopsies obtained from 67 patients showed all histological grades (1= predominantly hyaline; 2= fibrocartilage-hyaline mixture; 3= fibrocartilage; 4= no repair tissue) for both procedures and no significant difference between the cartilage repair techniques. Hence the ultrastructure of the repair tissue is not (only) dependent on the respective cartilage repair procedure and MFX might not only produce fibrocartilage and ACT might not only produce hyaline-like cartilage. ii) However, a comparison of the histological quality of the repair tissue in patients with and without treatment failure revealed that none of the patients with treatment failure in this study had hyaline-like cartilage. This finding suggests that repair cartilage, which is predominantly hyaline, may reduce the risk of subsequent failure [11]. Hence after all cartilage repair procedures the quantity and the quality of the repair tissue have to be assessed and together they might provide an objective outcome measure and a possible predictive value. MRI - what can we visualize? Morphological MRI The morphological shape of the repair tissue, the degree of repair filling (1), the integration of the cartilage repair tissue to the border zone (2), the structure of the surface (3), the structure of the whole repair tissue (4), the signal intensity (5), the constitution of the subchondral lamina (6), the the constitution of the subchondral bone (7), possible adhesions (8), and possible effusion (9) can be combined in the nine variables of the magnetic resonance observation of cartilage repair tissue (MOCART) scoring system [12] which is claimed to allow subtle and suitable assessment of the articular cartilage repair tissue. The MR assessment of the MOCART score is based on standard MR sequences, also recommended by the International Cartilage Repair Society [13]. Depending on the locality of the area of cartilage repair, the MR evaluation of the cartilage repair tissue is performed on sagittal, axial or coronal two-dimensional planes using high spatial resolution together with a slice thickness of 2–4 mm. Following the current standard procedure, the recommended MR sequences by the ICRS and the recommended sequences for the MOCART scoring visualize the area of cartilage repair and the adjacent cartilage as well as the surrounding structures in 2D. In contrast, new isovoxel sequences have the potential for high-resolution isotropic imaging with a voxel size down to 0.4mm3, and can be reformatted in every plane without any loss of spatial resolution. Hence the cartilage repair tissue can be visualized three-dimensionally (3D) in every plane, its subsequent classification and grading by an MR-based scoring system benefits and a new 3D-MOCART score can be presented at the ICRS 2009. Biochemical - dGERMIC and others Of the major macromolecules, glycosamnioglycans (GAG), are important to the cartilage tissueâ ™s biochemical and biomechanical function. GAG are the main source of fixed charge density (FCD) in cartilage, often decreased in reparative cartilage after cartilage repair [14]. Intravenously administered gadolinium diethylenetriamine pentaacetate anion, (Gd-DTPA2-) penetrates the cartilage through both the articular surface and the subchondral bone. The contrast equilibrates in inverse relation to the FCD, which is, in turn, directly related to the GAG concentration; therefore, T1, which is determined by the Gd-DTPA2- concentration, becomes a specific measure of tissue GAG concentration, suggesting that T1 mapping enhanced by delayed administration of Gd-DTPA2- (T1 dGEMRIC) has the potential for monitoring GAG content of cartilage in vivo [15, 16]. Besides standard inversion recovery (IR) evaluation, a new approach for fast T1 mapping has shown promising results and is increasing the clinical applicability of the dGEMRIC technique [17]. Since GAG content is responsible for cartilage function, particularly its tensile strength, the monitoring of the development of GAG content in cartilage repair tissues may provide information about the quality of the repair tissue. A recent study by our group showed dGEMRIC to be able to differentiate between different cartilage repair tissues with higher delta R1 values, and thus, lower GAG content for cartilage repair tissue after MFX compared to MACT [18]. As the mapping of the GAG concentration is desirable in the follow-up of cartilage repair procedures and the presented dGEMRIC technique has the limitation of contrast agent administration and a time delay before post-contrast MRI, a recently described technique for the assessment of GAG concentration in vivo by chemical exchange-dependent saturation transfer (CEST) may have potential in future applications on articular cartilage [19]. At high fields and ultra-high fields, sodium MR imaging might become a gold-standard in future approaches. Sodium MRI has been validated as direct and quantitative method of computing FCD and, hence, proteoglycan content. Achieving high enough signal-to-noise ratio, the sensitivity of sodium MRI is high enough for detecting small changes in proteoglycan content. Biochemical - T2 and others Changes in water and collagen content and tissue anisotropy are most frequently analyzed using the transverse relaxation time (T2) of cartilage [20]. The collagen fiber orientation, its three-dimensional organization and curvature can be reliably visualized by cartilage T2 mapping, however influencing its appearance at 55deg; (with respect to the main magnetic field (B0)) resulting in the magic angle [5, 21]. In healthy articular cartilage, an increase in T2 values from deep to superficial cartilage layers can be observed based on the anisotropy of collagen fibers running perpendicular to cortical bone in the deep layer of cartilage [22]. Histologically validated animal studies have shown this zonal increase in T2 values as a marker of hyaline or hyaline-like cartilage structure after cartilage repair procedures within the knee [23, 24]. To visualize this zonal variation is essential for cartilage T2 mapping and high enough spatial resolution has to be provided. In cartilage repair tissue, full-thickness T2 values, as zonal evaluation have been shown able to visualize cartilage repair maturation [25, 26]. Another study by our group further showed the ability of zonal T2 evaluation to differentiate cartilage repair tissue after MFX and MACT [27]. In addition to standard 2D multi-echo spin-echo T2 relaxation, T2*- weighted 3D gradient-echo articular cartilage imaging has shown reliable results in the evaluation of chondromalacia of the knee [28]. In recent studies, T2* mapping, with its potentially short scan times, was correlated to standard T2, and showed information comparable to that obtained for articular cartilage in the knee, but with overall lower T2* values (ms) [29, 30]. Furthermore, also for T2*, a clear zonal variation between deep and superficial cartilage layers can be shown. Another advanced approach to combine morphological and biochemical T2 cartilage imaging is based on a Double-Echo-Steady-State (DESS) sequence, which generates two signal echoes that are characterized by a different contrast behavior where the underlying T2 can be calculated. Further emerging biochemical MR techniques that are gaining more and more importance are, besides others, diffusion weighted imaging (DWI), magnetization transfer contrast (MTC) or T1rho. The cartilage composition or component visualized by these methodologies is not yet clearly validated. However initial studies are showing very promising results and additional information besides dGERMIC and T2 can be gained also in patients after cartilage repair. Concluding, a combination of morphological and biochemical MRI might, besides a sophisticated non-invasive follow-up examination, provide the chance to gain a predictive value for the future development and performance of the cartilage repair tissue and the joint., Today, there are several existing techniques for the treatment of cartilage injuries but still the evaluation of the quality of such repairs has not been good enough. Also, the classification of cartilage lesions in order to diagnose an early osteoarthritis is yet to date not satisfying. Today, one may classify OA by clinical symptoms and/or the degree of joint space narrowing and number and size of existing osteophytes (Altman 1991). An evaluation system that can in a more objective and qualitative way, describe the nature of the cartilage repair and cartilage morphology would be of great importance. An objective more secure way of OA classification would also be of great importance. Magnetic resonance (MR) imaging is a 3D imaging technique that can permit a direct delineation of cartilage structure. With MRI one may estimate the volume and thickness of cartilage securely and make compositional imaging of cartilage using quantitative MR as well as for semi quantitative scoring of cartilage disease For the surgeon it is possible with MRI to estimate the size, nature and location of lesions Preoperatively and post-surgery at certain intervals also evaluate the quality regarding filling of the defect, tissue integration and subchondral bone reaction. Is it possible with a cartilage repair to stop an osteoarthritic development? MRI may give such an answer and could be used to follow the structure of the surrounding cartilage post-repair comparing the cartilage in such a repaired joint with a joint with similar lesions but without cartilage defect repair. However, with the most MRI techniques used by normal hospitals worldwide, the MRI technique is not yet good enough. High qualities MRI are mostly found at university centres. Figueroa et al, 2007, studied one hundred ffteen chondral lesions in 82 patients which were found during the arthroscopic procedure. Most of them were single lesions (72%) located on the medial femoral condyle (32.2%) or medial patellae (22.6%); 62.6% of the lesions were classified as ICRS type II or II I-A, with an average surface of 1.99 cm2. The authors found a significant direct correlation between the patient's age and the size of the lesion. MRI sensitivity was 45% with a specificity of 100%. The sensitivity increased with deeper lesions (direct relation with the ICRS classification). The authors concluded that even though unenhanced MRI using a 1.5-Tesla magnet with conventional sequences (proton density-weighted, T1-weighted, and T2-weighted) is most accurate at revealing deeper lesions and defects at the patellae, that study shows that a considerable number of lesions will remain undetected until arthroscopy, which remains the gold standard. A step-forward have been the development of the MOCART classification system (Magnetic Resonance Observation of Cartilage Repair Tissue)(Marlovits et al, 2006). The system is an attempt of transferring the ICRS arthroscopic macroscopic post repair evaluation system to the evaluation of defect repair as seen on the MR images. The radiologists and the arthroscopists are subsequently focusing on the same issue; the success of the repair attempts. Two MR imaging techniques that are important in the evaluation of early structural damage in articular cartilage are quantitative T2 mapping and T1 mapping after Gd-DPTA2- administration (dGEMRIC). dGEMRIC technique can demonstrate the glycosamino-glycan(GAG) component of articular cartilage while T2 mapping focuses on changes noticed in the water and collagen content and the different tissue layers. With such techniques it is possible to both better compare the repair with the surrounding native cartilage and also follows the maturation process, especially as the maturation process is important when deciding for the rehabilitation protocols. Still today, when randomised study protocols are developed, biopsies of the repair tissues are included. However, such biopsies show a very little part of the repair tissue and with today's imaging techniques; biopsies should be avoided being invasive procedures with risk of morbidity for the patient. Important is the finding that normal articular cartilage demonstrates an increase in T2 values from the subchondral bone to the articular surface that has been correlated with type II collagen fibre matrix organization (anisotropy) in those zones (White et al, 2006). Perfect agreement was seen between organized T2 and histological findings of hyaline cartilage and between disorganized T2 and histological findings of fibrous reparative tissue. A significant trend of increasing T2 values (from deep to superficial) was found in hyaline cartilage while fibrous tissue sites had no significant change from bone to surface(White et al, 2006). Kurkijarvi et al (2007) found that according to T2 measurements, ACI repair tissue at 10–15 months differs from normal cartilage and probably lacks the preferential collagen arrangement of normal cartilage, while according to dGEMRIC a varying degree of proteoglycan replenishment takes place. Combining these two quantitative magnetic resonance imaging techniques enables a more comprehensive characterization of cartilage repair than before and in the future it will be of great interest to see randomised studies including such non-invasive methodologies in the evaluation of repairs from bone marrow stimulation techniques versus from different cell seeded scaffolds. Furthermore, for the diagnosis of osteoarthritis there is now the possibility to use spin-lock (T1RHO) magnetic resonance imaging (regatta, 2006). T1RHO is highly sensitive to changes in macro-molecular content, which may allow it to be used as a magnetic resonance marker of molecular changes in cartilage degeneration. T1rho has a higher dynamic range to measure even small macromolecular changes for detecting early pathology and subsequently useful to detect early OA compared to T2. The future will involve the above described imaging techniques for both the orthopaedic surgeons following the outcome of their surgeries as well as for the rheumatologists following their results of OA and RA treatments with chondroprotecive drugs and other pre-prosthetic treatments. New systems for cartilage injury classifications, cartilage repair assessments and OA definitions will be developed for the monitoring of success of treatments and for understanding of the epidemiology of disease. Furthermore to look forward for in the future is the use of high field systems (3 T and above) that significantly improve the sensitivity and specificity of imaging studies for the assessment of cartilage and cartilage disorders., In cartilage repair, similarly to other knee injuries and knee osteoarthritis, the relation of structural changes and clinical outcome is generally poor. It seems that pain and other clinical outcomes may be related to other factors than structures such as cartilage, ligaments and the menisci. Studies in knee osteoarthritis have shown quadriceps weakness and psychosocial factors to have a stronger association to pain than radiographic changes [1]. Similarly, impaired function seems to be largely explained by other factors than radiographic changes. McAlindon [2] found impaired function to be explained by quadriceps weakness, knee pain and older age and not by severity of radiographic changes. Traditionally, we consider pain as a result of an underlying patho-anatomic change. In knee injury and knee osteoarthritis causality is weak. Possible reasons for this weak association in knee osteoarthritis include radiographic changes being the result of a process and not mirroring the process in itself, the examination being performed in such a way that changes have not been registered, that radiographic changes which we think constitute the disease actually do not represent the full spectrum of the disease, or that the radiographic changes are not the reason for the symptoms. Much the same is seen in cartilage repair. Structural improvement is not necessarily followed by clinical improvement, and vice versa; clinical improvement may be registered without and structural improvement. In addition to lack of validity in evaluation measure, other underlying pain mechanisms than patho-anatomical lesions, reasons for the discrepancy seen in interventions such as cartilage repair include placebo effects. Placebo is very effective in the treatment of knee osteoarthritis. The size of the placebo effect is determined by the strength of the active treatment, the baseline disease severity, the route of delivery and the sample size of the study [3]. Surgery, as in cartilage repair, is associated with a larger placebo effect than a drug which is turn is associated with larger placebo effects than from non-pharmacological and non-surgical interventions. Although there is a correlation of increasing radiographic changes and pain at a group level in people with osteoarthritis, the association for the individual is weak. In population-based studies about half report knee pain [4–6]. In one of these studies, based on the Framingham cohort, only 15% of the individuals who reported knee pain had radiographic changes of osteoarthritis [6]. Since the prevalence of osteoarthritis increase with age, one could expect a stronger association with older age. However, only 21% of the subjects aged 51–74 who reported knee pain had radiographic osteoarthritis [6]. It has been suggested that the insensitivity of radiographic examination to detect cartilage changes and low sensitivity of the pain questions used could explain the weak correlation of radiographic osteoarthritis and pain. In population-based studies, pain has most often been defined as pain during most days of a month [2, 6]. In studies on osteoarthritis patients, most commonly a self-reported questionnaire evaluating pain during different activities during the last week is used. Although differently formulated questions have different ability to detect radiographic osteoarthritis [7] the differences are relatively small and most likely do not explain the discrepancy between pain and structure in osteoarthritis. In summary, the correlation of structure and pain is weak in cartilage repair as well as in other knee problems such as ligament deficiency, and in knee osteoarthritis. In design of clinical trials there is consensus within the communities of medicine, orthopedics and sports medicine that the primary outcome is the patient's self-report of pain and function. This is important to acknowledge also in the field of cartilage repair when interpreting the sometimes diverging results of evaluation of structure and clinical outcome., Articular cartilage is specialized connective tissue forming smooth surfaces between articulating bones in joints. Cartilage function is extremely demanding since human hip or knee joints should sustain loads up to several times body weight during normal daily activities, such as walking or stair climbing (Mow et al. 1992). The mechanical properties of cartilage are determined by the content, arrangement and interactions of the tissue constituents, i.e. three-dimensional collagen network, proteoglycans (PGs) and interstitial water (Mow et al. 1991). The balance between the mechanical demands and material properties of the tissue is essential for normal joint function. This homeostasis is disturbed in diseases such as osteoarthritis (OA, arthritis), where the balance between anabolic and catabolic activities of cartilage cells – chondrocytes – is distorted, and the collagen network and PGs become degraded with the consequence of impaired functional properties of the tissue. OA causes a high economical burden to the society nationally and internationally. Potentially, minor changes in the content and arrangement of tissue components may transmit to significant changes in cartilage biomechanics. Quantitative microscopic imaging techniques, e.g. polarized light microscopy (PLM) or magnetic resonance imaging (MRI) enable characterization of tissue structure. MRI is a clinical tool for the diagnosis of OA (Nissi et al. 2004). The limitation of MRI is the inability to directly quantitate functional (mechanical) changes of cartilage occurring, e.g., in early OA. Instead, theoretical modelling is needed to describe tissue function (DiSilvestro et al. 2001, Korhonen et al. 2003b). A realistic in vivo estimation of cartilage functional characteristics, stress/strain states and possible failure points in a joint would necessitate that the MRI-derived, depth-dependent tissue structure is incorporated into a theoretical model. Combination of MRI with realistic mechanical model could reveal sensitively early cartilage pathologies. Further, successfully conducted remodeling algorithm, showing for e.g. the development of collagen fibril network during OA progression, could provide a valuable tool for the evaluation of the effect of loading on cartilage development, progression of OA, and ideally prevention of OA. In previous studies we have developed anisotropic cartilage models and applied them for the 1)prediction of the mechanical behaviour of osteoarthritic articular cartilage and differentiation of PG loss from collagen degradation (Korhonen et al. 2003a),2)investigation of the role of superficial layer on the indentation response of cartilage (Julkunen et al. 2008a),3)investigation of the effect of anisotropy (mainly due to collagen) of the extracellular matrix on the mechanical signals experienced by chondrocytes (Korhonen et al. 2006),4)characterization of articular cartilage by combining microscopic analysis and finite element modelling (Julkunen et al. 2007)5)estimation of the mechanical properties of articular cartilage from MRI images (Julkunen et al. 2008b) Specifically, we found that the collagen and PG-specific MRI parameters correlated significantly with the corresponding mechanical parameters of articular cartilage, i.e. the fibril network modulus (collagen) and the non-fibrillar matrix modulus (PGs) (Julkunen et al. 2007). The study was based on the fibril-reinforced poroelastic model of cartilage, which has been shown to be one of the most realistic approaches to describe cartilage mechanics. We further developed the model and used only information on tissue composition to describe the mechanical behavior of cartilage (Julkunen et al. 2008c). Based on tissue fixed charge density, collagen content and fluid fraction the model was able to simulate stress-relaxation responses of cartilage. We believe that a technique which could predict cartilage development under certain loading conditions, or the progression of OA after a local cartilage damage would be important. A potential way to approach this problem is to 1) image joint and cartilage composition and structure using magnetic resonance imaging (MRI), 2) incorporate this information into a finite element model to predict tissue function from its composition, and, further, to estimate changes in tissue architecture, especially in collagen orientation, in loaded articular cartilage, and ultimately 3) simulate joint loads to investigate stresses and strains in cartilage, possible risk locations, and the development of collagen orientation in normal, degenerated or tissue-engineered cartilage. Prediction of cartilage development and progression of OA under spesific joint loads is a challenging task. Combining MRI analyses of tissue solid and fluid contents with the composition based theoretical model of cartilage enables estimation of cartilage mechanics from its composition. This would make a functional imaging technique for sensitive diagnosis of OA. Further, with succesfully conducted remodeling algorithm, it could be even possible to estimate cartilage development and progression of OA after a local damage several years ahead. This approach would help in decision making of clinical treatments and interventions for the prevention of OA. Importantly, the economic burden both for the society and people would be reduced significantly., The menisci of the knee joint are fibrocartilaginous structures that play critically important functions in load distribution, joint stability, shock absorption, and lubrication. The meniscus is classified as a fibrocartilage because of the rounded or oval shape of most of the cells in the inner tissue, and the partly fibrous appearance of the extra-cellular matrix (1). The meniscus has some ability to mount tissue repair processes. In striking contrast to hyaline articular cartilage, cells can and do migrate through the matrix of the meniscus in response to wounding (2,3). This suggests that the wounded meniscus can signal to the specialized cells in the environment to migrate towards the wound, and that the matrix of the tissue contains corridors that permit and may even promote cell migration. The meniscus has a vascular supply. How far the blood vessels penetrate into the meniscus, however, depends on the stage of development of the tissue. Essentially all of the tissue has a vascular supply in the newly born. At maturity, however, only about 15% of the outer radial distance has a vascular supply. Immunostaining and confocal microscopy demonstrates that type I collagen is found throughout the entire tissue of the immature and mature meniscus. Type II collagen, however, is restricted to the non-vascular, inner zone of the canine meniscus (4). No type II collagen was demonstrable in the juvenile meniscus, which had blood vessels throughout its structure. The dominant structural motifs in the meniscus are the circumferential bundles of collagen fibrils with occasional radial structures referred to as “tie fibers” or “tie sheaths”. Electron microscopic studies of normal canine meniscus demonstrates two separate matrix systems in the main body of the meniscus: (a) the “bundles” of circumferentially arranged bundles of collagen fibrils; and (b) a new system that we are calling the “peri-matrix” that is a part of and radiates out of the “tie fibers and that separates and enwraps the circumferential collagen fibrils. Cells reside in and appear to migrate through the peri-matrix. In the outer meniscus, the cells have a fibroblast appearance and show extending processes that enwrap the bundles of collagen fibrils. The wounded canine meniscus does have the capacity to emit signals that activate exogenous human mesenchymal stem cells. Taken together, these data show that the meniscus has a distinctive fibril architecture and perhaps signaling system that should facilitate repair of wounds in the tissue., Introduction The menisci are regarded as important structures for knee stability, shock absorption, and load distribution. Their location in the knee and the extreme forces that they can be subjected to, make them frequently susceptible to injury, especially in contact-sport activities. Due in large part to the limited vascularity of the meniscus, this tissue has little innate ability to heal spontaneously. Partial meniscectomy is still indicated if the lesion cannot be satisfactorily stabilized with sutures or anchors. Particularly troublesome are lesions to the inner portions of the meniscus where the tissue is not vascularized. Engineering tissue to repair an injured meniscus is an emerging strategy for restoring form and function of meniscal fibrocartilage. Specific considerations must be given to the type of cells necessary, the scaffold, and the physical forces within the microenvironment in which the meniscus is located. Although many efforts have been made for engineering articular cartilage, relatively few studies have been reported for engineering meniscal tissue or attempt to repair this construct by a cell-based strategy. Important considerations for engineering meniscal tissue include the material properties of the meniscal substance, the geometry of the construct, and the functional integrity of meniscal attachments. Cell Sources for Tissue Engineering The primary obstacle for engineering cartilage of any type is acquiring the appropriate numbers of chondrogenic cells for generating the tissue. The seeding density of cells capable of chondrogenesis in or onto a polymer carrier is a critical ingredient for successfully engineering cartilaginous tissues, but few would risk morbidity to normal meniscus to obtain cells for engineering new meniscal tissue. It is evident that the type and the number of cells and the physical environment, into which they are placed, are critical elements for successfully engineering meniscal cartilage tissue. Chondrocytes from other areas of the body could be used to generate meniscal tissue or used to induce meniscal repair. We have shown that articular chondrocytes embedded in fibrin glue are capable of inducing repair in meniscal lesions in an explant model in nude mice (1,2); also that articular chondrocytes are able of forming bonds between cartilage explants and meniscal lesions (3–6). Extending these findings to a large animal model, we have shown that articular chondrocytes seeded onto a devitalized cartilage matrix are capable of inducing a healing process in a bucket-handle lesion of the meniscus in swine (7). Other alternative cartilage sources for obtaining cells include the cartilage portion of ribs, which are expandable, or possibly small biopsies of ear cartilage. Chondrocytes from these sources have been shown to generate new cartilage matrix and are capable of forming bonds between cartilage discs (8). These alternative cell sources could be used to engineer reparative meniscal tissue. The use of other noncartilage cells with chondrogenic potential may permit the generation of cartilage given the correct polymer and appropriate conditions as well. Multiple experiments have shown that, under controlled in vitro conditions, mesenchymal stem cells (MSCs) can differentiate into bone, fat, tendon, muscle and cartilage-like tissues. MSCs can be used to regenerate connective tissues through tissue engineering techniques; additionally, they have a natural capacity to home to injured tissues and to participate to tissue healing. This second feature seems particularly interesting as long as MSCs not only provide a cell source for regeneration but they also secrete paracrine factors that enhance the potential for tissue repair, acting as “trophic mediators” (9). One of the first studies evaluating the potential of MSCs for meniscal repair was performed by Port et al. using MSCs cultured in fibrin clot. However, the use of MSCc in that study did not enhance meniscal healing (10). This was probably due to the absence of a scaffold providing a suitable environment to MSCs; and to the lack of a pre-culture time, which is known to improve the capacity of the engineered tissue to withstand the weight bearing forces (11). More recently, MSCs were delivered as a suspension by intra-articular injection after total meniscectomy and ACL resection in a study by Murphy et al., to develop an osteoarthritic knee (12). In this study, authors report a positive effect of MSCs to the injured joint with significant formation of neomeniscal tissue. This study suggests that there may be a therapeutic benefit associated with intra-articular injection of stem cells following traumatic injury to the knee, with a possible longer term effect determining a reduction or delay in the progression to osteoarthritis (12). MSCs seeded onto a hyaluronan-collagen-based scaffold were recently used to repair a critical-size defect in a rabbit model, with a tissue engineering approach (11). In their study, the authors removed the pars intermedia of the medial meniscus and replaced the resected section with an acellular biocompatible scaffold, or with the scaffold loaded with cultured MSCs. Menisci repaired with the acellular scaffold showed a fibrous, scar-like repair tissue, while those repaired with the engineered tissue demonstrated a significantly better filling and meniscal regeneration. Another interesting option to be exploited is the use of chondrocytes from allogeneic or xenogeneic sources for engineering cartilage. Whereas the use of allogeneic and xenogeneic cartilage “en bloc” did not always encounter secure clinical success, the use of isolated chondrocytes from these sources may perform satisfactorily. We have recently tested the potential of allogeneic ear and articular chondrocytes for repairing swine knee meniscus lesions. Ear autologous and allogeneic chondrocytes were seeded onto vicryl mesh and tested for meniscus repair in orthotopic lesion in the swine model. Articular autologous and allogeneic chondrocytes were also evaluated. It was demonstrated that all four cell populations possess repair potential for these lesions (13). Scaffolds for meniscus tissue engineering The other critical element for engineering meniscal cartilage is finding or developing suitable scaffold materials that permit or accelerate the formation of new extracellular matrix. A biomaterial used as scaffold for tissue engineering purposes should present many features: it should be biocompatible and possibly biodegradable allowing to be replaced by biologic tissue; it should have enough resiliency and resistance to withstand weigh-bearing while cells produce matrix; it should maintain structure, volume and shape in an in vivo orthotopic environment; it should promote cell differentiation and proliferation if seeded with cells, or promote cell migration if cell-free; it should allow diffusion of nutrients and catabolic remnants; it should be easily manageable and implantable by the surgeon (14). According to these features, many biomaterials have been evaluated for meniscal repair and tissue engineering both natural or synthetic (15). Natural materials used include: periosteal tissue, perichondral tissue, small intestine submucosa, and meniscal tissue. While these tissues have high biocompatibility, they cannot be adapted for tissue engineering techniques as they do not allow varying structure geometry and initial mechanical properties (15). Isolated tissue components such as collagens and proteoglycans (16) maintain the high biocompatibility of the natural tissues while allowing the creation of custom-made scaffold with definite pore dimension and geometry and, consequently, biomechanical features. However, these scaffolds have usually low biomechanical properties (15). On the other hand, biomimetic synthetic materials can be manufactured in custom-made shapes of any geometrical structure and porosity depending on the characteristic of the host tissue and of the cells seeded. In particular, it has been shown that optimal ingrowth and incorporation of a meniscal scaffold, macropore sizes must be in the range of 150–500 μm (17). To date, the most used synthetic materials include the following: polyglicolic acid (PGA), poly(L) lactic acid (PLLA), polyurethane, polyester, polytetrafuoroethylene, polycaprolactone (PCL), and various combinations of these polymers and other materials even the natural ones. The latter approach, namely coupling synthetic (PCL) and natural (hyaluronic acid) materials, has been recently evaluated in two large animal studies on partial and total meniscus tissue engineering with promising results (18,19). Cell-free techniques The rationale of using a cell-free biomaterial to replace part of the meniscus is based on scaffold repopulation by host cells from the synovium and the meniscal remnants, and subsequent tissue ingrowth. In fact, this approach could be considered to become cell-based after implantation. The collagen meniscus implant (CMI) is the first regenerative technique applied to meniscal repair in clinical practice (20–22). As long as an outer rim of menisci tissue is needed for CMI implantation, it is indicated only as partial meniscus regeneration and not as total meniscus regeneration. Satisfactory clinical results have been reported (23) while results from the imaging and histological standpoints are controversial as the CMI shrinks over time and showed no histological remnants 5 to 6 years after implantation (22) leaving predominantly scar tissue instead of fibrocartilage (24). However, CMI results in satisfactory improvement in pain, can be implantable arthroscopically, and it is the only method used thus far in the clinical setting. Small intestinal submucosa (SIS) has been applied to menisci regeneration in a dog model in a 12 months follow-up study (25). In that study, menisci receiving SIS in a posterior vascular lesion had more meniscus-like tissue filling the defects and significantly less cartilage damage than menisci receiving no implants. Conclusions The geometric complexity of the meniscus, the composition of the tissue, and the forces on the meniscus present many obstacles for engineering a substitute. Finding suitable cell sources, providing a blood supply from the periphery, and developing new polymers that can withstand the rigor of the mechanical forces in the knee are key to successfully engineering meniscal tissue. This requires the interaction and collaboration of several disciplines such as biology, chemistry, molecular biology, bio-engineering, material-and mechanical-engineering, genetics and surgery; that all need to focus on a common target for understanding the mechanisms occurring in the natural environment and for restoring or substituting the damaged tissue. At the present time, successful strategies are being developed using tissue-engineering approaches to heal meniscal tears. Clinical application of these approaches appears to be in the very near future.

Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12-13.1 (Refs 3,4). We have identified a new transcript, encoding a protein (bo, +AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes b o,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.

The management of critical limb ischemia due to below-the-knee disease remains challenging due to the frequent patient comorbidities, diffuse vascular involvement, and high rates of restenosis and disease progression. The BASIL study has established the substantial equivalence between bypass surgery and percutaneous transluminal angioplasty in this setting, at least at mid-term follow-up, but percutaneous techniques and devices have seen major developments since the publication of this pivotal trial in 2005. A major breakthrough has indeed been the introduction of drug-eluting balloons, which have several theoretical advantages in comparison to standard balloons and metallic stents for infra-popliteal lesions. Two clinical trials have already been reported with favorable results for the In.Pact Amphirion paclitaxel-eluting balloon, when employed for below-the-knee lesions. We hereby discuss the rationale for the use of drug-eluting balloons in this complex setting and the main findings of the study by Schmidt et al. and the DEBATE-BTK trial.

Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase essential for signaling via cytokine receptors that comprise the common gamma-chain (gammac), i.e., the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Jak3 is preferentially expressed in hemopoietic cells and is up-regulated upon cell differentiation and activation. Despite the importance of Jak3 in lymphoid development and immune function, the mechanisms that govern its expression have not been defined. To gain insight into this issue, we set out to characterize the Jak3 promoter. The 5'-untranslated region of the Jak3 gene is interrupted by a 3515-bp intron. Upstream of this intron and the transcription initiation site, we identified an approximately 1-kb segment that exhibited lymphoid-specific promoter activity and was responsive to TCR signals. Truncation of this fragment revealed that core promoter activity resided in a 267-bp fragment that contains putative Sp-1, AP-1, Ets, Stat, and other binding sites. Mutation of the AP-1 sites significantly diminished, whereas mutation of the Ets sites abolished, the inducibility of the promoter construct. Chromatin immunoprecipitation assays showed that histone acetylation correlates with mRNA expression and that Ets-1/2 binds this region. Thus, transcription factors that bind these sites, especially Ets family members, are likely to be important regulators of Jak3 expression.