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Bone marrow is one of the best characterized stem cell microenvironments that contains Mesenchymal Stem Cells (MSCs), a rare population of non-hematopoietic stromal cells. MSCs have been indicated as a new option for regenerative medicine because of their ability to differentiate into various lineages such as bone, cartilage and adipose tissue. However, isolation procedures are crucial for the functional activity of the transplanted cells. The use of concentrated bone marrow cells (BMCs) enables a cell population surrounded by its microenvironment (niche) to be implanted while avoiding all the complications related to the in vitro culture. The cells of the niche are able to regulate stem cell behavior through direct physical contact and secreting paracrine factors. The aim of this study was to characterize BMCs in vitro to evaluate their ability to differentiate toward mature cells and try to understand whether there are differences in the chondrogenic and osteogenic potential of cells from patients of different ages. Mononuclear Cells (MNCs) isolated by Ficoll were used as control. Both cell populations were grown in monolayers and differentiated with specific factors and analyzed by histological and molecular biology assays to evaluate the expression of some specific extracellular matrix molecules. The present investigations revealed the ability of BMCs to act as isolated cells. They are able to form colonies and differentiate toward chondrogenic and osteogenic lineages, the latter pathway appearing to be influenced by donor age. The results obtained by this study support the use of BMCs in clinical practice for the repair of osteochondral damage, which might be particularly useful for the one-step procedure allowing cells to be directly implanted in operating room.

Osteochondral lesions are considered a challenge for orthopedic surgeons. Currently, the treatments available are often unsatisfactory and unable to stimulate tissue regeneration. Tissue engineering offers a new therapeutic strategy, taking into account the role exerted by cells, biomaterial and growth factors in restoring tissue damage. In this light, Mesenchymal Stem Cells (MSCs) have been indicated as a fascinating tool for regenerative medicine thanks to their ability to differentiate into bone, cartilage and adipose tissue. However, in vitro-cultivation of MSCs could be associated with some risks such as de-differentiation/reprogramming, infection and contaminations of the cells. To overcome these shortcomings, a new approach is represented by the use of Bone Marrow Concentrate (BMC), that could allow the delivery of cells surrounded by their microenvironment in injured tissue. For this purpose, cells require a tridimensional scaffold that can support their adhesion, proliferation and differentiation. This study is focused on the potentiality of BMC seeded onto a hyaluronan-based scaffold (Hyaff-11) to differentiate into osteogenic lineage. This process depends on the specific interaction between cells derived from bone marrow (surrounded by their niche) and scaffold, that create an environment able to support the regeneration of damaged tissue. The data obtained from the present study demonstrate that BMC grown onto Hyaff-11 are able to differentiate toward osteogenic sense, producing specific osteogenic genes and matrix proteins.

Articular cartilage lesions represent a challenging problem for orthopaedic surgeons. The purpose of this study was to evaluate the effect of a new pulsed Nd:YAG High Intensity Laser Therapy on the regeneration of cartilage tissue in patients with traumatic lesions. Clinical, histological and immunohistochemical evaluations were performed. Ten patients affected by chondral lesions scheduled for ACI procedure, were enrolled into the study. During the chondrocyte expansion for ACI procedure, cartilage from five patients was treated by Nd:YAG High Intensity Laser Therapy (HILT group). No laser treatment was performed in the remaining patients, who were used as controls. Cartilage repair was assessed by clinicians using two different scores: Cartilage Repair Assessment (CRA) and Overall Repair Assessment (ORA). Cartilage biopsy specimens were harvested to perform histological and immunohistochemical analyses at T0 (before laser treatment) and T1 (at the end of the treatment). A significant decrease in cartilage depth was noticed in the HILT group at T1. Histological and immunohistochemical evaluations showed some regenerative processes in cartilaginous tissue in terms of high amount of proteoglycans, integration with adjacent articular cartilage and good cellular arrangement in the HILT group. By contrast, a not well organized cartilaginous tissue with various fibrous features in the control group at T0 and T1 was observed. In conclusion, the use of this new pulsed Nd:YAG HILT resulted promising in the treatment of moderate cartilage lesions markedly in the young patients.

Background: There are only a few studies concerning the cellular, biochemical, and genetic processes that occur during the remodeling of graft tissue after autologous chondrocyte transplantation. The purpose of the present study was to quantify the expression of genes encoding extracellular matrix proteins and regulatory factors that are essential for cell differentiation in cartilage biopsy specimens from patients who had this treatment two years previously. Methods: Two cartilage biopsy specimens from each of four patients who had been treated with autologous chondrocyte transplantation and from two multiorgan donors were used. Real-time reverse transcriptase-polymerase chain reaction analysis was performed to evaluate the expression of types I, II, and X collagen; aggrecan; cathepsin B; and early growth response protein-1 (Egr-1) and Sry-type high-mobility-group box transcription factor-9 (Sox-9) mRNAs. Immunohistochemical analysis for matrix proteins and regulatory proteins was carried out on paraffin-embedded sections. Results: Type-I collagen mRNA was expressed in all of the samples evaluated. Type-II collagen was present in autologous chondrocyte transplantation samples but at lower levels than in the controls. Type-X collagen messenger was undetectable. Aggrecan mRNA was present in all of the samples at lower levels than in the controls, while cathepsin-B messenger levels were higher and Egr-1 and Sox-9 mRNAs were expressed at lower levels. The immunohistochemical analysis showed slight positivity for type-I collagen in all of the sections. Type-II collagen was found in all of the samples with positivity confined inside the cells, while the controls displayed a positivity that was diffuse in the extracellular matrix. Cathepsin B was slightly positive in all of the samples, while the controls were negative. Egr-1 protein was particularly evident in the areas negative for type-II collagen. Sox-9 was positive in all samples, with evident localization in the superficial and middle layers. Conclusions: In biopsy specimens from autologous chondrocyte transplantation tissue at two years, there is evidence of the formation of new tissue, which displays varying degrees of organization with some fibrous and fibrocartilaginous features. Long-term follow-up investigations are needed to verify whether, once all of the remodeling processes are completed, the newly formed tissue will acquire the more typical features of articular cartilage. Clinical Relevance: Our data provide important further information about the nature of the new cartilage tissue formed two years after autologous chondrocyte transplantation, allowing a better interpretation of the clinical and histological findings.

Background: Fresh osteochondral allografts represent a treatment option for early ankle posttraumatic arthritis. Transplanted cartilage survivorship, integration, and colonization by recipient cells have not been fully investigated. The aim of this study was to evaluate the ability of recipient cells to colonize the allograft cartilage and to assess allograft cell phenotype. Methods: Seventeen ankle allograft samples were studied. Retrieved allograft cartilage DNA from fifteen cases was compared with recipient and donor constitutional DNA by genotyping. In addition, gene expression was evaluated on six allograft cartilage samples by means of real-time reverse transcription-polymerase chain reaction. Histology and immunohistochemistry were performed to support molecular observations. Results: Of fifteen genotyped allografts, ten completely matched to the host, three matched to the donor, and two showed a mixed profile. Gene expression analysis showed that grafted cartilage expressed cartilage-specific markers. Conclusions: The rare persistence of donor cells and the prevailing presence of host DNA in retrieved ankle allografts suggest the ingrowth of recipient cells into the allograft cartilage, presumably migrating from the subchondral bone, in accordance with morphological findings. The expression of chondrogenic markers in some of the samples argues for the acquisition of a chondrocyte-like phenotype by these cells. Clinical Relevance: To our knowledge, this is the first report describing the colonization of ankle allograft cartilage by host cells showing the acquisition of a chondrocyte-like phenotype.

INTRODUCTION: Osteochondral articular defects are a key concern in orthopaedic surgery. Current surgical techniques to repair osteochondral defects lead to poor subchondral bone regeneration and fibrocartilage formation, which is often associated with joint pain and stiffness. The objective of this pilot clinical study is to evaluate the performance and the intrinsic stability of a newly developed biomimetic osteochondral scaffold and to test the safety and the feasibility of the surgical procedure. METHODS: A gradient composite osteochondral scaffold based on type I collagen-hydroxyapatite was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles. Thirteen patients (15 defect sites) were treated with scaffold implantation from January 2007 to July 2007: four at the medial femoral condyle, two at the lateral femoral condyles, five at the patellas and four at the trochleas. The mean size of the defects was 2.8 cm(2) (range: 1.5-5.9 cm(2)). All patients were followed up prospectively. High-resolution magnetic resonance imaging (MRI) was used to determine "the early postoperative adherence rate" at 4-5 weeks and 25-26 weeks after scaffold implantation. Moreover, the magnetic resonance observation of cartilage repair tissue (MOCART) score was performed on every MRI. Two second-looks were performed at 6 months; cartilage repair was assessed using the International Cartilage Repair Society (ICRS) visual scoring system and histological and immunohistochemical analysis of the two biopsies was carried out. RESULTS: A completely attached graft and repair tissue were found in 13 of 15 lesions (86.7%). A partial detachment was observed in two patients (13.3%). No detached grafts were found. Complete filling of the cartilage defect and congruency of the articular surface were seen in 10 lesions (66.7%) with MRI evaluation at 6 months. The complete integration of the grafted cartilage was detected in eight lesions (53.3%). Subchondral bone changes (oedema or sclerosis) were found in eight defects (53.3%). Statistical analysis showed a significant improvement in the International Knee Documentation Committee (IKDC) subjective and objective scores from preoperative to 6 months' follow-up (p

Background: Severe post-traumatic ankle arthritis poses a reconstructive challenge in the young and active patient. Bipolar fresh osteochondral allograft (BFOA) may represent an intriguing alternative to arthrodesis and prosthetic replacement. The aim of this study was to describe a lateral trans-malleolar technique for BFOA, and to evaluate the results in a case series. Materials and Methods: From 2004 to 2006, 32 patients, mean age of 36.8 ± 8.4 years, affected by ankle arthritis underwent BFOA with a mean followup of 31.2 months. The graft was prepared by specifically designed jigs, including the talus and the tibia with the medial malleolus. The host surfaces were prepared by the same jigs through a lateral approach. The graft was placed and fixed with twist-off screws. Patients were evaluated clinically and radiographically at 2, 4, and 6 month after operation, and at a minimum 24 months followup. A biopsy of the grafted areas was obtained from 7 patients at 1-year followup for histological and immunohistochemical examination. Results: Preoperative AOFAS score was 33.1 ± 10.9 and postoperatively 69.5 ± 19.4 ( p < 0.0005). Six failures occurred. Cartilage harvests showed hyaline-like histology with a normal collagen component but low proteoglycan presence and a disorganized structure. Samples were positive for MMP-1, MMP-13 and Capsase-3. Conclusion: The use of BFOA represents an intriguing alternative to arthrodesis or arthroplasty. We believe precise allograft sizing, stable fitting and fixation and delayed weightbearing were key factors for a successful outcome. Further research regarding the immunological behavior of transplanted cartilage is needed. Level of Evidence: IV, Retrospective Case Series

The opportunity to apply autologous chondrocyte transplantation in repairing cartilage lesions in osteoarthritis (OA) is of great interest. To this end, chondrocytes from cartilage of these patients and from healthy donors were used to evaluate the expression of some extracellular matrix molecules once these cells were grown onto a hyaluronan-based scaffold already used in clinical practice. Constructs were analyzed by immunohistochemical and real-time PCR analyses. Chondrocytes from control and patients with OA cartilages expressed the same extracellular matrix molecules even if at different amount. These differences, which were appreciable both at protein and molecular levels, were not evident once the cells were grown onto Hyaff-11 scaffold. In this experimental culture condition, the cells derived from control and patients with OA showed a significant increase of collagen type II, Sox-9, and aggrecan and a decrease of collagen type I compared with chondrocytes grown in monolayer. On the other hand, MMPs were downregulated in both the cell types evaluated by the specific action of TIMP-1 which was highly expressed at molecular and protein levels in the two groups. The growth of chondrocytes onto Hyaff-11 membrane seems to erase the differences between the cells derived from normal and OA cartilages. The cells seem to benefit of the "hyaluronan" presence which is able to create an ideal environment for the expression of cartilage genes even in absence of specific growth factors. This is of particular relevance hypothesizing the use of tissue engineering therapeutical approach also in patients with OA.

Summary Objective To investigate the gene expression profile and the histological aspects of articular cartilage of patients affected by Morquio syndrome, a lysosomal storage disease characterized by the accumulation of glycosaminoglycans within the cells which result in abnormal formation and growth of the skeletal system. Method Articular cartilage samples were obtained from the femoral condyle of two siblings with Morquio syndrome during surgery performed to treat valgus knee. As controls, four biopsy samples of healthy cartilage were obtained from four different male multiorgan donors. A Real-Time Polymerase Chain reaction (RT-PCR) analysis was performed to evaluate the expression of type I and II collagens and aggrecan mRNAs. Histological and immunohistochemical analyses for some matrix proteins were carried out on paraffin embedded sections. Results Type I collagen mRNA mean level was higher in the samples of patients with Morquio syndrome compared to controls. Type II collagen and aggrecan mRNAs' mean expression was instead lower. The morphological appearance of the cartilage showed a poorly organized tissue structure with not homogeneously distributed cells that were larger compared to normal chondrocytes due to the presence inside the vacuoles of proteoglycans which were not metabolized. Chondrocytes were negative for collagen II immunostaining while the extracellular matrix was weakly positive. Collagen type I immunostaining was positive at cellular level. Keratan sulfate showed diffuse positivity and chondroitin-6-sulfate was present throughout the cartilaginous thickness. Conclusion In cartilage of patients with Morquio syndrome, a low expression of collagen type II and a high expression of collagen type I both at protein and molecular levels are evidentiated. This finding could give evidence of the reduction in ankle and knee joint movement observable in these patients.

The anterior cruciate ligament (ACL) is the most commonly injured tissue of the human knee. Its poor ability to regenerate after injury represents a challenge to ligament tissue engineering. An understanding of the molecular composition of the structures used for its repair is essential for clinical assessments and for the implementation of tissue engineering strategies. The objective of this study was to evaluate, both at gene and protein levels, the expression of characteristic molecules in human ACL, patellar, semitendinosus and gracilis tendons and in the ligament reconstructed with patellar or semitendinosus and gracilis tendons. We demonstrated that primary ACL and tendon tissues all express collagen I, II, Sox-9, tenascin-C and aggrecan. Collagen X expression was detected at very low levels or undetectable. Cathepsin B, MMP-1 and MMP-13 were expressed at higher levels in the ACL reconstructed by the two tendons, showing that a remodeling process occurs during “ligamentization”. Both our molecular and immunohistochemical evaluations did not reveal significative differences between the tendons and ligaments analyzed. However, ACL reconstructed with semitendinosus and gracilis tendon seems to present a higher expression of collagen type II when compared to that reconstructed with patellar tendon. This study could give a reasonable identification of genetic and protein markers specific to tendon/ligament tissues and be helpful in testing tissue engineering approaches for ACL reconstruction. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage repair. An ideal scaffold should possess a preformed three-dimensional shape, fix the cells to the damaged area and prevent their migration into the articular cavity. Furthermore, the constructs should have sufficient mechanical strength to facilitate handling in a clinical setting and stimulate the uniform spreading of cells and a phenotype re-differentiation process. The aim of this study was to verify the ability of an equine collagen membrane to support the growth of human chondrocytes and to allow the re-expression of their original phenotype. This ability was assessed by the evaluation of collagen type I, II and aggrecan mRNA expression by Real-Time PCR. Immunohistochemical analyses were performed to evaluate collagen type I, II and proteoglycans synthesis. Electron microscopy was utilized to highlight the structure of the biomaterial and its interactions with the cells. Our data indicate that human chondrocytes seeded onto a collagen membrane express and produce collagen type II and aggrecan and downregulate the production of collagen type I during the experimental times analyzed. These results provide an in vitro demonstration for the therapeutic potential of autologous chondrocyte transplantation by an equine collagen membrane as a delivery vehicle in a tissue-engineered approach towards the repair of articular cartilage defects.