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Osteoarthritis (OA) is a degenerative disease resulting in irreversible, progressive destruction of articular cartilage(1). The etiology of OA is complex and involves a variety of factors, including genetic predisposition, acute injury and chronic inflammation(2–4). Here we investigate the ability of resident skeletal stem-cell (SSC) populations to regenerate cartilage in relation to age, a possible contributor to the development of osteoarthritis(5–7). We demonstrate that aging is associated with progressive loss of SSCs and diminished chondrogenesis in the joints of both mice and humans. However, a local expansion of SSCs could still be triggered in the chondral surface of adult limb joints in mice by stimulating a regenerative response using microfracture (MF) surgery. Although MF-activated SSCs tended to form fibrous tissues, localized co-delivery of BMP2 and soluble VEGFR1 (sVEGFR1), a VEGF receptor antagonist, in a hydrogel skewed differentiation of MF-activated SSCs toward articular cartilage. These data indicate that following MF, a resident stem-cell population can be induced to generate cartilage for treatment of localized chondral disease in OA.

Implantation of allograft tissues has massively grown over the last years, especially in the fields related to sports medicine. Beside the fact that often no autograft option exists, autograft related disadvantages as donor-site morbidity and prolonged operative time are drastically reduced with allograft tissues. Despite the well documented clinical success for bone allograft procedures, advances in tissue engineering raised the interest in meniscus, osteochondral and ligament/tendon allografts. Notably, their overall success rates are constantly higher than 80%, making them a valuable treatment option in orthopaedics, especially in knee surgery. Complications reported for allografting procedures are a small risk of disease transmission, immunologic rejection, and decreased biologic incorporation together with nonunion at the graft-host juncture and, rarely, massive allograft resorption. Although allografting is a successful procedure, improved techniques and biological knowledge to limit these pitfalls and maximize graft incorporation are needed. A basic understanding of the biologic processes that affect the donor-host interactions and eventual incorporation and remodelling of various allograft tissues is a fundamental prerequisite for their successful clinical use. Further, the importance of the interaction of immunologic factors with the biologic processes involved in allograft incorporation has yet to be fully dissected. Finally, new tissue engineering techniques and use of adjunctive growth factors, cell based and focused gene therapies may improve the quality and uniformity of clinical outcomes. The aim of this review is to shed light on the biology of meniscus, osteochondral and ligament/tendon allograft incorporation and how collection and storage techniques may affect graft stability and embodiment.Level of evidence V.

The disk of hyaline cartilage that is interposed between the epiphysis and the metaphysis of each of the long bones is responsible for its elongation, and, thus, when the lower limbs are concerned, for increases in bodily height. This so-called growth plate is avascular, aneural, and alymphatic. It consists solely of chondrocytes and an extracellular matrix which the cells elaborate. The growth plate is architectonically striking in so far as the chondrocytes are aligned in strictly vertical columns, which represent the functional units of longitudinal bone growth. The growth process begins with the slow division of chondrocytes in the resting ("stem cell") zone and proceeds with their rapid proliferation in the adjacent zone. These cells then undergo a process of progressive enlargement, which culminates in the zone of terminal hypertrophy. The life history of any given cell is recapitulated in a vertical column. The neoformation of cartilage in the axial direction is synchronized with its destruction at the vascular invasion front of the metaphysis and results in an elongation of the bony trabeculae. The mechanism that governs the highly coordinated sequence of events that underlies the growth of the long bones is complex; it is subject to influence by genetic, hormonal, nutritional, environmental, and pathological factors.

Background: Two strains of guinea pig develop spontaneous osteoarthritis of the knee. Although the disease evolves at different rates in the two strains, it is not known whether these differences are reflected in the structure of the cartilage and cancellous bone. Questions/purposes: We determined whether the three-dimensional structure of the tibial-plateau cartilage and femoral cancellous bone differed between the two strains. Methods: Six Dunkin-Hartley and six GOHI/SPF guinea pigs were evaluated. The animals were sacrificed at 11months of age. The 24 proximal tibias were used for a stereologic histomorphometric analysis of the tibial-plateau cartilage. The 24 femurs were used for a site-specific, three-dimensional quantitative analysis of the cancellous bone by micro-CT. Results: Compared to the GOHI/SPF guinea pigs, the tibial-plateau cartilage of the Dunkin-Hartley strain had a larger lesion volume (3.8% versus 1.5%) and a thicker uncalcified cartilage layer (0.042 versus 0.035mm), but a thinner calcified cartilage zone (0.008 versus 0.01mm) and a thinner subchondral cortical bone plate (0.035 versus 0.039mm). The femoral cancellous bone in the Dunkin-Hartley strain had a lower bone mineral density (477 versus 509mg/cm3). However, the trabeculae were thicker (3.91 versus 3.53 pixels) and farther apart (7.8 versus 5.6 pixels). The osteoarthritic changes in the cartilage were topographically mirrored in the subchondral bone. They were most severe on the medial side of the joint, particularly in the anterior region. Conclusions: Spontaneous osteoarthritis in the guinea pig is associated with site-specific changes in the articular cartilage layer, which are topographically mirrored in the underlying subchondral bone. Clinical Relevance: Three-dimensional structural information not revealed by two-dimensional radiography may help characterize the stages of osteoarthritis

The onset and timing of the growth of children and adolescents occurs with considerable variability in cohorts of the same chronological age. The musculoskeletal system changes in proportion over time, and lever-arm changes, altered individual flexibility, and strength lead to age-specific injury patterns in youth sports. In sports, juniors are commonly grouped according to their chronological age. Early- and late-maturing children and adolescents might therefore not routinely be trained in relation to their biology. This not only represents a risk for overuse and injury but might limit their development in sports. To obtain information about the biological age of children is challenging. Numerous methods have been studied and validated. However, the implementation of these methods on a large scale is still to come. This report provides a brief overview of growth dynamics in relation to youth sports injuries and describes a few challenges for the future.

Osteoarthritis (OA) is a degenerative joint condition characterized by painful cartilage lesions that impair joint mobility. Current treatments such as lavage, microfracture, and osteochondral implantation fail to integrate newly formed tissue with host tissues and establish a stable transition to subchondral bone. Similarly, tissue-engineered grafts that facilitate cartilage and bone regeneration are challenged by how to integrate the graft seamlessly with surrounding host cartilage and/or bone. This review centers on current approaches to promote cartilage graft integration. It begins with an overview of articular cartilage structure and function, as well as degenerative changes to this relationship attributed to aging, disease, and trauma. A discussion of the current progress in integrative cartilage repair follows, focusing on graft or scaffold design strategies targeting cartilage-cartilage and/or cartilage-bone integration. It is emphasized that integrative repair is required to ensure long-term success of the cartilage graft and preserve the integrity of the newly engineered articular cartilage. Studies involving the use of enzymes, choice of cell source, biomaterial selection, growth factor incorporation, and stratified versus gradient scaffolds are therefore highlighted. Moreover, models that accurately evaluate the ability of cartilage grafts to enhance tissue integrity and prevent ectopic calcification are also discussed. A summary and future directions section concludes the review.

Mesenchymal stromal cells (MSCs), which reside within various tissues, are utilized in the engineering of cartilage tissue. Dexamethasone (DEX)--a synthetic glucocorticoid--is almost invariably applied to potentiate the growth-factor-induced chondrogenesis of MSCs in vitro, albeit that this effect has been experimentally demonstrated only for transforming-growth-factor-beta (TGF-β)-stimulated bone-marrow-derived MSCs. Clinically, systemic glucocorticoid therapy is associated with untoward side effects (e.g., bone loss and increased susceptibility to infection). Hence, the use of these agents should be avoided or limited. We hypothesize that the influence of DEX on the chondrogenesis of MSCs depends upon their tissue origin and microenvironment [absence or presence of an extracellular matrix (ECM)], as well as upon the nature of the growth factor. We investigated its effects upon the TGF-β1- and bone-morphogenetic-protein 2 (BMP-2)-induced chondrogenesis of MSCs as a function of tissue source (bone marrow vs. synovium) and microenvironment [cell aggregates (no ECM) vs. explants (presence of a natural ECM)]. In aggregates of bone-marrow-derived MSCs, DEX enhanced TGF-β1-induced chondrogenesis by an up-regulation of cartilaginous genes, but had little influence on the BMP-2-induced response. In aggregates of synovial MSCs, DEX exerted no remarkable effect on either TGF-β1- or BMP-2-induced chondrogenesis. In synovial explants, DEX inhibited BMP-2-induced chondrogenesis almost completely, but had little impact on the TGF-β1-induced response. Our data reveal that steroids are not indispensable for the chondrogenesis of MSCs in vitro. Their influence is context dependent (tissue source of the MSCs, their microenvironment and the nature of the growth-factor). This finding has important implications for MSC based approaches to cartilage repair.

Investigational devices for articular cartilage repair or replacement are considered to be significant risk devices by regulatory bodies. Therefore animal models are needed to provide proof of efficacy and safety prior to clinical testing. The financial commitment and regulatory steps needed to bring a new technology to clinical use can be major obstacles, so the implementation of highly predictive animal models is a pressing issue. Until recently, a reductionist approach using acute chondral defects in immature laboratory species, particularly the rabbit, was considered adequate; however, if successful and timely translation from animal models to regulatory approval and clinical use is the goal, a step-wise development using laboratory animals for screening and early development work followed by larger species such as the goat, sheep and horse for late development and pivotal studies is recommended. Such animals must have fully organized and mature cartilage. Both acute and chronic chondral defects can be used but the later are more like the lesions found in patients and may be more predictive. Quantitative and qualitative outcome measures such as macroscopic appearance, histology, biochemistry, functional imaging, and biomechanical testing of cartilage, provide reliable data to support investment decisions and subsequent applications to regulatory bodies for clinical trials. No one model or species can be considered ideal for pivotal studies, but the larger animal species are recommended for pivotal studies. Larger species such as the horse, goat and pig also allow arthroscopic delivery, and press-fit or sutured implant fixation in thick cartilage as well as second look arthroscopies and biopsy procedures.

Summary Aim The primary goal of this body of work is to suggest a standardized system for histopathological assessment of experimental surgical instability models of osteoarthritis (OA) in rabbits, building on past experience, to achieve comparability of studies from different centres. An additional objective is to review methodologies that have been employed in the past for assessing OA in rabbits with particular reference to the surgical anterior cruciate ligament transection (ACLT) model. Methods A panel of scientists and clinician-scientists with recognized expertise in assessing rabbit models of OA reviewed the literature to provide a critical appraisal of the methods that have been employed to assess both macroscopic and microscopic changes occurring in rabbit joint tissues in experimental OA. In addition, a validation of the proposed histologic histochemical grading system was performed. Results The ACLT variant of the surgical instability model in skeletally mature rabbits is the variation most capable of reproducing the entire range of cartilage, synovial and bone lesions recognized to be associated with OA. These lesions can be semiquantitatively graded using macroscopic and microscopic techniques. Further, as well as cartilage lesions, this ACLT model can produce synovial and bone lesions similar to that of human OA. Conclusions The ACLT variant of the surgical instability model in rabbits is a reproducible and effective model of OA. The cartilage lesions in this model and their response to therapy can be graded according to an adapted histological and histochemical grading system, though also this system is to some extent subjective and, thus, neither objective nor entirely reproducible.

Summary Objective This review focuses on histomorphometry for assessing the pathological changes in various compartments of the joint including cartilage, bone and synovium in animal models of osteoarthritis (OA). Methods Different methodological approaches are presented concerning sampling, embedding, sectioning, staining, mounting of stained sections and measurement of histomorphometric parameters using automated and semi-automated methods. Notes are provided describing some methods in greater detail. Results Histomorphometry allows a significant gain of objectivity, accuracy and reproducibility in the quantification of the main histological parameters which best characterize OA in the affected joint (cartilage thickness (CT), chondrocyte size and density, cartilage fissure, proteoglycan (PG) content, subchondral bone plate thickness (SBPT), thickness of synovial living cell layer) in animal models. Conclusion Use of histomorphometry could contribute to a better quantification of histological differences between control and OA animals. Contributing also to the introduction of normative data, it is a major advantage for therapeutic assessments in experimental OA and particularly for the analytical comparison of the efficacy of disease modifying OA drugs (DMOAD).

Matrilins are oligomeric extracellular matrix adaptor proteins mediating interactions between collagen fibrils and other matrix constituents. All four matrilins are expressed in cartilage and mutations in the human gene encoding matrilin-3 (MATN3) are associated with different forms of chondrodysplasia. Surprisingly, however, Matn3-null as well as Matn1- and Matn2-null mice do not show an overt skeletal phenotype, suggesting a dominant negative pathomechanism for the human disorders and redundancy/compensation among the family members in the knock-out situation. Here, we show that mice lacking both matrilin-1 and matrilin-3 develop an apparently normal skeleton, but exhibit biochemical and ultrastructural abnormalities of the knee joint cartilage. At the protein level, an altered SDS-PAGE band pattern and a clear up-regulation of the homotrimeric form of matrilin-4 were evident in newborn Matn1/Matn3 and Matn1 knock-out mice, but not in Matn3-null mice. The ultrastructure of the cartilage matrix after conventional chemical fixation was grossly normal; however, electron microscopy of high pressure frozen and freeze-substituted samples, revealed two consistent observations: 1) moderately increased collagen fibril diameters throughout the epiphysis and the growth plate in both single and double mutants; and 2) increased collagen volume density in Matn1(-/-)/Matn3(-/-) and Matn3(-/-) mice. Taken together, our results demonstrate that matrilin-1 and matrilin-3 modulate collagen fibrillogenesis in cartilage and provide evidence that biochemical compensation might exist between matrilins.

Summary Objective During postnatal development, mammalian articular cartilage acts as a surface growth plate for the underlying epiphyseal bone. Concomitantly, it undergoes a fundamental process of structural reorganization from an immature isotropic to a mature (adult) anisotropic architecture. However, the mechanism underlying this structural transformation is unknown. It could involve either an internal remodelling process, or complete resorption followed by tissue neoformation. The aim of this study was to establish which of these two alternative tissue reorganization mechanisms is physiologically operative. We also wished to pinpoint the articular cartilage source of the stem cells for clonal expansion and the zonal location of the chondrocyte pool with high proliferative activity. Methods The New Zealand white rabbit served as our animal model. The analysis was confined to the high-weight-bearing (central) areas of the medial and lateral femoral condyles. After birth, the articular cartilage layer was evaluated morphologically at monthly intervals from the first to the eighth postnatal month, when this species attains skeletal maturity. The overall height of the articular cartilage layer at each juncture was measured. The growth performance of the articular cartilage layer was assessed by calcein labelling, which permitted an estimation of the daily growth rate of the epiphyseal bone and its monthly length-gain. The slowly proliferating stem-cell pool was identified immunohistochemically (after labelling with bromodeoxyuridine), and the rapidly proliferating chondrocyte population by autoradiography (after labelling with 3 H-thymidine). Results The growth activity of the articular cartilage layer was highest 1 month after birth. It declined precipitously between the first and third months, and ceased between the third and fourth months, when the animal enters puberty. The structural maturation of the articular cartilage layer followed a corresponding temporal trend. During the first 3 months, when the articular cartilage layer is undergoing structural reorganization, the net length-gain in the epiphyseal bone exceeded the height of the articular cartilage layer. This finding indicates that the postnatal reorganization of articular cartilage from an immature isotropic to a mature anisotropic structure is not achieved by a process of internal remodelling, but by the resorption and neoformation of all zones except the most superficial (stem-cell) one. The superficial zone was found to consist of slowly dividing stem cells with bidirectional mitotic activity. In the horizontal direction, this zone furnishes new stem cells that replenish the pool and effect a lateral expansion of the articular cartilage layer. In the vertical direction, the superficial zone supplies the rapidly dividing, transit-amplifying daughter-cell pool that feeds the transitional and upper radial zones during the postnatal growth phase of the articular cartilage layer. Conclusions During postnatal development, mammalian articular cartilage fulfils a dual function, viz., it acts not only as an articulating layer but also as a surface growth plate. In the lapine model, this growth activity ceases at puberty (3–4 months of age), whereas that of the true (metaphyseal) growth plate continues until the time of skeletal maturity (8 months). Hence, the two structures are regulated independently. The structural maturation of the articular cartilage layer coincides temporally with the cessation of its growth activity – for the radial expansion and remodelling of the epiphyseal bone – and with sexual maturation. That articular cartilage is physiologically reorganized by a process of tissue resorption and neoformation, rather than by one of internal remodelling, has important implications for the functional engineering and repair of articular cartilage tissue.

Objective To compare the potential of bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7) and transforming growth factor β1 (TGFβ1) to effect the chondrogenic differentiation of synovial explants by analyzing the histologic, biochemical, and gene expression characteristics of the cartilaginous tissues formed. Methods Synovial explants derived from the metacarpal joints of calves were cultured in agarose. Initially, BMP-2 was used to evaluate the chondrogenic potential of the synovial explants under different culturing conditions. Under appropriate conditions, the chondrogenic effects of BMP-2, BMP-7, and TGFβ1 were then compared. The differentiated tissue was characterized histologically, histomorphometrically, immunohistochemically, biochemically, and at the gene expression level. Results BMP-2 induced the chondrogenic differentiation of synovial explants in a dose- and time-dependent manner under serum- and dexamethasone-free conditions. The expression levels of cartilage-related genes increased in a time-dependent manner. BMP-7 was more potent than BMP-2 in inducing chondrogenesis, but the properties of the differentiated tissue were similar in each case. The type of cartilaginous tissue formed under the influence of TGFβ1 differed in terms of both cell phenotype and gene expression profiles. Conclusion The 3 tested members of the TGFβ superfamily have different chondrogenic potentials and induce the formation of different types of cartilaginous tissue. To effect the full differentiation of synovial explants into a typically hyaline type of articular cartilage, further refinement of the stimulation conditions is required. This might be achieved by the simultaneous application of several growth factors.

The articular cartilage layer of synovial joints is commonly lesioned by trauma or by a degenerative joint disease. Attempts to repair the damage frequently involve the performance of autologous chondrocyte implantation (ACI). Healthy cartilage must be first removed from the joint, and then, on a separate occasion, following the isolation of the chondrocytes and their expansion in vitro, implanted within the lesion. The disadvantages of this therapeutic approach include the destruction of healthy cartilage-which may predispose the joint to osteoarthritic degeneration-the necessarily restricted availability of healthy tissue, the limited proliferative capacity of the donor cells-which declines with age-and the need for two surgical interventions. We postulated that it should be possible to induce synovial stem cells, which are characterized by high, age-independent, proliferative and chondrogenic differentiation capacities, to lay down cartilage within the outer juxtasynovial space after the transcutaneous implantation of a carrier bearing BMP-2 in a slow-release system. The chondrocytes could be isolated on-site and immediately used for ACI. To test this hypothesis, Chinchilla rabbits were used as an experimental model. A collagenous patch bearing BMP-2 in a slow-delivery vehicle was sutured to the inner face of the synovial membrane. The neoformed tissue was excised 5, 8, 11 and 14 days postimplantation for histological and histomorphometric analyses. Neoformed tissue was observed within the outer juxtasynovial space already on the 5th postimplantation day. It contained connective and adipose tissues, and a central nugget of growing cartilage. Between days 5 and 14, the absolute volume of cartilage increased, attaining a value of 12 mm(3) at the latter juncture. Bone was deposited in measurable quantities from the 11th day onwards, but owing to resorption, the net volume did not exceed 1.5 mm(3) (14th day). The findings confirm our hypothesis. The quantity of neoformed cartilage that is deposited after only 1 week within the outer juxtasynovial space would yield sufficient cells for ACI. Since the BMP-2-bearing patches would be implanted transcutaneously in humans, only one surgical or arthroscopic intervention would be called for. Moreover, most importantly, sufficient numbers of cells could be generated in patients of all ages.

Integrin alpha10beta1 is a collagen-binding integrin expressed on chondrocytes. In order to unravel the role of the alpha10 integrin during development, we generated mice carrying a constitutive deletion of the alpha10 integrin gene. The mutant mice had a normal lifespan and were fertile but developed a growth retardation of the long bones. Analysis of the skeleton revealed defects in the growth plate after birth characterized by a disturbed columnar arrangement of chondrocytes, abnormal chondrocyte shape and reduced chondrocyte proliferation. Electron microscopy of growth plates from newborn mice revealed an increased number of apoptotic chondrocytes and reduced density of the collagen fibrillar network compared to these structures in control mice. These results demonstrate that integrin alpha10beta1 plays a specific role in growth plate morphogenesis and function.

A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.

Objective: It has been suggested that chondrocyte death by apoptosis may play a role in the pathogenesis of cartilage destruction in osteoarthritis, but the results of in-vivo and in-vitro investigations have been conflicting. To investigate further the cell death in our in-vitro model for traumatic joint injury, we performed a quantitative analysis by electron microscopy (EM) of cell morphology after injurious compression. For comparison, the TUNEL assay was also performed. Design: Articular cartilage explant disks were harvested from newborn calf femoropatellar groove. The disks were subjected to injurious compression (50% strain at a strain rate of 100%/s), incubated for 3 days, and then fixed for quantitative morphological analysis. Results: By TUNEL, the cell apoptosis rate increased from 7±2% in unloaded controls to 33±6% after injury ( P =0.01; N =8 animals). By EM, the apoptosis rate increased from 5±1% in unloaded controls to 62±10% in injured cartilage ( P =0.02, N =5 animals). Analysis by EM also identified that of the dead cells in injured disks, 97% were apoptotic by morphology. Conclusions: These results confirm a significant increase in cell death after injurious compression and suggest that most cell death observed here was by an apoptotic process.

β1 integrins are highly expressed on chondrocytes, where they mediate adhesion to cartilage matrix proteins. To assess the functions of β1 integrin during skeletogenesis, we inactivated the β1 integrin gene in chondrocytes. We show here that these mutant mice develop a chondrodysplasia of various severity. β1-deficient chondrocytes had an abnormal shape and failed to arrange into columns in the growth plate. This is caused by a lack of motility, which is in turn caused by a loss of adhesion to collagen type II, reduced binding to and impaired spreading on fibronectin, and an abnormal F-actin organization. In addition, mutant chondrocytes show decreased proliferation caused by a defect in G1/S transition and cytokinesis. The G1/S defect is, at least partially, caused by overexpression of Fgfr3, nuclear translocation of Stat1/Stat5a, and up-regulation of the cell cycle inhibitors p16 and p21. Altogether these findings establish that β1-integrin-dependent motility and proliferation of chondrocytes are mandatory events for endochondral bone formation to occur.

The cartilage extracellular matrix is composed of a dense collagen network that entraps a range of other specialized proteins important for the proper formation and function of the tissue. Loss of two abundant cartilage components, type II collagen and perlecan, has drastic effects on skeletal development. Both collagen II and perlecan mutants have severe and lethal chondrodysplasia characterized by disorganized growth plate, lack of collagen network, defective endochondral bone formation, and abnormal intervertebral disk development. To test whether the reduced collagen density in the perlecan-null cartilage is due to enhanced activity of collagen-degrading proteinases, we have analyzed gelatinase expression and activity in the mutant tissue. Immunohistochemical analysis revealed a weak, but clear, expansion of MMP-9 deposition into the hypertrophic zone of the perlecan-null growth plate. However, in situ and SDS-PAGE zymography showed that the activity of gelatinases (MMP-2 and MMP-9) is not altered in perlecan-null cartilage, suggesting that they are not primarily linked to the reduced fibrillar network observed in the mutant. Likewise, intercrossing of perlecan mutants onto an MMP-9-null background could not rescue the ultrastructural abnormalities of the perlecan-deficient cartilage.

Objective : Induction of growth-factor-based repair in full-thickness articular cartilage defects can be impaired by the upgrowth of blood vessels and new bone into the cartilaginous compartment. We postulated that if an antiangiogenic factor (suramin) is included in the chondrogenic matrix applied to the cartilaginous compartment of a full-thickness defect, vascular upgrowth and therefore bone formation will be inhibited (functional barrier principle). Design : Full-thickness defects were created in miniature pigs and the bony portion filled with a chondrogenic matrix. The cartilaginous compartment was filled with the same matrix which additionally contained suramin, either in a free form or in free and liposome-encapsulated forms. Animals were sacrificed 8 weeks after surgery and the extent to which bone tissue had encroached on the cartilaginous compartment was graded semiquantitatively using light microscopy. Results : In 63% of the control defects, bone represented more than 50% of the repair tissue present. In 10% of the defects treated with free suramin, bone upgrowth was completely inhibited; in 55%, osseous tissue occupied 1–10% of the cartilaginous space and in the other 35%, it represented 11–50% of the repair tissue present in this compartment. In 69% of the defects treated with free and liposome-encapsulated suramin, bone upgrowth into the cartilaginous compartment was completely inhibited; in the remaining 31%, osseous tissue occupied no more than 1–10% of this space. Conclusions : To be effective, an antiangiogenic factor needs to be present at a sustained level throughout the chondrogenic treatment course.

Articular cartilage exhibits anisotropic mechanical properties when subjected to tension. However, mechanical anisotropy of mature cartilage in compression is poorly known. In this study, both confined and unconfined compression tests of cylindrical cartilage discs, taken from the adult human patello-femoral groove and cut either perpendicular (normal disc) or parallel (tangential disc) to the articular surface, were utilized to determine possible anisotropy in Young's modulus, E, aggregate modulus, Ha, Poisson's ratio, v and hydraulic permeability, k, of articular cartilage. The results indicated that Ha was significantly higher in the direction parallel to the articular surface as compared with the direction perpendicular to the surface ( Ha = 1.237 ± 0.486 MPa versus Ha = 0.845 ± 0.383 MPa, p = 0.017, n = 10). The values of Poisson's ratio were similar, 0.158 ± 0.148 for normal discs compared with 0.180 ± 0.046 for tangential discs. Analysis using the linear biphasic model revealed that the decrease of permeability during the offset compression of 0–20 per cent was higher ( p = 0.015, n = 10) in normal (from 25.5 × 10− 15 to 1.8 × 10−15 m4/N s) than in tangential (from 12.3 × 10− 15 to 1.3 × 10− 15 m4/N s) discs. Based on the results, it is concluded that the mechanical characteristics of adult femoral groove articular cartilage are anisotropic also during compression. Anisotropy during compression may be essential for normal cartilage function. This property has to be considered when developing advanced theoretical models for cartilage biomechanics.

Objective Data pertaining to the quantitative structural features and organization of normal articular cartilage are of great importance in understanding its biomechanical properties and in attempting to establish this tissue's counterpart by engineering in vitro . A comprehensive set of such baseline data is, however, not available for humans. It was the purpose of the present study to furnish the necessary information. Design The articular cartilage layer covering the medial femoral condyle of deceased persons aged between 23 and 49 years was chosen for the morphometric analysis of cell parameters using confocal microscopy in conjunction with unbiased stereological methods. The height of the hyaline articular cartilage layer, as well as that of the calcified cartilage layer and the subchondral bone plate, were also measured. Results The mean height of the hyaline articular cartilage layer was found to be 2.4mm, the volume density of chondrocytes therein being 1.65%, the number of cells per mm 3 of tissue 9626 and the mean cell diameter 13μm. Other estimators (including matrix mass per cell and cell profile density) were also determined. Conclusions A comparison of these normal human quantitative data with those published for experimental animals commonly used in orthopaedic research reveals substantial differences, consideration of which in tissue engineering strategies destined for human application are of paramount importance for successful repair. Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved .

Indentation testing is a widely used technique for nondestructive mechanical analysis of articular cartilage. Although cartilage shows an inhomogeneous, layered structure with anisotropic mechanical properties, most theoretical indentation models assume material homogeneity and isotropy. In the present study, quantitative polarized light microscopy (PLM) measurements from canine cartilage were utilized to characterize thickness and structure of the superficial, collageneous tissue layer as well as to reveal its relation to experimental indentation measurements. In addition to experimental analyses, a layered, transversely isotropic finite element (FE) model was developed and the effect of superficial (tangential) tissue layer with high elastic modulus in the direction parallel to articular surface on the indentation response was studied. The experimental indentation stiffness was positively correlated with the relative thickness of the superficial cartilage layer. Also the optical retardation, which reflects the degree of parallel organization of collagen fibrils as well as collagen content, was related to indentation stiffness. FE results indicated effective stiffening of articular cartilage under indentation due to high transverse modulus of the superficial layer. The present results suggest that indentation testing is an efficient technique for the characterization of the superficial degeneration of articular cartilage.

Chondrocytes are known to sense and respond to mechanical and physicochemical stimuli by multiple regulatory pathways, including upstream signaling, transcription, translation, posttranslational modifications, and vesicular transport. Due to the complexity of identifying the biophysical phenomena that occur during cartilage loading in vivo, the regulatory mechanisms that govern chondrocyte mechanotransduction are not fully understood. Recent studies have shown that fluid flow during dynamic compression of cartilage explants can stimulate proteoglycan and protein synthesis. In this study, we examined the effect of deformations of cell and extracellular matrix on chondrocyte biosynthesis. We used tissue shear loading, since tissue shear causes little volumetric deformation and can thereby decouple fluid flow from cell and matrix deformation. Shear loading was applied over a wide range of frequencies, 0.01-1.0 Hz, using 1-3% sinusoidal shear strain amplitudes, and the resulting proteoglycan and protein syntheses were measured using radiolabel incorporation. In addition, quantitative autoradiography was used to investigate spatial variations in matrix biosynthesis and to correlate these variations with the spatial profiles of biophysical stimuli. Our data show that tissue shear loading at 1-3% strain amplitude stimulated the synthesis of protein by approximately 50% and proteoglycans by approximately 25% at frequencies between 0.01 and 1.0 Hz. The relatively uniform patterns of biosynthesis in the radial and vertical directions within cylindrical explants revealed by autoradiography suggest that the stimulatory effect was associated with the relatively uniform deformation caused by simple shear loading. These results suggest that chondrocytes can respond to tissue shear stress-initiated pathways for the production of collagen and proteoglycan, which include deformation of cells and pericellular matrix, even in the absence of macroscopic tissue-level fluid flow.

Mechanical overloading of cartilage has been implicated in the initiation and progression of osteoarthrosis. Our objectives were to identify threshold levels of strain rate and peak stress at which sub-impact loads could induce cartilage matrix damage and chondrocyte injury in bovine osteochondral explants and to explore relationships between matrix damage, spatial patterns of cell injury, and applied loads. Single sub-impact loads characterized by a constant strain rate between 3×10−5 and 0.7 s −1 to a peak stress between 3.5 and 14 MPa were applied, after which explants were maintained in culture for four days. At the higher strain rates, matrix mechanical failure (tissue cracks) and cell deactivation were most severe near the cartilage superficial zone and were associated with sustained increased release of proteoglycan from explants. In contrast, low strain rate loading was associated with cell deactivation in the absence of visible matrix damage. Furthermore, cell activity and proteoglycan synthesis were suppressed throughout the cartilage depth, but in a radially dependent manner with the most severe effects at the center of cylindrical explants. Results highlight spatial patterns of matrix damage and cell injury which depend upon the nature of injurious loading applied. These patterns of injury may also differ in terms of their long-term implications for progression of degradative disease and possibilities for cartilage repair.

Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.

Chondrogenesis in cartilage development and repair and cartilage degeneration in arthritis can be regulated by mechanical-load-induced physical factors such as tissue deformation, interstitial fluid flow and pressure, and electrical fields or streaming potentials. Previous animal and tissue explant studies have shown that time-varying dynamic tissue loading can increase the synthesis and deposition of matrix molecules in an amplitude-, frequency-, and spatially dependent manner. To provide information on the cell-level physical factors which may stimulate chondrocytes to increase production and export of aggrecan, the main proteoglycan component of the cartilage matrix, we characterized local changes in aggrecan synthesis within cyclically loaded tissue explant disks and compared those changes to values of predicted local physical factors. Aggrecan synthesis following a 23-h compression/radiolabel protocol was measured with a spatial resolution of approximately 0.1 mm across the 1.5-mm radius of explanted disks using a quantitative autoradiography method. A uniform stimulation of aggrecan synthesis was observed at an intermediate frequency of 0.01 Hz, while, at a higher frequency of 0.1 Hz, stimulation was only seen at peripheral radial positions. Profiles of radial solid matrix deformation and interstitial fluid pressure and velocity predicted to be occurring across the radius of the disk during sinusoidal loading were estimated using a composite poroelastic model. Tissue regions experiencing high interstitial fluid velocities corresponded to those displaying increased aggrecan synthesis. These results reinforce the role of load-induced flow of interstitial fluid in the stimulation of aggrecan production during dynamic loading of cartilage.

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

This article reviews the experimental and clinical strategies currently in use or under development for the treatment of articular cartilage lesions. The vast majority of protocols under investigation pertain to the treatment of full-thickness defects (i.e., those which penetrate the subchondral bone and trabecular–bone spaces) rather than partial-thickness ones (i.e., those which are confined to the substance of articular cartilage tissue itself). This bias probably reflects the circumstance that partial-thickness defects do not heal spontaneously whereas full-thickness ones below a critical size do, albeit transiently. And it is, of course, a seemingly easier task to manipulate a process which is readily set in train than it is to overcome an induction-problem which Nature herself has not solved. Indeed, the reasons for this inert state of partial-thickness defects have only recently been elucidated, and these are briefly discussed. However, the main body of this review deals with the various transplantation concepts implemented for the repair of full-thickness defects. These fall into two broad categories: tissue-based (entailing the grafting of perichondrial, periosteal, cartilage or bone-cartilage material) and cell-based (utilizing chondroblasts, chondrocytes, periochondrial cells or mesenchymal stem cells). Cell-based systems are further subdivided according to whether cells are transplanted within a matrix (biodegradable, non-biodegradable or synthetic) or free in suspension. Thus far, the application of cell suspensions has always been combined with the grafting of a periosteal flap. The strengths and weaknesses of each concept are discussed.

Skeletal elements are formed either by replacing a preformed cartilagenous matrix template in a process called endochondral ossification or directly from mesenchyme by a process known as membranous ossification. Longitudinal growth of bones is achieved by growth plates where calcified cartilage is converted into bone. To investigate the role of extracellular matrix as well as intracellular signaling pathways in the formation and growth of bone, the genes coding for type II collagen and cyclic guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinase (cGK) II, were disrupted. It is demonstrated that loss of Col2a1 or cGKII led to abnormal endochondral ossification and skeletal development. In cGKII −/− mice, bones derived by membranous ossification developed normally while bones derived by endochondral ossification were shortened. This growth defect was not associated with a general metabolic disturbance. In Col2a1 knockout mice, endochondral ossification was completely absent, whereas membraneous ossification was not affected. Despite the defects in bone formation, invasion of blood vessels into bone cavities and formation of bone marrow occurred in Col2a1-null mice. Taken together, the phenotypes of these two knockout mice show that chondrocytes need a well-functioning extracellular matrix scaffold and a normal cGMP-signaling system for endochondral ossification to form a normal skeleton.

The effects of mechanical injury on the metabolism of cartilage matrix are of interest for understanding the pathogenesis of osteoarthrosis and the development of strategies for cartilage repair. The purpose of the present study was to examine the effects of injury on matrix turnover in a calf articular cartilage explant system for which the effects of mechanical loading on cell activity and the cell-mediated pathways of matrix metabolism are already well characterized. New methods of quantitative autoradiography were used in combination with established biochemical and biomechanical techniques for the analysis of cell and matrix responses to acute mechanical injury, with particular attention to the processes of localized matrix turnover in the cell-associated matrices of individual chondrocytes. Matrix deposition and turnover around cells in control explants was spatially dependent, with the highest rates of proteoglycan deposition and tunrtover arid the lowest rates of collagen deposition (as indicated by [3H]proline autoradiography) occurring in the pericellular matrix. Injurious compression was associated with (a) an abrupt decrease in the tensile load-carrying capacity of the collagen matrix, apparently associated with mechanical failure of the tissue, (b) a considerable but subtotal decrease in cell viability, marked by the emergence of an apparently inactive cell population interspersed within catabolically active but abnormally large cells, and (c) sustained, elevated rates of protcoglycan turnover, particularly in the cell-associated matrices of apparently viable cells, which involved the increased release of aggregating species in addition to a spectrum of degradation fragments that were also in controls. These results may represent an in vitro model for the responses of chondrocytes and the cartilage extracellular matrix to mechanical injury.

Knowledge of matrix assembly is necessary to understand the pathogenesis of disease processes and to find solutions for repair of articular cartilage lesions. The influence of growth factors on matrix assembly is largely unknown. We investigated whether, and to what degree, insulin-like growth factor (IGF-1) and transforming growth factor beta-2 (TGF beta-2) influence proteoglycan synthesis and accumulation in the cell-associated matrix compartment (consisting of pericellular and territorial matrix) compared to the further-removed matrix compartment (consisting of the interterritorial matrix).Bovine articular chondrocytes were cultured in alginate beads for day 13. The effect of addition of 25 ng/ml IGF-1 or 25 ng/ml TGF beta-2 during the last 7 days in culture was determined. Cell-associated and further-removed matrix compartments were separated by centrifugation after sodium citrate/EDTA treatment. The amount of DNA, the total amount of proteoglycans and the amount of newly synthesized proteoglycans were analyzed biochemically. Morphometric analysis on electron micrographs was used to calculate the volumes of the cell-associated and further-removed matrix components.It was demonstrated in control beads that 25 +/- 8% of the proteoglycans were laid down in the cell-associated matrix compartment compared with 75 +/- 8% in the further-removed matrix compartment. The cell-associated matrix compartment in intact beads could be recognized in electron microscopy by a delineation of dense amorph material. Morphometric evaluation showed a relative volume of the cell-associated matrix compartment of 5.2 +/- 0.6% compared with 91.3 +/- 0.8% of the further-removed matrix compartment and 3.5 +/- 0.3% of the area occupied by cells. Combination of biochemical and morphometric results showed that the concentration of proteoglycans in the cell-associated matrix compartment was 3.63 +/- 0.32 mg/ml. By adding IGF-1 or TGF beta-2, the amount of both total accumulated proteoglycans and newly synthesized [35S]proteoglycans at day 13 in culture increased. In addition to an overall rise in proteoglycan content, IGF-1 significantly increased (24%) the percentage of proteoglycans laid down in the cell-associated matrix compartment while not changing the relative volume of this compartment (5.2 +/- 0.8%). This leads to a 82% (P0.05) increase in the proteoglycan concentration in the cell-associated matrix compartment compared to control beads. In contrast, TGF beta-2 significantly decreased (24%) the relative amount of proteoglycans in the cell-associated matrix compartment which was paralleled by a reduction of the relative volume from 5.2 +/- 0.6 to 3.6 +/- 1.4%. This leads to a significant increase of 87% of the proteoglycan concentration in the cell-associated matrix compartment.This study demonstrates that both IGF-1 and TGF beta-2 significantly but differently influence proteoglycan synthesis and accumulation in the cell-associated matrix compartment of cultured bovine chondrocytes in alginate. Both growth factors increase the concentration of proteoglycans in the cell-associated matrix compartment. However, addition of TGF beta-2 to bovine articular chondrocytes cultured in alginate beads for 13 days results in a significant reduction of the relative volume of the pericellular matrix compartment compared to controls, indicating differences in assembly of the matrix.

BACKGROUND Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2) induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2) and transforming growth factor beta 1 (TGF-s1) were investigated. METHODOLOGY/PRINCIPAL FINDINGS Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml) for 4 (or 6) weeks. FGF-2 (10 ng/ml) or TGF-s1 (10 ng/ml) was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs) only when it was present during the first week of culturing alone. TGF-s1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2), but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume. CONCLUSIONS/SIGNIFICANCE TGF-s1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of clinical repair, our findings will be of importance in fine-tuning the stimulation protocol for the optimal chondrogenic differentiation of synovial explants.

Summary Objective Marked differences exist between human knee and ankle joints regarding risks and progression of osteoarthritis (OA). Pathomechanisms of degenerative joint disease may therefore differ in these joints, due to differences in tissue structure and function. Focusing on structural issues, which are design goals for tissue engineering, we compared cell and matrix morphologies in different anatomical sites of adult human knee and ankle joints. Methods Osteochondral explants were acquired from knee and ankle joints of deceased persons aged 20–40 years and analyzed for cell, matrix and tissue morphology using confocal and electron microscopy (EM) and unbiased stereological methods. Morphological variations disclosing an association between joint type (knee vs ankle) and biomechanical role (convex vs concave articular surfaces) were identified by a 2-way analysis of variance (ANOVA) and a post-hoc analysis. Results Knee cartilage exhibited higher cell densities in the superficial zone than ankle cartilage. In the transitional zone, higher cell densities were observed in association with convex vs concave articular surfaces, without significant differences between knee and ankle cartilage. Highly uniform cell and matrix morphologies were evident throughout the radial zone in the knee and ankle, regardless of tissue biomechanical role. Throughout the knee and ankle cartilage sampled, chondron density was remarkably constant at approximately 4.2 × 10 6 chondrons/cm 3 . Conclusion Variation in cartilage cell and matrix morphologies with changing joint and biomechanical environments suggests that tissue structural adaptations are performed primarily by the superficial and transitional zones. Data may aid the development of site-specific cartilage tissue engineering, and help to identify conditions where OA is likely to occur.

The efficacy of biological therapeutics against cartilage degradation in osteoarthritis is restricted by the limited transport of macromolecules through the dense, avascular extracellular matrix. The availability of biologics to cell surface and matrix targets is limited by steric hindrance of the matrix, and the microstructure of matrix itself can be dramatically altered by joint injury and the subsequent inflammatory response. We studied the transport into cartilage of a 48 kDa anti-IL-6 antigen binding fragment (Fab) using an in vitro model of joint injury to quantify the transport of Fab fragments into normal and mechanically injured cartilage. The anti-IL-6 Fab was able to diffuse throughout the depth of the tissue, suggesting that Fab fragments can have the desired property of achieving local delivery to targets within cartilage, unlike full-sized antibodies which are too large to penetrate beyond the cartilage surface. Uptake of the anti-IL-6 Fab was significantly increased following mechanical injury, and an additional increase in uptake was observed in response to combined treatment with TNFα and mechanical injury, a model used to mimic the inflammatory response following joint injury. These results suggest that joint trauma leading to cartilage degradation can further alter the transport of such therapeutics and similar-sized macromolecules., National Institute of Arthritis and Musculoskeletal and Skin Diseases (U.S.) (Grant AR45779), National Institute of Arthritis and Musculoskeletal and Skin Diseases (U.S.) (Grant AR60331), Janssen Pharmaceutical Ltd. (Research and Development Grant)

Bovine articular cartilage was vitrified by high-pressure freezing. On the one hand vitrified samples were cryosectioned and investigated by cryoelectron microscopy in an unstained frozen hydrated state. On the other hand, they were freeze substituted in pure acetone, ethanol, or methanol, respectively, and subsequently embedded in Epon. Ultrathin Epon sections were poststained with uranyl acetate and lead citrate. The resulting ultrastructural representation was different for every protocol. The evaluation of the combined results provides evidence for a distinct water-rich layer surrounding collagen fibrils in articular cartilage extracellular matrix, which has not been recognized before. The possible composition and function of this layer is discussed.

Maintenance of superficial structural integrity is essential for the load-bearing function of articular cartilage. In this study, we used atomic force microscopy (AFM) to image the 3-D surface and subsurface morphology of fresh bovine humeral head articular cartilage maintained in physiological solution. Complementary ultrastructural data were obtained by transmission electron microscopy (TEM) of cryo-processed samples. The surface irregularities observed in previous scanning electron microscopic studies were not apparent with AFM. The most superficial layer, typically 200-500 nm thick, consisted of acellular and nonfibrous tissue. Occasionally, it exhibited local discontinuities through which the underlying network of collagen fibrils, oriented parallel to the surface and displaying the characteristic periodic banding, could been seen. Local variations in force-curve measurements indicate the existence of differences in micromechanical properties along the articular surface. AFM thus furnishes a new method for characterizing the surface structure and properties of freshly excised articular cartilage in physiologically relevant conditions. It confirms the existence of an amorphous, nonfibrous articular surface which may be vital for its normal lubrication and wearing properties in vivo.