Chondrocytes Directly Transform into Bone Cells in Mandibular Condyle Growth.

For decades, it has been widely accepted that hypertrophic chondrocytes undergo apoptosis prior to endochondral bone formation. However, very recent studies in long bone suggest that chondrocytes can directly transform into bone cells. Our initial in vivo characterization of condylar hypertrophic chondrocytes revealed modest numbers of apoptotic cells but high levels of antiapoptotic Bcl-2 expression, some dividing cells, and clear alkaline phosphatase activity (early bone marker). Ex vivo culture of newborn condylar cartilage on a chick chorioallantoic membrane showed that after 5 d the cells on the periphery of the explants had begun to express Col1 (bone marker). The cartilage-specific cell lineage-tracing approach in triple mice containing Rosa 26(tdTomato) (tracing marker), 2.3 Col1(GFP) (bone cell marker), and aggrecan Cre(ERT2) (onetime tamoxifen induced) or Col10-Cre (activated from E14.5 throughout adult stage) demonstrated the direct transformation of chondrocytes into bone cells in vivo. This transformation was initiated at the inferior portion of the condylar cartilage, in contrast to the initial ossification site in long bone, which is in the center. Quantitative data from the Col10-Cre compound mice showed that hypertrophic chondrocytes contributed to ~80% of bone cells in subchondral bone, ~70% in a somewhat more inferior region, and ~40% in the most inferior part of the condylar neck (n = 4, P < 0.01 for differences among regions). This multipronged approach clearly demonstrates that a majority of chondrocytes in the fibrocartilaginous condylar cartilage, similar to hyaline cartilage in long bones, directly transform into bone cells during endochondral bone formation. Moreover, ossification is initiated from the inferior portion of mandibular condylar cartilage with expansion in one direction.

Cartilage regeneration by gene therapy.

Damage of articular cartilage is a frequent clinical problem and is commonly considered to be irreversible. Full-thickness defects may lead to the formation of fibrous repair tissue of minor mechanical quality, while partial-thickness lesions hardly show any repair response. Surgical approaches often fail to restore the articular surface, facing the problem of incomplete chondrogenesis or rapid degradation of the repair tissue. However, advances in molecular biology have revealed the potential of growth factors, differentiation factors, and cytokines in directing cellular differentiation and metabolic activity. Anabolic factors including members of the TGF-beta superfamily, IGF-1, FGF, or HGF have proven their potential to stimulate chondrogenesis and synthesis of cartilage-specific matrix components, allowing the formation of a hyaline cartilage-like repair tissue in experimental studies. In addition, anti-catabolic or anti-inflammatory molecules, such as IL-4, IL-10, IL-1Ra, and TNFsR may also exert beneficial effects by inhibiting excessive cartilage degradation. Although it is questionable whether regeneration of hyaline cartilage implying a complete restoration of the articular surface by a tissue that is identical with the original can ever be achieved, all these molecules have been considered as suitable tools for cartilage repair. The transfer of the respective genes into the joint, possibly in combination with the supply of chondroprogenitor cells, might be an elegant method to achieve a sustained delivery of such therapeutic factors at the required location in vivo. This review focuses on the therapeutic molecules, the suitability of different viral and non-viral vectors for intraarticular gene transfer and the lessons learned from gene therapy studies on various animal models.

Bone regeneration in critical size defects by cell-mediated BMP-2 gene transfer: a comparison of adenoviral vectors and liposomes.

Large bone defects resulting from nonunion fractures or tumour resections are common clinical problems. Recent studies have shown bone morphogenetic protein-2 (BMP-2) gene transfer using adenoviral vectors to be a promising new therapeutic approach. However, comparative studies of different vectors are required to identify the optimal system for possible clinical trials. This study compares the use of liposome-mediated and adenoviral gene transfer for the generation of autologous BMP-2-producing bone marrow stromal cells (BMSC). Primary BMSC isolated from the rat femur were treated ex vivo with either an adenovirus or a liposome carrying human BMP-2 cDNA. The genetically modified cells were evaluated in vitro and transplanted into critical size defects in the rat mandible in vivo. BMSC treated with a reporter gene vector or untreated BMSC served as controls. The newly formed tissue was analysed by in situ hybridization, radiography and immunohistochemistry. Both groups of genetically modified cells produced BMP-2 for at least 2 weeks, and markers of new bone matrix such as osteopontin and osteocalcin were observed within 2 weeks following gene transfer. In the liposome group, the critical size defects were found completely healed at 6 weeks after the gene transfer, whereas the more efficient adenoviral gene transfer allowed for complete bone healing within 4 weeks. None of the three control groups showed bone healing, not even after 8 weeks. Thus, both liposome-mediated and adenoviral BMP-2 gene transfer to primary BMSC are suitable methods to achieve the healing of critical size bone defects in rats. As liposomes have proven sufficient for this purpose and offer several advantages over any other vector, such as ease of preparation, theoretically no limitation of the size of the DNA, and less immunological and safety problems, they may represent the best vector system for future clinical trials of bone regeneration by BMP-2 gene therapy.

Sequential expression of annexin A5 in the vasculature and skeletal elements during mouse development.

Annexin A5 (annexin V, anchorin CII) represents the prototype member of the large annexin family, characterized by its ability to interact with phospholipids in a calcium-dependent manner and to form calcium-specific ion channels. Despite intense biochemical analysis, the in vivo expression and function of this annexin during mouse development, still remains unclear. Immunohistochemistry, in situ hybridization and reporter gene expression were used to define expression of annexin A5 during early mouse development. First, annexin A5 expression is associated with the developing vascular system. Later, expression is detected within the notochord and found in parallel to the differentiation of cartilage and bone. Therefore, expression of the Anxa5 gene may represent a novel marker characterizing cell lineages involved in the development of the vascular as well as the skeletal system.

Changes in the expression of annexin A5 gene during in vitro chondrocyte differentiation: influence of cell attachment.

Several lines of evidence indicate that annexin A5, a membrane-associated protein with calcium-channel activity, plays a key role in cartilage calcification during endochondral ossification. As a major constituent of cartilage matrix vesicles, which are released from microvilli of hypertrophic chondrocytes, it is involved in calcium uptake necessary for the initial stages of cartilage calcification. Little is known, however, concerning transcriptional regulation of the annexin A5 gene during chondrocyte differentiation. Here, we report on changes in annexin A5 expression by measuring mRNA and protein levels during in vitro differentiation of chick sternal chondrocytes to the hypertrophic phenotype. Terminal differentiation of mature sternal chondrocytes was achieved in the presence of sodium ascorbate in high-density cultures growing either in monolayer or over agarose as cell aggregates. Differentiation of chondrocytes to hypertrophic cells was followed by morphological analysis and by the onset of type X collagen expression. High expression levels of annexin A5 mRNA were detected in chondrocytes freshly isolated from the sterna by enzymatic digestion and subsequently in cells growing in monolayer, but annexin A5 gene transcription was rapidly downregulated when cells were grown in suspension as aggregates over agarose. However, protein levels did not decrease probably due to its low turnover rate. In suspension culture, annexin A5 mRNA reappeared after 3 weeks concomitantly with segregation of the aggregates into single cells and onset of chondrocyte hypertrophy. The downregulation of annexin A5 expression in cells growing as matrix-rich aggregates was reverted when extracellular matrix components were removed and cells were reseeded onto tissue culture plastic, suggesting that cell spreading, formation of focal contacts and stress fibers stimulated annexin A5 expression in proliferating as well as in hypertrophic chondrocytes.

Role of growth factors in rabbit articular cartilage repair by chondrocytes in agarose.

OBJECTIVE: Novel approaches to intervention in joint diseases consist of the replacement of diseased cartilage by in vitro engineered, viable cells or graft tissues. Two major obstacles remain to be overcome: (1) Hyaline cartilage in vitro often loses differentiated traits. (2) Grafts frequently are not integrated satisfactorily into host cartilage and/or the tissue is remodelled in situ into functionally inferior fibrocartilage. Therefore, we have explored the possibility whether chondrocytes embedded into agarose gels provided better graft tissues in a repair model of full thickness defects in rabbit joint cartilage. DESIGN: Experimental defects of knee joint cartilage was filled with articular chondrocytes cultured in agarose gels. Chondrocytes in vitro either remained unstimulated or were treated with several growth factors. Repair of the defects was assessed by histology and was scored between 0 (no healing) and 1 (perfect healing) as judged by the follwing parameters: intensity of proteoglycan staining, organization of the superficial zone, ossification at the border between repair cartilage and subchondral bone, tidemark formation in the repaired area, arrangement of chondrocytes, and integration of repair cartilage into host. RESULTS: Treatment of chondrocyte cultures with bFGF had a stabilizing effect on the differentiated state of the cells in implanted grafts whereas bone morphogenetic proteins stimulated ingrowth of subchondral bone reducing repair cartilage thickness and preventing normal tide mark formation; TGF-beta did not significantly affect evaluation parameters in comparison with untreated controls. CONCLUSION: Growth factor treatment resulted in an ambiguous quality of graft development. Only FGF had a clear beneficial effect to the graft tissues after 1 month. Further studies are required to define the precise conditions and sequence of growth factor treatment of in vitro engineered cartilage which benefits graft quality.

Fibroblast-mediated delivery of growth factor complementary DNA into mouse joints induces chondrogenesis but avoids the disadvantages of direct viral gene transfer.

OBJECTIVE: To assess the advantages and disadvantages of a direct adenoviral and a cell-mediated approach to the induction of cartilage formation in joints by transfer of growth factor genes. METHODS: Adenoviral vectors carrying insulin-like growth factor 1 (IGF-1) or bone morphogenetic protein 2 (BMP-2) complementary DNA were constructed and applied to primary human and murine chondrocytes or fibroblasts. Transgene expression was quantified by enzyme-linked immunosorbent assay. Direct injection of these vectors or AdLacZ, a reporter gene vector, into mouse knee joints was compared with the transplantation of syngeneic fibroblasts (infected ex vivo with the same vectors) with respect to virus spread, immune response, and cartilage formation by use of histologic, immunohistochemical, and molecular analyses. RESULTS: AdIGF-1 and AdBMP-2 efficiently infected all cell types tested. Human cells secreted biologically relevant levels of protein over a period of at least 28 days. Direct transfer of AdLacZ into mouse knee joints resulted in positively stained synovial tissues, whereas AdLacZ-infected fibroblasts settled on the surface of the synovial membranes. Inadvertent spread of vector DNA into the liver, lung, and spleen was identified by nested polymerase chain reaction in all mice that had received the vector directly; this rarely occurred following fibroblast-mediated gene transfer. Direct injection of AdBMP-2 induced the synthesis of new cartilage in periarticular mesenchyme, accompanied by extensive osteophyte formation. When AdBMP-2 was administered by injecting ex vivo-infected fibroblasts, cartilage formation was observed only in regions near the injected cells. AdIGF-1 treatment did not lead to morphologic changes. Importantly, fibroblast-mediated gene transfer avoided the strong immune response to adenovirus that was elicited following direct application of the vector. CONCLUSION: Our results indicate that cell-mediated gene transfer provides sufficient BMP-2 levels in the joint to induce cartilage formation while avoiding inadvertent vector spread and immune reactions.

The integrin alpha 7 cytoplasmic domain regulates cell migration, lamellipodia formation, and p130CAS/Crk coupling.

The integrin alpha(7)beta(1) is the major laminin-binding integrin in skeletal, heart, and smooth muscle and is a receptor for laminin-1 and -2. It mediates myoblast migration on laminin-1 and -2 and thus might be involved in muscle development and repair. Previously we have shown that alpha(7)B as well as the alpha(7)A and -C splice variants induce cell motility on laminin when transfected into nonmotile HEK293 cells. In this study we have investigated the role of the cytoplasmic domain of alpha(7) in the laminin-induced signal transduction of alpha(7)beta(1) integrin regulating cell adhesion and migration. Deletion of the cytoplasmic domain did not affect assembly of the mutated alpha(7)Deltacyt/beta(1) heterodimer on the cell surface or adhesion of alpha(7)Deltacyt-transfected cells to laminin. The motility of these cells on the laminin-1/E8 fragment, however, was significantly reduced to the level of mock-transfected cells; lamellipodia formation and polarization of the cells were also impaired. Adhesion to the laminin-1/E8 fragment induced tyrosine phosphorylation of the focal adhesion kinase, paxillin, and p130(CAS) as well as the formation of a p130(CAS)-Crk complex in wild-type alpha(7)B-transfected cells. In alpha(7)BDeltacyt cells, however, the extent of p130(CAS) tyrosine formation was reduced and formation of the p130(CAS)-Crk complex was impaired, with unaltered levels of p130(CAS) and Crk protein levels. These findings indicate adhesion-dependent regulation of p130(CAS)/Crk complex formation by the cytoplasmic domain of alpha(7)B integrin after cell adhesion to laminin-1/E8 and imply alpha(7)B-controlled lamellipodia formation and cell migration through the p130(CAS)/Crk protein complex.

Coexpression of alpha and beta subunits of prolyl 4-hydroxylase stabilizes the triple helix of recombinant human type X collagen.

We have reported previously on the expression of recombinant human type X collagen (hrColX) in HEK 293 and HT 1080 cells by using the eukaryotic expression vector pCMVsis (in which CMV stands for cytomegalovirus). Several stably transfected clones secreted full-length triple-helical hrColX molecules in large amounts, but the secreted collagen was underhydroxylated, with a hydroxyproline-to-proline ratio of 0.25 and a melting temperature (T(m)) of 31 degrees C. By comparison, native chicken type X procollagen has a T(m) of 46 degrees C. To stabilize the triple helix of hrColX, an hrColX-expressing clone (A6/16) was co-transfected with both alpha and beta subunits of human prolyl 4-hydroxylase. Clones were selected that secreted proalpha1(X) collagen chains with an apparent molecular mass of 75 kDa and an increased hydroxyproline-to-proline ratio of close to 0.5. As a result of enhanced prolyl hydroxylation, the T(m) of the hrColX was increased to 41 degrees C as measured by CD analysis at various temperatures. The CD spectra indicated a minimum ellipticity at 198 nm and a peak at 225 nm at 20 degrees C, confirming the presence of a triple helix. The same T(m) of 41 degrees C was measured for the triple-helical core fragments of hrColX of 60-65 kDa that were retained after brief digestion with chymotrypsin/trypsin at increasing temperatures. This shows that the human cell line HEK-293 is suitable for the simultaneous expression of three genes and the stable production of substantial amounts of recombinant, fully hydroxylated type X collagen over several years.

Laminin alpha4 and integrin alpha6 are upregulated in regenerating dy/dy skeletal muscle: comparative expression of laminin and integrin isoforms in muscles regenerating after crush injury.

The expression of laminin isoforms and laminin-binding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin alpha2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and "preactivated" myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin alpha1 was not detectable in skeletal muscle. Laminin alpha2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin alpha2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin alpha chains in early myogenic differentiation, such as laminin alpha4 and alpha5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin beta1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin alpha3 was not expressed in uninjured or regenerating muscle, while integrin alpha6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin alpha6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin alpha6 occurred on some newly formed myotubes. Integrin alpha7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin alpha7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin alpha7 negative. No marked difference was observed in integrin alpha7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin alpha4 and integrin alpha6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin alpha4 and integrin alpha6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin alpha2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.

The role of extracellular and cytoplasmic splice domains of alpha7-integrin in cell adhesion and migration on laminins.

The major laminin-binding integrin of skeletal, smooth, and heart muscle is alpha7beta1-integrin, which is structurally related to alpha6beta1. It occurs in three cytoplasmic splice variants (alpha7A, -B, and -C) and two extracellular forms (X1 and X2) which are developmentally regulated and differentially expressed in skeletal muscle. Previously, we have shown that ectopic expression of the alpha7beta-integrin splice variant in nonmotile HEK293 cells specifically induced cell locomotion on laminin-1 but not on fibronectin. To investigate the specificity and the mechanism of the alpha7-mediated cell motility, we expressed the three alpha7-chain cytoplasmic splice variants, as well as alpha6A- and alpha6B-integrin subunits in HEK293 cells. Here we show that all three alpha7 splice variants (containing the X2 domain), as well as alpha6A and alpha6B, promote cell attachment and stimulate cell motility on laminin-1 and its E8 fragment. Deletion of the cytoplasmic domain (excluding the GFFKR consensus sequence) from alpha7B resulted in a loss of the motility-enhancing effect. On laminin-2/4 (merosin), the predominant isoform in mature skeletal muscle, only alpha7-expressing cells showed enhanced motility, whereas cells transfected with alpha6A and alpha6B neither attached nor migrated on laminin-2. Adhesion of alpha7-expressing cells to both laminin-1 and laminin-2 was specifically inhibited by a new monoclonal antibody (6A11) specific for alpha7. Expression of the two extracellular splice variants alpha7X1 and alpha7X2 in HEK293 cells conferred different motilities on laminin isoforms: Whereas alpha7X2B promoted cell migration on both laminin-1 and laminin-2, alpha7X1B supported motility only on laminin-2 and not on laminin-1, although both X1 and X2 splice variants revealed similar adhesion rates to laminin-1 and -2. Fluorescence-activated cell sorter analysis revealed a dramatic reduction of surface expression of alpha6-integrin subunits after alpha7A or -B transfection; also, surface expression of alpha1-, alpha3-, and alpha5-integrins was significantly reduced. These results demonstrate selective responses of alpha6- and alpha7-integrins and of the alpha7 splice variants to laminin-1 and -2 and indicate differential roles in laminin-controlled cell adhesion and migration.

[Current status of autologous chondrocyte transplantation]. / Aktueller Stellenwert der autologen Chondrozytentransplantation.

PURPOSE: Autogenous chondrocyte transplantation (ACT) is a promising but disputable new method for the treatment of full thickness hyaline cartilage defects. Neither long-term follow-up studies nor prospective randomised comparative clinical trials exist to measure the outcome. METHODS: Several bioengeneering companies have emerged (Genzyme, Verigen, Co.don, etc.) and commercialised this new method of ACT which has generated enormous interest. Each company uses its own (secret) protocol for culturing human cartilage cells. These protocols vary considerably from each other with regard to culture media, enzymes and other additives (e.g. antibiotics) used. However, no quality control requirements for the culturing process of cartilage cells have been proposed which the companies have to fulfill. At the time of surgery the treating surgeon has to assume (and hope) that the cultured cells are viable, sterile, active and potent but a control mechanism does not exist. RESULTS: Considering the enormous price for ACT which the patients and the insurance companies are asked to pay quality control standards should be developed with the following informations given: cell viability, grade of differentiation, cell morphology, secretion levels of essential matrix components (collagen type II, IX, X and XI, Aggrecan) and the total cell number. None of the above information is currently provided by any of the companies involved. CONCLUSION: The widespread clinical use of this technique cannot be recommended at this stage as the scientific proof for reproducibility of ACT has not been given so far. The operative technique of ACT is demanding and should only be used by surgeons having attended special practical training courses and workshops to minimise technical failure rates. Clinical results should be documented in a uniform standardised way using established outcome scores and modern diagnostic measures (e.g. intraoperative biomechanical testing of repair tissue). Further controlled clinical trials, experimental research and the establishment of quality control standards are required.

Immunohistochemical analysis of type-X-collagen expression in osteoarthritis of the hip joint.

Conflicting data have been reported on the spatial distribution of type X-collagen expression in osteoarthritis, and no concise data exist on a possible correlation between type X-collagen expression and clinical and radiological alterations. Well defined clinical and radiological data were compared with histopathological and immunohistochemical findings to investigate the expression of type-X collagen in osteoarthritis of the hip joint. Femoral heads were obtained in toto from 11 patients undergoing routine hip arthroplasty for femoral neck fractures (n = 3) or osteoarthritis (n = 8) and from 13 patients (age: 12 days to 69 years) without any evidence of hip-joint pathology. Whole coronal sections from the femoral head were decalcified for routine histology and immunohistochemical analysis with use of type-specific monoclonal antibodies to type-X collagen. Our results demonstrate that type-X collagen is consistently found in osteoarthritic cartilage and is absent from normal adult cartilage (including the region of calcified cartilage). Except for the occurrence of type-X collagen in the middle zone of articular cartilage in advanced stages of osteoarthritis, there is no specific change in the staining pattern or intensity for the collagen during osteoarthritis, particularly when the staining is related to clinical and radiological parameters. Hardly more than 20% of the extracellular matrix stained for type-X collagen; therefore, we suggest that, in most cases, this type of collagen may not play a direct biomechanical role in the weakening of osteoarthritic cartilage but rather may contribute indirectly to a disturbance of the disc biomechanics by altering matrix-molecule interaction. However, expression of type-X collagen may indicate a change in chondrocyte phenotype that consistently coincides with the formation of chondrocyte clusters, one of the first alterations in osteoarthritis visible on histologic examination.

Localization of type X collagen in the intervertebral disc of mature beagle dogs.

Type X collagen expression in intervertebral disc of young adult beagle dogs (n = 10) was studied. Type X collagen was immunostained mainly pericellularly in the central area of the vertebral endplate, but interterritorial staining there was also present. Annulus fibrosus and nucleus pulposus did not usually stain for type X collagen. However, immunostaining of nucleus pulposus for type X collagen with a simultaneous expression of collagen alpha1(X) mRNA was observed in one dog. A weak staining was observed in two other animals with a weak collagen alpha1(X) mRNA signal. In annulus fibrosus, lamellar staining was observed in two dogs. In three animals, type X collagen mRNAs were observed in the outer edge of the annulus fibrosus, but immunohistochemical staining did not always correlate with in situ hybridization signals. In conclusion, intervertebral disc type X collagen was mainly expressed in the cartilaginous endplate. In some apparently healthy animals there was type X collagen expression in the nucleus pulposus and also in the annulus fibrosus.

Variation with age in the pattern of type X collagen expression in normal and scoliotic human intervertebral discs.

The distribution and expression of type X collagen, a calcium-binding collagen, which is a marker of hypertrophic chondrocytes and thought to be involved in cartilage calcification, was examined in situ in nondegenerate (grade I or II) human discs taken at autopsy over a wide age range (fetal->80 years) and also in scoliotic discs removed at surgery. In the fetal vertebral column, type X collagen was strongly expressed in the hypertrophic chondrocytes of the endplate, but was not seen in other areas. In the cartilaginous endplate of adults, it was found over the whole age range examined, with intensity increasing with age. In the disc matrix itself, type X collagen was demonstrated around individual cells from all individuals older than 50 years, but not in any fetal or autopsy disc from individuals younger than 40 years. In scoliotic discs, however, focal type X collagen expression was seen in 3/8 patients younger than 40 years including one 12-year-old. No type X collagen was found in the outer annulus in any autopsy or scoliotic disc, supporting the idea that cells of the outer annulus are phenotypically distinct from cells of the inner annulus and the nucleus. Our results demonstrate for the first time that type X collagen is a possible gene product of the intervertebral disc cells and a potential biochemical component of the disc matrix. They indicate that with age or in scoliosis, some cells from the inner annulus or nucleus of the disc differentiate to the hypertrophic chondrocyte phenotype. This might be the initiating event for the abnormal calcification described in aged and scoliotic discs in other studies.

Type X collagen, a natural component of mouse articular cartilage: association with growth, aging, and osteoarthritis.

OBJECTIVE: To perform a systematic study on the production and deposition of type X collagen in developing, aging, and osteoarthritic (OA) mouse articular cartilage. METHODS: Immunohistochemistry was employed to define the distribution of type X collagen and Northern analyses to determine the messenger RNA levels as an indicator of the synthetic activity of the protein. RESULTS: Type X collagen was observed in the epiphyseal and articular cartilage of mouse knee joints throughout development and growth. Type X collagen deposition in the transitional zone of articular cartilage became evident toward cessation of growth, at the age of 2-3 months. The most intense staining for type X collagen was limited to the tidemark, the border between uncalcified and calcified cartilage. Northern analysis confirmed that the type X collagen gene is also transcribed by articular cartilage chondrocytes. Intense immunostaining was observed in the areas of OA lesions, specifically, at sites of osteophyte formation and surface fibrillation. Type X collagen deposition was also seen in degenerating menisci. CONCLUSION: This study demonstrates that type X collagen is a natural component of mouse articular cartilage throughout development, growth, and aging. This finding and the deposition of type X collagen at sites of OA lesions suggest that type X collagen may have a role in providing structural support for articular cartilage.

Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits.

OBJECTIVE: Durable healing of full-thickness articular cartilage defects has been considered for a long time as a highly desirable, but unlikely event to occur. In recent years, conflicting reports on the outcome of in vitro and in vivo studies on chondrocyte and cartilage grafting into animal and human joints have raised new arguments for and against controlled repair of articular cartilage following injury. Some of the problems result from insufficient characterization of implant and repair tissue, and from too short follow up phases. Here we describe a new approach to repair articular cartilage defects in rabbit knees by allografting chondrocytes cultured in agarose gels. DESIGN: The implants were monitored over 6-18 months and graded histologically, immunohistochemically, and electron microscopically, using a grading scale based on seven evaluation criteria. Control implants of pure agarose produced poor fibrous substitute tissue, insufficient healing and incomplete filling of the cartilage defects. After transplantation of allogenic chondrocytes embedded in agarose, the quality of the newly formed repair cartilage was superior with respect to type II collagen and proteoglycan content and cellular architecture when compared with untreated defects. Superficial fibrillation and degradation were significantly diminished or prevented. RESULTS: New subchondral bone formed at the level of the previous subchondral bone. In most cases the repair tissue merged with the host articular cartilage; normal calcified cartilage was the only tissue zone that did not participate in the integration of the transplant. By gross evaluation 24% of grafts showed an extent of recovery never observed in controls. The best results were obtained after 18 months when 47% of the grafts (N = 15) developed a morphologically stable hyaline cartilage. CONCLUSION: These studies demonstrate that agarose-embedded chondrocyte may prove a valuable tool for controlled repair of articular cartilage defects.