Articular Cartilage Formation Research Articles

Regeneration of cartilage tissue by combination of canine chondrocyte and a hybrid mesh scaffold

Canine chondrocytes isolated from articular cartilage were cultured in a PLGA-collagen hybrid mesh in DMEM containing 10% FBS in vitro for 10 days, and subsequently implanted subcutaneously in the dorsum of nude mouse. The formation of neo-cartilage tissue was examined. HE staining showed a spherical morphology of chondrocytes in the center of the explants and a fibroblast-like morphology in the periphery of the explants. The safranin O/fast green staining showed that the central part was positively stained for GAG while the periphery was negatively stained. The toluidine blue staining showed a similar result. The histological results demonstrated spherical chondrocytes surrounded by abundant cartilaginous matrices in the center of the explants. The fibro-tissue in the periphery became denser and the central cartilage tissue became thinner as the implantation period increased, suggesting the effects such as invasion of host fibroblasts and diffusion of soluble factors on the degeneration of newly formed cartilage tissue. Gene expression study using reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that the expressions of type II collagen and aggrecan were detected and up-regulated, while that for type I collagen was down-regulated when the cells were cultured in the hybrid mesh. These results suggest the formation of articular cartilage in the subcutaneous dorsum of nude mouse.

Human monoclonal rheumatoid factors augment arthritis in mice by the activation of T cells.

In order to investigate the in vivo role of rheumatoid factor (RF), the effects of the administration of human monoclonal (m) IgM-RF and IgG-RF on the development of arthritis in mice were examined. The administration of human mRFs into mice immunized with type II collagen (CII) markedly enhanced the clinical score and paw swelling. The severity of arthritic joint disease with a marked infiltration of lymphoid cells, proliferation of synovial membrane, pannus formation and destruction of articular cartilage was significantly enhanced in both groups receiving RF (RF-enhanced arthritis). Skin ulcers were also observed in some of these RF-enhanced arthritis mice, whereas no such signs were observed in CII-immunized mice without mRFs. Both IgM-RF and IgG-RF increased CII-specific IgG antibodies in circulation, and the severity of arthritis correlated with the production of high titres of anti-CII antibodies. In vivo treatment of RF-enhanced arthritis mice with an anti-CD4 MoAb or an anti-CD8 MoAb inhibited the induction and progression of arthritis in these mice. Administration of RF to severe combined immunodeficient (SCID) mice with arthritis developed by the transfer of spleen cells from CII-immunized mice, prolonged the arthritis and enhanced the severity. This murine model of RF-enhanced arthritis may provide a useful tool for analysing the pathogenesis of rheumatoid arthritis in RF-positive patients.

Developmental expression patterns of Beta-ig (βIG-H3) and its function as a cell adhesion protein

Beta-ig is a secretory protein embodied by fasciclin I-like repeats containing sequences that might bind integrins and glycosaminoglycans in vivo. Expression of Beta-ig is responsive to Transforming Growth Factor-β and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating Beta-ig as an ECM adhesive protein of developmental processes. The spatiotemporal distribution of Beta-ig during various stages of murine development was examined and its ability to support adhesion of various cell types assessed. In situ hybridization of mouse embryos (E12.5–E18.5) indicated a prominent, distinct expression pattern for Beta-ig message in connective tissue. Beta-ig transcripts were abundantly expressed during mesenchymal cell condensation in areas of axial, craniofacial and appendicular primordial cartilage from E12.5–E14.5. Beginning at E15.5, Beta-ig transcripts appeared in collagen-rich tissues, including dura mater and corneal stroma. During E16.5–E18.5, Beta-ig transcripts were observed in proliferating chondrocytes and areas of endochondral ossification in joint and articular cartilage formation. Connective tissues expressed Beta-ig transcripts within the nasal septum and surrounding cartilage primordia, and in the pericardium, optic cup, kidney, ovary, esophagus, diaphragm, bronchi, trachea and corneal epithelium, and during cardiac valve formation. These patterns of expression indicate that Beta-ig may be involved in tissue morphogenesis. Cells derived from mesenchyme attached onto a substratum comprised of purified recombinant Beta-ig. Taken together, the results indicate that Beta-ig is expressed principally in collagen-rich tissues where it may interact with cells and ECM molecules, perhaps playing a role in tissue morphogenesis.

The distribution of Notch receptors and their ligands during articular cartilage development.

We examined the distribution of Notch family members and their ligands during the development of articular cartilage and the growth plate. Notch 1 was expressed by the chondrocytes of the developing articular surface but became increasingly restricted to the deeper layers after birth whilst expression of this family member was restricted to hypertrophic chondrocytes in the growth plate. Notch 2 and 4, Delta and Jagged 2 showed a broadly similar distribution, being present throughout the articular cartilage during development and becoming increasingly restricted to deeper layers with age. Hypertrophic chondrocytes within the growth plate also expressed Notch 2 and 4, Delta and Jagged 2 (which was also expressed in prehypertrophs). Notch 3 and Jagged 1 were absent from developing articular cartilage but were present in deeper layers at later time points (> 1 month) and both receptor and ligand were expressed in hypertrophic chondrocytes at all ages examined. These results highlight the complex Notch signalling interactions that result in the formation of the heterogeneous articular cartilage and allow for the co-ordinated ossification and elongation of the growth plate. Mechanisms by which these processes are controlled are discussed in light of recent advances in the understanding of Notch signalling pathways.

Developmentally regulated alternative splicing of the alpha1(XI) collagen chain: spatial and temporal segregation of isoforms in the cartilage of fetal rat long bones.

Type XI collagen is a component of the heterotypic collagen fibrils of fetal cartilage and is required to maintain the unusually thin diameter of these fibrils. The mature matrix form of the molecule retains an N-terminal variable region whose structure is modulated by alternative exon splicing that is tissue-specific and developmentally regulated. In the alpha1(XI) chain, antibodies to two of the peptides, p6b and p8, encoded by the alternatively spliced exons localized these epitopes to the surface of the collagen fibrils and were used to determine the pattern of isoform expression during the development of rat long bones (humerus). Expression of the p6b isoform was restricted to the periphery of the cartilage underlying the perichondrium of the diaphysis, a pattern that appears de novo at embryonic Day (E) 14. P8 isoforms appeared to be associated with early stages of chondrocyte differentiation and were detected throughout prechondrogenic mesenchyme and immature cartilage. After E16, p8 isoforms gradually disappeared from the diaphysis and then from the epiphysis preceding chondrocyte hypertrophy, but were highly evident at the periarticular joint surface, where ongoing chondrogenesis accompanies the formation of articular cartilage. The spatially restricted and differentiation-specific distribution of alpha1(XI) isoforms is evidence that Type XI collagen participates in skeletal development via a mechanism that may be distinct from regulation of fibrillogenesis.

Development of epiphyseal structure and function in Didelphis virginiana (Marsupiala, Didelphidae)

This study addressed the question of how the epiphyses of growing mammals change their external shape and internal architecture during postnatal development. Ontogenetic transformations in the external form and internal structure of the fore- and hindlimb epiphyses were examined in a mixed cross-sectional sample of Didelphis virginiana using two methods: morphometric analysis of linear epiphyseal dimensions and histological staining of serially sectioned epiphyses. Metric data indicate that Virginia opossums are born with relatively short hindlimbs and long forelimbs, but by the time they are weaned their hindlimbs are longer than their forelimbs. Functional integration of the locomotor system in D. virginiana involves a decoupling of fore- and hindlimb growth rates so that between birth and weaning, femoral length, diaphyseal cross-sectional area, and articular surface area increase at a significantly faster rate than the corresponding humeral dimensions. Histological results demonstrate that these differences in growth rate are reflected in morphology of the humeral and femoral growth plate and epiphyseal cartilages. The humeral cartilages exhibit a level of cellular organization characteristic of more mature limb elements at earlier developmental stages compared to the femoral cartilages, which assume this anisotropic structure relatively later in postnatal development. Results presented here also reveal that the formation of articular cartilage and the initiation of epiphyseal ossification in D. virginiana are both correlated with the development of independent positional behaviors prior to weaning. These histological data, therefore, suggest that mechanical loading associated with the postnatal onset of locomotor and postural development may provide an important stimulus for the progression of ossification and the formation of articular cartilage in the epiphyses of growing mammals. J. Morphol. 239:283–296, 1999. © 1999 Wiley-Liss, Inc.

Development of epiphyseal structure and function inDidelphis virginiana (Marsupiala, Didelphidae)

This study addressed the question of how the epiphyses of growing mammals change their external shape and internal architecture during postnatal development. Ontogenetic transformations in the external form and internal structure of the fore- and hindlimb epiphyses were examined in a mixed cross-sectional sample of Didelphis virginiana using two methods: morphometric analysis of linear epiphyseal dimensions and histological staining of serially sectioned epiphyses. Metric data indicate that Virginia opossums are born with relatively short hindlimbs and long forelimbs, but by the time they are weaned their hindlimbs are longer than their forelimbs. Functional integration of the locomotor system in D. virginiana involves a decoupling of fore- and hindlimb growth rates so that between birth and weaning, femoral length, diaphyseal cross-sectional area, and articular surface area increase at a significantly faster rate than the corresponding humeral dimensions. Histological results demonstrate that these differences in growth rate are reflected in morphology of the humeral and femoral growth plate and epiphyseal cartilages. The humeral cartilages exhibit a level of cellular organization characteristic of more mature limb elements at earlier developmental stages compared to the femoral cartilages, which assume this anisotropic structure relatively later in postnatal development. Results presented here also reveal that the formation of articular cartilage and the initiation of epiphyseal ossification in D. virginiana are both correlated with the development of independent positional behaviors prior to weaning. These histological data, therefore, suggest that mechanical loading associated with the postnatal onset of locomotor and postural development may provide an important stimulus for the progression of ossification and the formation of articular cartilage in the epiphyses of growing mammals. J. Morphol. 239:283-296, 1999. © 1999 Wiley-Liss, Inc.

Chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of rat tibial articular cartilage.

The present study was designed to investigate whether or not chondrocytes in articular cartilage express type I collagen in vivo under physiological conditions. Expressions of the gene and the phenotype of type I collagen were examined in rat tibial articular cartilage in the knee joint during development. Knee joints of Wistar rats at 1, 5, and 11 weeks postnatal were fixed in 4% paraformaldehyde with or without 0.5% glutaraldehyde and decalcified in 10% EDTA. After the specimens were embedded in paraffin and serial sections made, adjacent sections were processed for immunohistochemistry and in situ hybridization for type I collagen. The epiphysis of the tibia was composed of cartilage in week- 1 rats. Formation of articular cartilage was in progress in week 5 as endochondral ossification proceeded and was completed in week 11. Anti-type I collagen antibody stained only the superficial area of the epiphysis in week 1, but the immunoreactivity was expanded into the deeper region of the articular cartilage with development in weeks 5 and 11. Hybridization signals for pro-alpha 1 (I) collagen were seen in some of chondrocytes in the epiphysis of the week-1 tibia. The most intense signals were identified in chondrocytes in week 5 and the signals appeared weaker in week 11. The present study demonstrated that chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of the articular cartilage.

Distribution and expression of cartilage oligomeric matrix protein and bone sialoprotein show marked changes during rat femoral head development.

Distribution and sites of synthesis of a cartilage extracellular matrix protein, cartilage oligomeric matrix protein (COMP), and of a bone extracellular matrix protein, bone sialoprotein (BSP), were studied in the femoral head of growing Wistar rats from day 14 to day 60 by immunocytochemistry and in situ hybridization. This period includes formation of the secondary ossification center and differentiation of articular cartilage. At early stages, immunoreactivity for COMP was pronounced throughout the cartilage. The localization of COMP was predominantly territorial in the center of the immature femoral head and in the growth plate at all ages studied. In the superficial parts, a shift from a uniform extracellular matrix staining at day 14 to an interterritorial localization at day 33 to day 60 was seen, apparently concurrent with formation of articular cartilage. COMP staining, representing cartilage remnants, also extended into the center of the trabecular bone in the primary spongiosa. In the secondary ossification center, the staining for COMP decreased at the onset of calcification. The protein was only synthesized by chondrocytes, as shown by in situ hybridization. The highest level of COMP mRNA was detected in chondrocytes in the central region of the growth plate. In the layer corresponding to the articular cartilage of the femoral head, mRNA levels for COMP were low from day 14 to day 33 but were increased on day 60. This shows substantial synthesis in the developing articular cartilage. Immunoreactivity for BSP was detected in bone trabeculae of primary spongiosa. In situ hybridization showed the highest levels of BSP mRNA in regions of newly formed bone. BSP mRNA was detected in hypertrophic chondrocytes in the secondary ossification center as early as day 18, well before the appearance of immunochemically detectable BSP. Interestingly, simultaneous expression of COMP and BSP mRNA was seen after day 18 in hypertrophic chondrocytes of the growth plate and later also in hypertrophic chondrocytes close to the mineralization zone of the articular cartilage.

Atraumatic osteolysis of the distal clavicle: MR findings.

The purpose of this study was to describe the MRI appearance in atraumatic osteolysis of the distal clavicle (AODC). We retrospectively evaluated MRI, medical records, ancillary diagnostic imaging studies and clinical course in five men and two women (mean age, 39 years) in whom the final clinical diagnosis of AODC was established. None of the patients had significant shoulder injury, but all participated in activities involving repetitive strain of the acromioclavicular (AC) joint. In three of these patients, we performed follow-up MRI (ranging from 5 1/2 to 15 months after the initial MRI). In all seven patients, signal intensity changes within the intramedullary portion of the distal clavicle on MRI were consistent with diffuse bone marrow edema. Marrow edema was most conspicuous on STIR imaging and occasionally could be misinterpreted as normal marrow signal patterns on spin-echo imaging. Cortical thinning or irregularity of the distal clavicle was seen in six cases and tiny subchondral cysts were seen in three, corresponding to subtle cystic changes on shoulder radiography. Limited bone scans obtained in two patients showed markedly increased uptake of radiotracer at the distal clavicle and AC joint. Histologic examination in one case showed disruption of articular cartilage, subchondral cysts, and metaplastic bone formation with increased osteoclastic activity. Follow-up MRI in three patients who were asymptomatic following conservative therapy showed normalization of marrow signal intensity. Atraumatic osteolysis of the distal clavicle is a relatively uncommon but important cause of shoulder pain. Particularly when the clinical history is suggestive of repetitive AC joint stress, MRI of the distal clavicle should be examined closely for marrow edema, cortical irregularity, and cystic changes. Such abnormalities may be especially conspicuous when STIR imaging techniques are used.

Insulin-like growth factor I accelerates recovery of articular cartilage proteoglycan synthesis in culture after inhibition by interleukin 1.

Interleukin 1 (IL-1) is a cytokine which induces cartilage proteoglycan (PG) depletion by inhibiting PG synthesis and increasing PG breakdown. Insulin-like growth factor I (IGF-I), in contrast, is known to promote matrix formation. We examined the effects of both mediators in a bovine tissue culture model. IL-1 dose-dependently inhibited PG formation of articular cartilage [half-maximal effect (EC50) at 4 ng/ml], while PG synthesis was increased by IGF-I (EC50 = 15 ng/ml). After inhibition of PG formation with IL-1 for 2 days and subsequent removal of free IL-1, addition of IGF-I dose-dependently accelerated restoration of the original rate of synthesis with a half-maximal effect at 20 ng/ml and a maximal effect at 50 ng/ml. The IGF-I concentration required to elicit a half-maximal effect on cartilage PG synthesis remained constant in the absence or presence of IL-1. We therefore conclude that inhibition of cartilage PG synthesis by IL-1 is not effected by damage to the IGF receptor. Synovial fluid (SF) of 40 patients with rheumatoid arthritis (RA) was found to contain 64 +/- 6 ng IGF-I/ml (mean +/- SEM). The reported effects of IGF-I in vitro therefore occurred at concentrations comparable to those present in joints in vivo. IL-1 beta was detectable (> 0.5 pg/ml) in 38 of 40 RA-SF samples (mean 28 +/- 6 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

The role of mechanical loading histories in the development of diarthrodial joints.

The possible role of mechanical loading history in chondroosseous development at the ends of long bones is explored using two-dimensional finite element models of chondroepiphyses. Loading histories are characterized in terms of discrete loading cases defined by joint contact pressure distributions and an associated number of loading cycles. An osteogenic stimulus throughout the chondroepiphyses is calculated following the theory that cyclic octahedral shear stresses promote endochondral ossification and cyclic compressive dilatational stresses inhibit ossification. The resulting distributions for the osteogenic stimulus predict the appearance of the secondary ossific nucleus and the shape of the developing bony epiphysis. The zone of Ranvier and the formation of articular cartilage and the growth plate are also predicted by the models. These findings are consistent with the hypothesis that tissue stress histories constitute an important influence during skeletal morphogenesis. Further study and testing of the concepts introduced in this study are appropriate.

In vivo Requirement for Silicon in Articular Cartilage and Connective Tissue Formation in the Chick

Studies were undertaken to determine further effects of silicon deficiency in the chick. The diet and experimental conditions were the same as those used in previous studies to demonstrate the essentiality of silicon for growth and development. Skeletal and other abnormalities involving glycosaminoglycans in formation of articular cartilage and comb connective tissue were found to be associated with silicon deficiency. The bones of 1 day-old deutectomized cockerels fed a silicon supplemented diet and killed at 4 weeks of age had significantly greater amounts of articular cartilage and water as compared with the silicon deficient group and also a greater proportion of hexosamine in the cartilage. The greater water content in bones of the silicon supplemented chicks coincided with a larger content of glycosaminoglycans in the articular cartilage. A similar relationship was obtained in cockerel comb. In addition to larger amounts of connective tissue and of total hexosamine in combs of the supplemented group, a higher percentage of hexosamine and a higher silicon content was found. These findings provide the first evidence for a requirement for silicon in articular cartilage and connective tissue formation and that the site of action of silicon is in the glycosaminoglycan-protein complexes of the ground substance.