Adult Articular Cartilage Research Articles

Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis.

To study the changes in patterns of gene expression exhibited by human chondrocytes as they dedifferentiate into fibroblastic cells in culture in order to better understand the mechanisms that control this process and its relationship to the phenotypic changes that occur in chondrocytes during the development of osteoarthritis (OA). Human fetal epiphyseal chondrocytes (HFCs) were cultured either on poly-(2-hydroxyethyl methacrylate)-coated plates (differentiated HFC cultures) or in plastic tissue culture flasks as monolayers (dedifferentiated HFC cultures). Following 11 days of culture under either condition, poly(A+) RNA was isolated from the two cell populations and subjected to a gene expression analysis using a microarray containing approximately 5,000 known human genes and approximately 3,000 expressed sequence tags (ESTs). A > or =2-fold difference in the expression of 62 known genes and 6 ESTs was observed between the two cell types. The differences in expression of several of the genes detected by the microarray hybridization were confirmed by Northern analyses. Two transcription factor genes, TWIST and HIF-1alpha, and a cellular adhesion protein gene, cadherin 11, were markedly regulated in response to differentiation and dedifferentiation. Expression of these genes was also detected in adult normal and OA cartilage and chondrocytes. Analysis of the gene expression profile of HFCs revealed a complex pattern of gene expression, including many genes not yet reported to be expressed by chondrocytes. Chondrocytes in monolayer become dedifferentiated, acquiring a fibroblast-like appearance and changing their pattern of gene expression from one of expression of chondrocyte-specific genes to one that resembles a fibroblastic or chondroprogenitor-like pattern. Changes in gene expression associated with the process of dedifferentiation of HFCs in vitro were observed in a wide variety of genes, including genes encoding extracellular matrix proteins, transcription factors, and growth factors. At least 3 of the genes that were regulated in response to dedifferentiation were also found to be expressed in adult normal and OA articular cartilage and chondrocytes.

Scaffolds for tissue engineering of cartilage.

Articular cartilage lesions resulting from trauma or degenerative diseases are commonly encountered clinical problems. It is well-established that adult articular cartilage has limited regenerative capacity, and, although numerous treatment protocols are currently employed clinically, few approaches exist that are capable of consistently restoring long-term function to damaged articular cartilage. Tissue engineering strategies that focus on the use of three-dimensional scaffolds for repairing articular cartilage lesions offer many advantages over current treatment strategies. Appropriate design of biodegradable scaffold conduits (either preformed or injectable) allow for the delivery of reparative cells bioactive factors, or gene factors to the defect site in an organized manner. This review seeks to highlight pertinent design considerations and limitations related to the development, material selection, and processing of scaffolds for articular cartilage tissue engineering, evidenced over the last decade. In particular, considerations for novel repair strategies that use scaffolds in combination with controlled release of bioactive factors or gene therapy are discussed, as are scaffold criteria related to mechanical stimulation of cell-seeded constructs. Furthermore, the subsequent impact of current and future aspects of these multidisciplinary scaffold-based approaches related to in vitro and in vivo cartilage tissue engineering are reported herein.

Expression of collagen and aggrecan genes in normal and osteoarthritic murine knee joints

Objective The STR/ort mouse strain develops osteoarthritis (OA) of the medial tibial cartilage whilst CBA mice do not develop this disease. We investigated whether changes occur in the expression of genes encoding major extracellular matrix proteins in the connective tissue of the murine knee joint in OA.Design Expression of the genes encoding collagens II (Col2α1), X (Col10α1), α2(XI) (Col11α2) and aggrecan (Agc) was detected in skeletally mature and immature male mice of the CBA and STR/ort strains by in situ hybridization.ResultsCol2α1 was expressed by chondrocytes of the tibial and patella–femoral cartilage and by the meniscal cartilage in all young mice (4–9 weeks) but only in the patella–femoral cartilage in older mice of both strains (36–45 weeks). In contrast Col2α1 was expressed by growth plate chondrocytes of both species at all ages. Similarly, Col2α1 transcripts were detected in cruciate ligament cells in both strains at all ages. Col10α1 transcripts were detected in cruciate ligament cells in both strains at all ages. Col10α1 expression was evident in the hypertrophic chondrocytes in the growth plate of young CBA and STR mice, but was not active in these cells in mature animals. However,Col10α1 was transcribed in articular chondrocytes of the tibia, meniscal and patella–femoral cartilages of all ages, in normal and osteoarthritic mice. Transcripts were also present in ligament of some mature animals.Col11α2 followed a similar pattern of expression in CBA cartilages to Col2α1, being active in adult growth plate but generally inactive in adult articular cartilages. Young CBA and STR/ort mice expressedCol11α2 in articular cartilage and very strongly throughout the growth plate. Agc expression was detected in all articular cartilages at all ages in both strains. Interestingly, transcripts for all four genes were absent in tibial articular chondrocytes located close to osteoarthritic lesions in STR/ort mice, indicating that these cells are unable to synthesize matrix proteins. Adult STR/ort mice also showed evidence of tissue remodeling around the periphery of the knee joint. Cells in remodeling areas actively transcribed Col2α1, Col10α1,Col11α2 and Agc.Conclusion It is unlikely that OA develops in STR/ort mice because of failure to express major proteins in joint tissue. However, once lesions develop in articular cartilage neighbouring chondrocytes fail to express genes encoding several matrix proteins.

The C5 Domain of Col6A3 Is Cleaved Off from the Col6 Fibrils Immediately after Secretion

In articular cartilage, type VI collagen is concentrated in the pericellular matrix compartment. During protein synthesis and processing at least the α3(VI) chain undergoes significant posttranslational modification and cleavage. In this study, we investigated the processing of type VI collagen in articular cartilage. Immunostaining with a specific polyclonal antiserum against the C5 domain of α3(VI) showed strong cellular staining seen in nearly all chondrocytes of articular cartilage. Confocal laser-scanning microscopy and immunoelectron microscopy allowed localization of this staining mainly to the cytoplasm and the immediate pericellular matrix. Double-labeling experiments showed a narrow overlap of the C5 domain and the pericellular mature type VI collagen. Our results suggest that at least in human adult articular cartilage the C5 domain of α3(VI) collagen is synthesized and initially incorporated into the newly formed type VI collagen fibrils, but immediately after secretion is cut off and is not present in the mature pericellular type VI matrix of articular cartilage.

Crosslinking by advanced glycation end products increases the stiffness of the collagen network in human articular cartilage: a possible mechanism through which age is a risk factor for osteoarthritis.

Age is an important risk factor for osteoarthritis (OA). During aging, nonenzymatic glycation results in the accumulation of advanced glycation end products (AGEs) in cartilage collagen. We studied the effect of AGE crosslinking on the stiffness of the collagen network in human articular cartilage. To increase AGE levels, human adult articular cartilage was incubated with threose. The stiffness of the collagen network was measured as the instantaneous deformation (ID) of the cartilage and as the change in tensile stress in the collagen network as a function of hydration (osmotic stress technique). AGE levels in the collagen network were determined as: Nepsilon-(carboxy[m]ethyl)lysine, pentosidine, amino acid modification (loss of arginine and [hydroxy-]lysine), AGE fluorescence (360/460 nm), and digestibility by bacterial collagenase. Incubation of cartilage with threose resulted in a dose-dependent increase in AGEs and a concomitant decrease in ID (r = -0.81, P < 0.001; up to a 40% decrease at 200 mM threose), i.e., increased stiffness, which was confirmed by results from the osmotic stress technique. The decreased ID strongly correlated with AGE levels (e.g., AGE fluorescence r = -0.81, P < 0.0001). Coincubation with arginine or lysine (glycation inhibitors) attenuated the threose-induced decrease in ID (P < 0.05). Increasing cartilage AGE crosslinking by in vitro incubation with threose resulted in increased stiffness of the collagen network. Increased stiffness by AGE crosslinking may contribute to the age-related failure of the collagen network in human articular cartilage to resist damage. Thus, the age-related accumulation of AGE crosslinks presents a putative molecular mechanism whereby age is a predisposing factor for the development of OA.

Tissue transglutaminase localization and activity regulation in the extracellular matrix of articular cartilage

Tissue transglutaminase (tTG) catalyzes a Ca 2+-dependent transglutaminase (TGase) activity which cross-links proteins and stabilizes many tissues [C.S. Greenberg et al. FASEB J. 5 (1991) 3071]. Because cartilage is subjected to great stress in vivo, an enzyme that strengthens and stabilizes tissue could play an integral role in maintaining cartilage integrity. The purpose of this study was to determine if active tTG is present in the extracellular matrix (ECM) of adult human osteoarthritic articular cartilage. Using a TGase activity assay along with immunolabeling for tTG of cartilage sections, TGase activity and tTG immunoreactivity were localized in the ECM in cartilage sections, predominantly in the superficial layer. Previous in vitro studies have demonstrated that the Mg-GTP complex inhibits the TGase activity of tTG [T.S. Lai et al. J. Biol. Chem. 273 (1998) 1776]. To investigate the in situ regulation of the TGase activity of tTG, a TGase activity assay was done with a dose response of GTP, measuring incorporation of fluorescein cadaverine. TGase activity was inhibited by GTP in a similar manner as in vitro. These results not only confirm tTG presence in the ECM, but also indicate tTG as the major TGase activity of the ECM. Secondly, the study provides a possible mechanism by which extracellular tTG is regulated in vivo.

Elastic energy storage in human articular cartilage: estimation of the elastic modulus for type II collagen and changes associated with osteoarthritis.

The viscoelastic mechanical properties of normal and osteoarthritic articular were analyzed based on data reported by Kempson [in: Adult Articular Cartilage (1973)] and Silver et al. (Connect. Tissue Res., 2001b). Results of the analysis of tensile elastic stress-strain curves suggest that the elastic modulus of cartilage from the superficial zone is approximately 7.0 GPa parallel and 2.21 GPa perpendicular to the cleavage line pattern. Collagen fibril lengths in the superficial zone were found to be approximately 1265 microm parallel and 668 microm perpendicular to the cleavage line direction. The values for the elastic modulus and fibril lengths decreased with increased extent of osteoarthritis. The elastic modulus of type II collagen parallel to the cleavage line pattern in the superficial zone approaches that of type I collagen in tendon, suggesting that elastic energy storage occurs in the superficial zone due to the tensile pre-tension that exists in this region. Decreases in the elastic modulus associated with osteoarthritis reflect decreased ability of cartilage to store elastic energy, which leads to cartilage fibrillation and fissure formation. We hypothesize that under normal physiological conditions, collagen fibrils in cartilage function to store elastic energy associated with weight bearing and locomotion. Enzymatic cleavage of cartilage proteoglycans and collagen observed in osteoarthritis may lead to fibrillation and fissure formation as a result of impaired energy storage capability of cartilage.

Adenomatous polyposis coli (APC), beta-catenin, and cadherin are expressed in human bone and cartilage.

Members of the cadherin and catenin families are involved in chondrogenesis and catenin gene mutations have been detected in malignant tumours of bone. This study was undertaken to assess in detail expression of cadherin, beta-catenin and the associated tumour suppressor gene product APC in bone and cartilage at different stages of human skeletal maturity and in non-neoplastic and neoplastic osteoarticular disease. Immunohistochemical staining of formalin-fixed paraffin-embedded normal and osteoarthritic adult articular cartilage, fetal growth plate and a series of tumours of bone and cartilage was undertaken with a panel of antibodies against APC, beta-catenin, and pan-cadherin. This study demonstrated expression of APC, beta-catenin and cadherin in normal and diseased bone and cartilage. APC was present both in osteoblasts and osteoclasts but not in osteocytes. Although only weak APC staining of occasional growth plate hypertrophic chondrocytes and normal articular chondrocytes was seen, APC staining was increased in osteoarthritic articular cartilage. beta-catenin and pan-cadherin staining was strongly positive in osteoclasts and osteoblasts, with expression being lost when bone cells differentiated into osteocytes. Expression of APC, beta-catenin and pan-cadherin in bone tumours was similar to that of non-neoplastic adult tissues. These findings suggest previously unrecognized roles for APC in regulation of function of chondrocytes, osteoblasts and osteoclasts and support the view that catenin-cadherin interactions are important in regulation of bone cell activity. Abnormalities of expression or function of these molecules may be important in formation of bone tumours and their clinical behaviour.

Paracrine interactions of chondrocytes and macrophages in cartilage degradation: articular chondrocytes provide factors that activate macrophage-derived pro-gelatinase B (pro-MMP-9).

Cells of the monocyte/macrophage lineage are involved in the development of inflammatory joint diseases such as rheumatoid arthritis. This disease is characterized by cartilage degradation and synovial membrane inflammation with a progressive loss of joint function. The pathological processes are still not well understood. Therefore it would be interesting to develop a suitable experimental in vitro model system for defined studies of monocyte/macrophage and chondrocyte interactions at the molecular level. For that purpose we cocultured chondrocytes from adult human articular cartilage with human monocytes and macrophages for defined periods of time in agarose without addition of serum. We performed zymographic and western blot analysis of culture medium, completed by quantitative RT-PCR of each chondrocyte, monocyte and macrophage RNA, respectively. The reliability of the newly established coculture systems is confirmed by causing a clear decrease of intact aggrecan in the coculture medium plus concurrent appearance of additional smaller fragments and a reduction of chondrocyte aggrecan and collagen II gene expression in the presence of monocytes. In culture medium from cocultures we detected active forms of the matrix metalloproteinases MMP-1, MMP-3 and MMP-9 accompanied by induction of gene expression of MMP-1, membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in chondrocytes. No gene expression of MMP-9 was detectable in chondrocytes, the enzyme was solely expressed in monocytes and macrophages and was downregulated in the presence of chondrocytes. Our results suggest that MMP-9 protein in coculture medium originated from monocytes and macrophages but activation required chondrocyte-derived factors. Because addition of plasmin, a partial activator of pro-MMP-3 and pro-MMP-1, enhanced the activation of pro-MMP-9 and pro-MMP-1 in cocultures but not in monocultured macrophages, and the presence of MMP-3 inhibitor II prevented pro-MMP-9 activation, we assumed a stepwise activation process of pro-MMP-9 that is dependent on the presence of at least MMP-3 and possibly also MMP-1.

NG2/HMPG modulation of human articular chondrocyte adhesion to type VI collagen is lost in osteoarthritis.

NG2/human melanoma proteoglycan (HMPG) is a chondroitin sulphate proteoglycan (CSPG), expressed by chondrocytes in fetal and in normal and osteoarthritic (OA) adult articular cartilage. NG2/HMPG is a receptor for extracellular matrix proteins, including type VI collagen, and regulates beta1 integrin binding to fibronectin. This study was undertaken to identify whether NG2/HMPG had similar activities in human articular chondrocytes (HACs). Normal and OA adult HAC adhesion to fibronectin, type II or type VI collagen was assessed using a methylene blue assay. The requirement for integrins, NG2/HMPG, and integrin-associated signalling molecules was investigated using anti-beta1 integrin and anti-HMPG antibodies and pharmacological inhibitors of signalling molecules. The adhesion of normal and OA HACs to fibronectin, type II and type VI collagen was beta1 integrin-dependent. Normal HAC adhesion to type VI collagen was stimulated by anti-HMPG antibodies. This effect was inhibited by pertussis toxin. Anti-HMPG antibodies had no effect on OA chondrocyte adhesion to type VI collagen, or on normal and OA cell adhesion to fibronectin and type II collagen. The results show that NG2/HMPG modulates integrin-mediated interactions of normal HACs with type VI collagen. Loss of this activity may be of importance in the progression of osteoarthritis.

Static compression of articular cartilage can reduce solute diffusivity and partitioning: implications for the chondrocyte biological response

Chondrocytes depend upon solute transport within the avascular extracellular matrix of adult articular cartilage for many of their biological activities. Alterations to bioactive solute transport may, therefore, represent a mechanism by which cartilage compression is transduced into cellular metabolic responses. We investigated the effects of cartilage static compression on diffusivity and partitioning of a range of model solutes including dextrans of molecular weights 3 and 40 kDa, and tetramethylrhodamine (a 430 Da fluorophore). New fluorescence methods were developed for real-time visualization and measurement of transport within compressed cartilage explants. Experimental design allowed for multiple measurements on individual explants at different compression levels in order to minimize confounding influences of compositional variations. Results demonstrate that physiological levels of static compression may significantly decrease solute diffusivity and partitioning in cartilage. Effects of compression were most dramatic for the relatively high molecular weight solutes. For 40 kDa dextran, diffusivity decreased significantly ( p<0.01) between 8% and 23% compression, while partitioning of 3 and 40 kDa dextran decreased significantly ( p<0.01) between free-swelling conditions and 8% compression. Since diffusivity and partitioning can influence pericellular concentrations of bioactive solutes, these observations support a role for perturbations to solute transport in mediating the cartilage biological response to compression.

Articular cartilage and changes in Arthritis: Collagen of articular cartilage

The extracellular framework and two-thirds of the dry mass of adult articular cartilage are polymeric collagen. Type II collagen is the principal molecular component in mammals, but collagens III, VI, IX, X, XI, XII and XIV all contribute to the mature matrix. In developing cartilage, the core fibrillar network is a cross-linked copolymer of collagens II, IX and XI. The functions of collagens IX and XI in this heteropolymer are not yet fully defined but, evidently, they are critically important since mutations in COLIX and COLXI genes result in chondrodysplasia phenotypes that feature precocious osteoarthritis. Collagens XII and XIV are thought also to be bound to fibril surfaces but not covalently attached. Collagen VI polymerizes into its own type of filamentous network that has multiple adhesion domains for cells and other matrix components. Collagen X is normally restricted to the thin layer of calcified cartilage that interfaces articular cartilage with bone.

Gene expression by human articular chondrocytes cultured in alginate beads.

Culture of articular chondrocytes in alginate beads offers several advantages over culture in monolayer; cells retain their phenotype for 8 months or longer. Earlier studies of chondrocytes cultured in alginate concentrated on collagen and proteoglycan synthesis. However, gene expression by in situ hybridization (ISH) has not been investigated. The purposes of the present study on human chondrocytes were (a) to modify the ISH procedure for the alginate beads to examine the mRNA expression of alpha1 (II) procollagen, aggrecan, and two matrix metalloproteinases (MMP-3 and MMP-8) thought to be involved in cartilage matrix degradation, and (b) to compare expression in cultured chondrocytes with that in chondrocytes of intact human cartilage. The modifications made for ISH include the presence of CaCl2 and BaCl2 in the fixation and washing steps and exclusion of cetyl pyridinium chloride. By ISH we show that aggrecan, MMP-3, and MMP-8 are continuously expressed during 8 months of culture. The alpha1 (II) procollagen gene is expressed only during the first 2 months of culture and after 3 months its expression is undetectable, which is consistent with its absence in adult articular cartilage. By Western blotting, Type II collagen protein had been synthesized and deposited in both the cell-associated and further-removed matrix compartments at 7 and 14 days of culture. These data indicate that chondrocytes cultured in alginate beads could be preserved for immunohistochemistry and ISH and that culture of human chondrocytes in alginate beads may serve as a good model for studying cartilage-specific phenotype as well as factors that influence cartilage matrix turnover.

Chondrogenesis in cartilage repair is induced by members of the transforming growth factor-beta superfamily.

The authors recently reported on the principle of an intrinsic repair strategy for partial-thickness articular cartilage defects, which is based on the introduction of a biocompatible and biodegradable matrix loaded with a free chemotactic and mitogenic agent (transforming growth factor-beta 1, at low concentration) and a liposome-encapsulated chondrogenic factor (transforming growth factor-beta 1 at high concentration). In the current study, the potential of other members of the transforming growth factor-beta superfamily (transforming growth factor-beta 2, transforming growth factor-beta 3, bone morphogenetic protein-2 and bone morphogenetic protein-13), and of insulinlike growth factor-1, epidermal growth factor, transforming growth factor-alpha, and Tenascin-C, to induce chondrogenesis within the authors' adult miniature pig articular cartilage defect model, was evaluated. The degree of chondrogenic tissue differentiation was assessed 6 weeks after surgery, on a semiquantitative basis, histologic assessment of cell morphologic features, and intercellular matrix staining being used as the relevant criteria. All selected members of the transforming growth factor-beta superfamily were efficacious in inducing chondrogenic tissue transformation, whereas the other signaling substances tested were not. When encapsulated at high activity levels, bone morphogenetic proteins were less prone than transforming growth factor-beta 1, transforming growth factor-beta 2, and transforming growth factor-beta 3 to evoke undesired side effects as a result of incidental leakage into the joint cavities and subsynovial connective tissue spaces, and therefore they are potentially more suitable candidates for use in human patients.

Role of uppermost superficial surface layer of articular cartilage in the lubrication mechanism of joints

The uppermost superficial surface layer of articular cartilage, the 'lamina splendens' which provides a very low friction lubrication surface in articular joints, was investigated using atomic force microscopy (AFM). Complementary specimens were also observed under SEM at -10 degrees C without dehydration or sputter ion coating. Fresh adult pig osteochondral specimens were prepared from the patellas of pig knee joints and digested with the enzymes, hyaluronidase, chondroitinase ABC and alkaline protease. Friction coefficients between a pyrex glass plate and the osteochondral specimens digested by enzymes as well as natural (undigested) specimens were measured, using a thrust collar apparatus. Normal saline, hyaluronic acid (HA) and a mixture of albumin, globulin, HA (AGH) were used as lubrication media. The surface irregularities usually observed in SEM studies were not apparent under AFM. The articular cartilage surface was resistant to hyaluronidase and also to chondroitinase ABC, but a fibrous structure was exhibited in alkaline protease enzymes-digested specimens. AFM analysis revealed that the thickness of the uppermost superficial surface layer of articular cartilage was between 800 nm and 2 microm in adult pig articular cartilage. The coefficient of friction (c.f.) was significantly higher in chondroitinase ABC and alkaline protease enzymes digested specimens. Generally, in normal saline lubrication medium, c.f. was higher in comparison to HA and AGH lubrication media. The role of the uppermost, superficial surface layer of articular cartilage in the lubrication mechanism of joints is discussed.

Del1: A New Protein in the Superficial Layer of Articular Cartilage

Articular cartilage contains four distinct zones, extending from the surface to the subchondral bone. Freshly isolated chondrocytes from the superficial zone of articular cartilage retain a collagenase-P-resistant cell-associated matrix. In the studies described here, the protein Del1 was identified as a component of the cell-associated matrix of superficial zone chondrocytes from adult bovine articular cartilage. Very little Del1 was associated with freshly isolated deep zone chondrocytes. Western blot analysis of articular cartilage cell and tissue extracts using polyclonal antibodies specific for Del1 showed Del1 was present in an insoluble cell-associated fraction. Extracts of the superficial zone of articular cartilage were found to be enriched in Del1 compared to the deeper layers of the tissue. Immunohistochemical staining of full-thickness articular cartilage with anti-Del1 antibodies also showed an enrichment of Del1 in the superficial zone. These observations are the first to describe the protein Del1 in a nonendothelial, nonfetal tissue.

Preservation of Articular Cartilage for Electron Microscopy

Water makes up 60-80% of the wet weight of articular cartilage. The remaining parts of this tissue are made up of a variety of cells, generally referred to as chondrocytes, and an extracellular matrix of, collagen fibrils (types II, IX and XI), proteoglycans. The collagens and proteoglycans, with associated water, are assembled into a structure that is capable of withstanding considerable loading.The micro-structure of adult rabbit articular cartilage has been described, based upon ultrastructural studies. The tissue is arranged into zones, mainly based on the general orientation of collagen fibrils (See Figure 1). The collagen framework in the radial zone forms a continuous area of radially oriented and tightly bound collagen fibrils, in which are embedded at regular interval, tubular inclusions of proteoglycans (Figure 2). in rabbit tibial plateau tissue these inclusions have diameter of 1-3 fim and can be continuous from the calcified to the tangential zone.

Evidence of chondrocyte repopulation in adult ovine articular cartilage following cryoinjury and long-term transplantation

Objective To characterize the response of articular chondrocytes to a specific cryoinjury that leads to cluster formation following long-term transplantation.DesignOsteochondral dowels from 20 adult sheep were cryopreserved to optimize the recovery of chondrocytes immediately after thawing. The dowels were transplanted as allografts and observed at 3 and 12 months. Chondrocyte distribution and viability was assessed using paravital dyes after transplantation. Chondrocyte phenotype was assessed by in situ hybridization and immunohistochemistry to detect type II collagen. An anticentrosome antibody was used to identify cells undergoing cell cycle progression towards mitosis.Results All cryopreserved grafts showed the presence of spheroidal clusters of chondrocytes 1 year after transplantation while the host cartilage adjacent to the graft appeared morphologically normal. The average size of the clusters increased from four cells at 3 months to 12 cells at 1 year. The chondrocytes in the clusters displayed newly formed type II collagen protein and mRNA. Some cells within clusters were observed with two centrosomes, indicative of cells progressing through the S phase of the cell cycle.Conclusion Adult articular chondrocytes retain the ability to repopulate the matrix, an ability which is demonstrated with this specific cryoinjury. This may be an initial stage of cartilage regeneration.

Variations in site and levels of expression of chondrocyte nucleotide pyrophosphohydrolase with aging.

The aim of this study was to identify changes in cartilage intermediate layer protein/nucleotide pyrophosphohydrolase (CILP/NTPPH) expression in articular cartilage during aging. Adult (3-4 years old) and young (7-10 days old) porcine articular hyaline cartilage and fibrocartilage were studied by Northern blot analysis, in situ hybridization, and immunohistochemistry using a complementary DNA (cDNA) probe encoding porcine CILP/NTPPH and antibody to a synthetic peptide corresponding to a CILP/NTPPH sequence. Northern blot analysis of chondrocytes showed lower expression of CILP/NTPPH messenger RNA (mRNA) in young cartilage than in adult cartilage. In adult cartilage, extracellular matrix from the surface to the middeep zone was immunoreactive for CILP/NTPPH, especially in the pericellular matrix surrounding the middeep zone chondrocytes. In young cartilage, chondrocytes were moderately immunoreactive for CILP/NTPPH throughout all zones except the calcified zone. The matrix of young cartilage was negative except in the superficial zone. In young cartilage, CILP/NTPPH mRNA expression was undetectable. In adult cartilage, chondrocytes showed strong mRNA expression for CILP/NTPPH throughout middeep zones. Protein and mRNA signals were not detectable below the tidemark. CILP/NTPPH secretion into matrix around chondrocytes increases with aging. In this extracellular site it may generate inorganic pyrophosphate and contribute to age-related calcium pyrophosphate dihydrate crystal deposition disease.

Modulation of bovine articular chondrocyte gene expression in vitro by oxygen tension

Objective Adult articular cartilage is a physiologically hypoxic tissue with a proposed gradient of oxygen tension ranging from <10% oxygen at the cartilage surface to <1% in the deepest layers. This gradient may be disturbed during diseases of the joint, for example in rheumatoid arthritis when synovial fluid pO2falls. We investigated whether changes in oxygen tension modulate gene expression in articular chondrocytes.Design Bovine articular chondrocytes were cultured in alginate beads in medium maintained at <0.1, 5, 10 or 20% oxygen. A modified RNA arbitrarily primed polymerase chain reaction (RAP-PCR) technique was used to identify several genes whose mRNA abundance in articular chondrocytes was dependent upon oxygen tension. Northern hybridization slot blots were used to quantify changes in mRNA level relative to a housekeeping gene, β-actin.Results Genes found by RAP-PCR to undergo up-regulation in hypoxia included TIMP-1 and integrin-linked kinase. Collagen V mRNA levels were down-regulated in hypoxic chondrocytes. This led us to examine mRNA levels for various cytokines, matrix structural molecules and β1 integrin. Interleukin 1β, transforming growth factor β and connective tissue growth factor were all up-regulated by low oxygen tensions, as was β1 integrin. Collagen II (COL2A1) was down-regulated by hypoxia but aggrecan mRNA levels remained unchanged. The mRNA levels for GAPDH, the archetypal hypoxia responsive gene, were not modulated in articular chondrocytes by changes in oxygen tension.ConclusionsOxygen tension modulates the abundance of mRNAs encoding structural molecules, several cytokines, β1 integrin and integrin-linked kinase in articular chondrocytes. This may be important during disease progression. Chondrocytes are unusual in their response to hypoxia, presumably because they exist physiologically in a low oxygen environment.