Retinoic acid induced proteoglycan release and cartilage resorption in rat bone cultures are age dependent and inhibited by EDTA

Summary In explanted humeri of late fetal rats, retinoic acid was found to induce the release of proteoglycan followed by cartilage resorption. Tissue breakdown, which was demonstrated by losses of DNA, RNA, and protein, coincided with the appearance of necrotic cells. In control humeri chondrocytes were the main cell type, but in humeri treated for 4 days with retinoic acid the surviving cells were chondroblastlike. Sensitivity of proteoglycan release and tissue breakdown to retinoic acid decreased with age.The proteinase inhibitors cysteine, Trasylol, and soya and lima bean trypsin inhibitors did not antagonize the effects of retinoic acid. Phenylmethanesulfonyl fluoride suppressed cartilage resorption more effectively than proteoglycan release, while pepstatin merely suppressed cartilage resorption. The inhibition by EDTA of both the release of proteoglycan and cartilage resorption induced by retinoic acid was dose dependent. Zn²⁺ abolished these effects, whereas Mn²⁺ only relieved the release of proteoglycan induced by retinoic acid; this indicates that these two effects of retinoic acid are not necessarily linked.In view of our recent demonstration that the release of proteoglycan induced by retinoic acid requires RNA and protein synthesis, we suggest that the degradation of proteoglycans in response to retinoic acid is dependent upon continued synthesis of metalloproteinases.     

Retinoic Acid-Induced Cartilage Resorption

The addition of retinoic acid to fetal rat bones in culture induces the release of proteoglycans followed by cartilage resorption. In this system retinoic acid markedly suppressed ³H-leucine and ³H-mannose incorporation into acid-precipitable macromolecules, and specifically changed the ³H-leucine incorporation pattern as revealed by gel electrophoresis. Tunicamycin, which selectively inhibits glycosylation of the asparagine residues in proteins, prevented the cartilage cell degradation in response to retinoic acid. Inhibitors of DNA synthesis did not affect the retinoic acid-induced changes indicating that cell division was not required for the cartilage degradation processes induced by retinoic acid. In consideration of our previous and present demonstrations that retinoic acid-induced cartilage resorption required RNA, protein, and glycoprotein synthesis and specifically changed the protein synthesis pattern, we suggest that retinoic acid may exert its action by altering gene expression.     

Distribution of newly synthesized aggrecan in explant cultures of bovine cartilage treated with retinoic acid.

This paper describes temporal changes in the metabolism and distribution of newly synthesized aggrecan and the organization of the extracellular matrix when explant cultures of articular cartilage maintained in the presence of fetal calf serum were exposed to retinoic acid for varying periods of time. Explant cultures of articular cartilage were incubated with radiolabeled sulfate prior to exposure to retinoic acid. The radiolabeled and chemical aggrecan present in the tissue and appearing in the culture medium was studied kinetically. Changes in the localization of radiolabeled aggrecan within the extracellular matrix were monitored by autoradiography in relation to type VI collagen distribution in the extracellular matrix. In control cultures where tissue levels of aggrecan remain constant the newly synthesized aggrecan remained closely associated with the territorial matrix surrounding the chondrocytes. Exposure of cultures to retinoic acid for the duration of the experiment, resulted in the extensive loss of aggrecan from the tissue and the redistribution of the remaining radiolabeled aggrecan from the chondron and territorial matrix into the inter-territorial matrix. These changes preceded alterations in the organization of type VI collagen in the extracellular matrix that involved the remodeling of the chondron and the appearance of type VI collagen in the inter-territorial matrix; there was also evidence of chondrocyte proliferation and clustering. In cartilage explant cultures exposed to retinoic acid for 24 h there was no loss of aggrecan from the matrix but there was an extensive redistribution of the radiolabeled aggrecan into the inter-territorial matrix. This work shows that maintenance of the structure and organization of the extracellular matrix that comprises the chondron and pericellular microenvironment of chondrocytes in articular cartilage is important for the regulation of the distribution of newly synthesized aggrecan monomers within the tissue.     

The effect of retinoic acid on proteoglycan biosynthesis in bovine articular cartilage cultures

The addition of retinoic acid to adult bovine articular cartilage cultures produces a concentration-dependent decrease in both proteoglycan synthesis and the proteoglycan content of the tissue. Total protein synthesis was not affected by the presence of retinoic acid, indicating that the inhibition of proteoglycan synthesis was not due to cytotoxicity. The proteoglycans synthesized in the presence of retinoic acid were similar in hydrodynamic size, ability to form aggregates with hyaluronate, and glycosaminoglycan composition to those of control cultures. However, the presence of larger glycosaminoglycan chains suggests that the core protein was substituted with fewer but longer glycosaminoglycan chains. In cultures maintained with retinoic acid, a decreased ratio of the large proteoglycan was synthesized relative to the small proteoglycan compared to that measured in control cultures. In cultures maintained with retinoic acid for 1 day and then switched to medium with 20% (v/v) fetal calf serum, the rate of proteoglycan synthesis and hexuronate contents increased within 5 days to levels near those of control cultures. Within 2 days of switching to medium with 20% (v/v) fetal calf serum, the relative proportions of the proteoglycan species were similar to those produced in cultures maintained in medium with 20% (v/v) fetal calf serum throughout. The rate of proteoglycan synthesis by bovine articular cartilage cultures exhibited an exponential decay following exposure to retinoic acid, with estimated half-lives of 11.5 and 5.3 h for tissue previously maintained in medium alone or containing 20% (v/v) fetal calf serum, respectively. The addition of 1 mM benzyl beta-D-xyloside only partially reversed the retinoic acid-mediated inhibition of proteoglycan synthesis. This indicates that the inhibition of proteoglycan synthesis by retinoic acid was due to both a decreased availability of xylosylated core protein and a decreased capacity of the chondrocytes to synthesize chondroitin sulfate chains.     

Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures.

The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular cartilage explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular cartilage explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by explant cultures of articular cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular cartilage explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.     

The effect of retinoic acid on proteoglycan turnover in bovine articular cartilage cultures

This paper describes proteoglycan catabolism by adult bovine articular cartilage treated with retinoic acid as a means of stimulating the loss of this macromolecule from the extracellular matrix of cartilage. Addition of retinoic acid (10(-12)-10(-6) M) to adult bovine articular cartilage which had been labeled with [35S]sulfate for 6 h after 5 days in culture, resulted in a dose-dependent increase in the rate of loss of 35S-labeled proteoglycans from the matrix of the tissue. Concomitant with this loss was a decrease in the proteoglycan content of the tissue. Incubation of cultures treated with 1 microM retinoic acid, at 4 degrees C, or with 0.5 mM cycloheximide, resulted in a significant decrease in the rate of retinoic acid-induced loss of proteoglycans and demonstrated cellular involvement in this process. Analysis of the 35S-labeled proteoglycans remaining in the matrix showed that the percentage of radioactivity associated with the small proteoglycan species extracted from the matrix of articular cartilage explants labeled with [35S]sulfate after 5 days in culture was 15% and this increased to 22% in tissue maintained in medium alone. In tissue treated with 1 microM retinoic acid for 6 days, the percentage of radioactivity associated with the small proteoglycan was 58%. Approximately 93% of the 35S-labeled proteoglycans released into the medium of control and retinoic acid-treated cultures was recovered in high density fractions after CsCl gradient centrifugation and eluted on Sepharose CL-2B as a broad peak with a Kav of 0.30-0.37. Less than 17% of these proteoglycans was capable of aggregating with hyaluronate. These results indicate that in both control and retinoic acid-treated cultures the larger proteoglycan species is lost to the medium at a greater rate than the small proteoglycan species. The effect of retinoic acid on proteoglycan turnover was shown to be reversible. Cartilage cultures maintained with retinoic acid for 1 day then switched to medium with 20% (v/v) fetal calf serum for the remainder of the culture period exhibited decreased rates of loss of 35S-labeled proteoglycans from the matrix and increased tissue hexuronate contents to levels near those observed in tissue maintained in medium with 20% (v/v) fetal calf serum throughout. Furthermore, following switching to 20% (v/v) fetal calf serum, the relative proportions of the 35S-labeled proteoglycan species remaining in the matrix of these cultures were similar to those of control cultures.     

TIMP-3 inhibits aggrecanase-mediated glycosaminoglycan release from cartilage explants stimulated by catabolic factors

Aggrecanases are considered to play a key role in the destruction of articular cartilage during the progression of arthritis. Here we report that the N-terminal inhibitory domain of tissue inhibitor of metalloproteinases 3 (N-TIMP-3), but not TIMP-1 or TIMP-2, inhibits glycosaminoglycan release from bovine nasal and porcine articular cartilage explants stimulated with interleukin-1alpha or retinoic acid in a dose-dependent manner. This inhibition is due to the blocking of aggrecanase activity induced by the catabolic factors. Little apoptosis of primary porcine chondrocytes is observed at an effective concentration of N-TIMP-3. These results suggest that TIMP-3 may be a candidate agent for use against cartilage degradation.     

Short- and long-term exposure of articular cartilage to curcumin or quercetin inhibits aggrecan loss

The aim of this study was to determine if curcumin and quercetin inhibit induced aggrecan loss from bovine articular cartilage explants given that these polyphenols have been shown to suppress the expression of matrix-degrading enzymes. The kinetics of loss of ³⁵S-aggrecan and the loss of total aggrecan in cartilage explants maintained in catabolic medium containing either 1 μM retinoic acid or 50 ng/ml interleukin (IL)-1α were studied in the presence of either 1–25 μM curcumin or 10–50 μM quercetin. The reversibility of catabolism of ³⁵S-aggrecan was also studied in catabolically stimulated cultures treated with 25 μM curcumin or 50 μM quercetin for the initial 4–5 days of culture followed by 10–15 days of culture in catabolic medium in the absence of either polyphenol. Curcumin and quercetin suppressed ³⁵S-aggrecan and total aggrecan loss from the explants in a dose-dependent manner. When the exposure of explants to curcumin or quercetin was limited to the first 4–5 days of culture, the suppression of ³⁵S-aggrecan loss was maintained in the extended culture period when the tissue was stimulated with either retinoic acid or IL-1α. Quercetin suppressed IL-1α-stimulated expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4. Curcumin suppressed retinoic acid stimulated expression of ADAMTS-5, and both polyphenols suppressed basal expression of ADAMTS-5. The ability of curcumin and quercetin to protect cartilage from stimulated aggrecan loss and to maintain this protection posttreatment may, at least in part, be due to the suppression of gene expression of ADAMTS-4 and -5.     

Inhibition and reversion of chondrogenesis by retinoic acid in rat limb bud cell cultures

Summary Mesenchyme cells derived from embryonic rat limb buds cultured at high density differentiated into chondrocytes. The degree of chondrogenesis was assessed by alcian blue staining, a stain specific for cartilage matrix. The addition of retinoic acid on day 1 of culture inhibited chondrogenesis in a dose-dependent fashion. When retinoic acid was added to the cultures on day 5, the cartilage nodules, consisting of newly differentiated cartilage cells, disappeared during the following 6 days. Coinciding with this process the histochemically demonstrable alkaline phosphatase activity, localized in the internodular areas, also disappeared. This indicated that retinoic acid not only inhibited chondrogenesis but also induced resorption of cartilage cells and that at least two cell types were affected, the cartilage cells and the cells bearing alkaline phosphatase.Actinomycin D and cycloheximide, inhibitors of RNA and protein synthesis, suppressed the retinoic acid effect in day 5 limb bud cell cultures. This result indicated that the effect of retinoic acid required RNA and protein synthesis and is compatible with the view that vitamin A may act in a hormone-like way.     

Detection of cartilage matrix degradation by autofluorescence lifetime

Cartilage is a vital organ to maintain joint function. Upon arthritis, proteolytic enzymes initiate degradation of cartilage extracellular matrix (ECM) resulting in eventual loss of joint function. However, there are only limited ways of non-invasively monitoring early chemical changes in cartilage matrix. Here we report that the autofluorescence decay profiles of cartilage tissue are significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A compact multidimensional fluorometer coupled to a fibre-optic probe was developed for single point measurements of AFL and applied to cartilage that was treated with different proteinases. Upon treating cartilage with bacterial collagenase, trypsin or matrix metalloproteinase 1, a significant dose and time dependent decrease of AFL was observed. Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may contribute to future diagnosis of cartilage defects as well as monitoring the efficacy of anti-joint therapeutic agents.     

Chondrocyte-mediated depletion of articular cartilage proteoglycans in vitro.

The degradation of proteoglycan was examined in cultured slices of pig articular cartilage. Pig leucocyte catabolin (10 ng/ml) was used to stimulate the chondrocytes and induce a 4-fold increase in the rate of proteoglycan loss from the matrix for 4 days. Material in the medium of both control and depleted cultures was mostly a degradation product of the aggregating proteoglycan. It was recovered as a very large molecule slightly smaller than the monomers extracted with 4M-guanidinium chloride and lacked a functional hyaluronate binding region. The size and charge were consistent with a very limited cleavage or conformational change of the core protein near the hyaluronate binding region releasing the C-terminal portion of the molecule intact from the aggregate. The 'clipped' monomer diffuses very rapidly through the matrix into the medium. The amount of proteoglycan extracted with 4M-guanidinium chloride decreased during culture from both the controls and depleted cartilage, and the average size of the molecules initially remained the same. However, the proportion of molecules with a smaller average size increased with time and was predominant in explants that had lost more than 70% of their proteoglycan. All of this material was able to form aggregates when mixed with hyaluronate, and glycosaminoglycans were the same size and charge as normal, indicating either that the core protein had been cleaved in many places or that larger molecules were preferentially released. A large proportion of the easily extracted and non-extractable proteoglycan remained in the partially depleted cartilage and the molecules were the same size and charge as those found in the controls. There was no evidence of detectable glycosidase activity and only very limited sulphatase activity. A similar rate of breakdown and final distribution pattern was found for newly synthesized proteoglycan. Increased amounts of latent neutral metalloproteinases and acid proteinase activities were present in the medium of depleted cartilage. These were not thought to be involved in the breakdown of proteoglycan. Increased release of proteoglycan ceased within 24h of removal of the catabolin, indicating that the effect was reversible and persisted only while the stimulus was present.     

Distinguishing aggrecan loss from aggrecan proteolysis in ADAMTS-4 and ADAMTS-5 single and double deficient mice

Aggrecan loss from mouse cartilage is predominantly because of ADAMTS-5 activity; however, the relative contribution of other proteolytic and nonproteolytic processes to this loss is not clear. This is the first study to compare aggrecan loss with aggrecan processing in mice with single and double deletions of ADAMTS-4 and -5 activity (Deltacat). Cartilage explants harvested from single and double ADAMTS-4 and -5 Deltacat mice were cultured with or without interleukin (IL)-1alpha or retinoic acid and analyzed for (i) the kinetics of (35)S-labeled aggrecan loss, (ii) the pattern of (35)S-labeled aggrecan fragments released into the media and retained in the matrix, (iii) the pattern of total aggrecan fragments released into the media and retained in the matrix, and (iv) specific cleavage sites within the interglobular and chondroitin sulfate-2 domains. The loss of radiolabeled aggrecan from ADAMTS-4/-5 Deltacat cartilage was less than that from ADAMTS-4, ADAMTS-5, or wild-type cartilage under nonstimulated conditions. IL-1alpha and retinoic acid stimulated radiolabeled aggrecan loss from wild-type and ADAMTS-4 Deltacat cartilage, but there was little effect on ADAMTS-5 cartilage. Proteolysis of aggrecan contributed most to its loss in wild-type, ADAMTS-4, and ADAMTS-5 Deltacat cartilage explants. The pattern of proteolytic processing of aggrecan in these cultures was consistent with that occurring in cartilage pathologies. Retinoic acid, but not IL-1alpha, stimulated radiolabeled aggrecan loss from ADAMTS-4/-5 Deltacat cartilage explants. Even though there was a 300% increase in aggrecan loss from ADAMTS-4/-5 Deltacat cartilage stimulated with retinoic acid, the loss was not associated with aggrecanase cleavage but with the release of predominantly intact aggrecan consistent with the phenotype of the ADAMTS-4/-5 Deltacat mouse. Our results show that chondrocytes have additional mechanism for the turnover of aggrecan and that when proteolytic mechanisms are blocked by ablation of aggrecanase activity, nonproteolytic mechanisms compensate to maintain cartilage homeostasis.     

Chondrocytes isolated from tibial dyschondroplasia lesions and articular cartilage revert to a growth plate-like phenotype when cultured in vitro

We report here a comparative study of the development and behavior of chondrocytes isolated from normal growth plate tissue, tibial dyschondroplasic lesions, and from articular cartilage. The objective of these studies was to determine whether the properties exhibited by chondrocytes in dysplasic lesions or in articular cartilage were due to their cellular phenotype, their environment, or both. We had previously analyzed the electrolytes and amino acid levels in the extracellular fluid of avian growth plate chondrocytes. Using these data, we constructed a culture medium (DATP5) in which growth plate cells essentially recapitulate their normal behavior in vivo. Here, we used DATP5 to examine the behavior of chondrocytes isolated from lesions of tibial dyschondroplasia (TD). We found that once isolated from lesion and grown in this supportive medium, dysplasic chondrocytes behaved essentially like normal growth plate cells. These findings suggest that the cause of TD is local factors operating in vivo to prevent these cells from developing normally. With respect to articular chondrocytes, our data indicate that they more closely retain normal protein and proteoglycan synthesis when grown in serum-free media. These cells readily induced mineral formation in vitro, both in the presence and absence of serum. However, in serum-containing media, mineralization was significantly enhanced when the cells were exposed to retinoic acid (RA) or osteogenic protein-1 (OP-1). Our studies support previous work indicating the presence of autocrine factors produced by articular chondrocytes in vivo that prevent mineralization and preserve matrix integrity. The lack of inhibitory factors and the presence of supporting factors are likely reasons for the induction of mineralization by articular chondrocytes in vitro.     

Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis

A bovine cartilage explant system was used to evaluate the effects of injurious compression on chondrocyte apoptosis and matrix biochemical and biomechanical properties within intact cartilage. Disks of newborn bovine articular cartilage were compressed in vitro to various peak stress levels and chondrocyte apoptotic cell death, tissue biomechanical properties, tissue swelling, glycosaminoglycan loss, and nitrite levels were quantified. Chondrocyte apoptosis occurred at peak stresses as low as 4.5 MPa and increased with peak stress in a dose-dependent manner. This increase in apoptosis was maximal by 24 h after the termination of the loading protocol. At high peak stresses (>20 MPa), greater than 50% of cells apoptosed. When measured in uniaxial confined compression, the equilibrium and dynamic stiffness of explants decreased with the severity of injurious load, although this trend was not significant until 24-MPa peak stress. In contrast, the equilibrium and dynamic stiffness measured in radially unconfined compression decreased significantly after injurious stresses of 12 and 7 MPa, respectively. Together, these results suggested that injurious compression caused a degradation of the collagen fibril network in the 7- to 12-MPa range. Consistent with this hypothesis, injurious compression caused a dose-dependent increase in tissue swelling, significant by 13-MPa peak stress. Glycosaminoglycans were also released from the cartilage in a dose-dependent manner, significant by 6- to 13-MPa peak stress. Nitrite levels were significantly increased above controls at 20-MPa peak stress. Together, these data suggest that injurious compression can stimulate cell death as well as a range of biomechanical and biochemical alterations to the matrix and, possibly, chondrocyte nitric oxide expression. Interestingly, chondrocyte programmed cell death appears to take place at stresses lower than those required to stimulate cartilage matrix degradation and biomechanical changes. While chondrocyte apoptosis may therefore be one of the earliest responses to tissue injury, it is currently unclear whether this initial cellular response subsequently drives cartilage matrix degradation and changes in the biomechanical properties of the tissue.     

Recombinant human interleukin-1 stimulates human articular cartilage to undergo resorption and human chondrocytes to produce both tissue- and urokinase-type plasminogen activator

Cytokines capable of stimulating cartilage resorption have frequently been identified as 'interleukin-1 (IL-1)-like' peptides. In this study for the first time we have employed homogeneous recombinant IL-1 alpha and IL-1 beta in an all-human culture system to define the effects of IL-1 on articular cartilage and chondrocytes in culture. Recombinant IL-1 (10-100 U/ml) could stimulate cartilage resorption, although the maximum degree of tissue breakdown rarely reached the levels obtained when cartilage was treated with crude mononuclear-cell conditioned medium or all-trans retinoic acid (1 microM) over a similar time course. Levels of plasminogen activator (PA) activity, a neutral proteinase which may contribute to cartilage destruction in arthritis, increased markedly in the cartilage/chondrocyte culture supernatants and in the chondrocyte cell layers in response to the stimulation of cultures with recombinant IL-1 (1-100 U/ml). Elevated levels of PA activity were detectable after 4-8 h stimulation of the chondrocytes with IL-1 while characterization of the PA activities indicated that both types of PA activity were expressed, viz. urokinase-type PA (u-PA) and tissue-type PA (t-PA). Both IL-1 alpha and IL-1 beta could elicit these responses and their effects were comparable for a given dose. These studies show definitively that pure IL-1, free from contaminating cytokines, is capable of inducing human cartilage resorption and stimulating the expression of two types of PA activity by chondrocytes. In contrast to IL-1, retinoic acid increased the detectable levels of only u-PA in the chondrocyte cell layers. Chondrocyte u-PA may have an important role in cartilage degradative processes since it is one of the few neutral proteinases now known to be increased in activity in retinoid-stimulated cartilage.     

Fourier transform infrared imaging spectroscopy investigations in the pathogenesis and repair of cartilage

Significant complications in the management of osteoarthritis (OA) are the inability to identify early cartilage changes during the development of the disease, and the lack of techniques to evaluate the tissue response to therapeutic and tissue engineering interventions. In recent studies several spectroscopic parameters have been elucidated by Fourier transform infrared imaging spectroscopy (FT-IRIS) that enable evaluation of molecular and compositional changes in human cartilage with progressively severe OA, and in repair cartilage from animal models. FT-IRIS permits evaluation of early-stage matrix changes in the primary components of cartilage, collagen and proteoglycan on histological sections at a spatial resolution of ∼6.25 μm. In osteoarthritic cartilage, the collagen integrity, monitored by the ratio of peak areas at 1338 cm⁻¹/Amide II, was found to correspond to the histological Mankin grade, the gold standard scale utilized to evaluate cartilage degeneration. Apparent matrix degradation was observable in the deep zone of cartilage even in the early stages of OA. FT-IRIS studies also found that within the territorial matrix of the cartilage cells (chondrocytes), proteoglycan content increased with progression of cartilage degeneration while the collagen content remained the same, but the collagen integrity decreased. Regenerative (repair) tissue from microfracture treatment of an equine cartilage defect showed significant changes in collagen distribution and loss in proteoglycan content compared to the adjacent normal cartilage, with collagen fibrils demonstrating a random orientation in most of the repair tissue. These studies demonstrate that FT-IRIS is a powerful technique that can provide detailed ultrastructural information on heterogeneous tissues such as diseased cartilage and thus has great potential as a diagnostic modality for cartilage degradation and repair.     

Chondromodulin I and pleiotrophin gene expression in bovine cartilage and epiphysis.

Pleiotrophin and chondromodulin-I are low molecular weight proteins that are abundant (20 microg/g tissue) in fetal cartilage and difficult to detect in adult cartilage. We characterized their gene and protein expression patterns to gain a better understanding of their roles in the regulation of limb development and growth. In order to compare and contrast the relative amounts of the respective mRNA species within the developing epiphysis, a competitive PCR assay was developed. The results showed that the mRNAs for both proteins were abundant in fetal cartilage and while present in adult cartilage, were at 20-60-fold lower levels. Northern blotting revealed gradients of mRNA for both of these proteins in growth plate cartilage, with the highest levels in the resting zone, and the lowest in the hypertrophic zone. In contrast to pleiotrophin, chondromodulin-1 is down-regulated by retinoic acid with a pattern of expression similar to collagen type II and link protein, and may play a more specific role than pleiotrophin in modulating the chondrocyte phenotype.     

Fourier transform infrared imaging spectroscopy investigations in the pathogenesis and repair of cartilage

Significant complications in the management of osteoarthritis (OA) are the inability to identify early cartilage changes during the development of the disease, and the lack of techniques to evaluate the tissue response to therapeutic and tissue engineering interventions. In recent studies several spectroscopic parameters have been elucidated by Fourier transform infrared imaging spectroscopy (FT-IRIS) that enable evaluation of molecular and compositional changes in human cartilage with progressively severe OA, and in repair cartilage from animal models. FT-IRIS permits evaluation of early-stage matrix changes in the primary components of cartilage, collagen and proteoglycan on histological sections at a spatial resolution of approximately 6.25 microm. In osteoarthritic cartilage, the collagen integrity, monitored by the ratio of peak areas at 1338 cm(-1)/Amide II, was found to correspond to the histological Mankin grade, the gold standard scale utilized to evaluate cartilage degeneration. Apparent matrix degradation was observable in the deep zone of cartilage even in the early stages of OA. FT-IRIS studies also found that within the territorial matrix of the cartilage cells (chondrocytes), proteoglycan content increased with progression of cartilage degeneration while the collagen content remained the same, but the collagen integrity decreased. Regenerative (repair) tissue from microfracture treatment of an equine cartilage defect showed significant changes in collagen distribution and loss in proteoglycan content compared to the adjacent normal cartilage, with collagen fibrils demonstrating a random orientation in most of the repair tissue. These studies demonstrate that FT-IRIS is a powerful technique that can provide detailed ultrastructural information on heterogeneous tissues such as diseased cartilage and thus has great potential as a diagnostic modality for cartilage degradation and repair.     

Mechanisms involved in cartilage proteoglycan catabolism.

The increased catabolism of the cartilage proteoglycan aggrecan is a principal pathological process which leads to the degeneration of articular cartilage in arthritic joint diseases. The consequent loss of sulphated glycosaminoglycans, which are intrinsic components of the aggrecan molecule, compromises both the functional and structural integrity of the cartilage matrix and ultimately renders the tissue incapable of resisting the compressive loads applied during joint articulation. Over time, this process leads to irreversible cartilage erosion. In situ degradation of aggrecan is a proteolytic process involving cleavage at specific peptide bonds located within the core protein. The most well characterised enzymatic activities contributing to this process are engendered by zinc-dependent metalloproteinases. In vitro aggrecanolysis by matrix metalloproteinases (MMPs) has been widely studied; however, it is now well recognised that the principal proteinases responsible for aggrecan degradation in situ in articular cartilage are the aggrecanases, two recently identified isoforms of which are members of the 'A Disintegrin And Metalloproteinase with Thrombospondin motifs' (ADAMTS) gene family. In this review we have described: (i) the development of monoclonal antibody technologies to identify catabolic neoepitopes on aggrecan degradation products; (ii) the use of such neoepitope antibodies in studies designed to characterise and identify the enzymes responsible for cartilage aggrecan metabolism; (iii) the biochemical properties of soluble cartilage aggrecanase(s) and their differential expression in situ; and (iv) model culture systems for studying cartilage aggrecan catabolism. These studies have clearly established that 'aggrecanase(s)' is primarily responsible for the catabolism and loss of aggrecan from articular cartilage in the early stages of arthritic joint diseases that precede overt collagen catabolism and disruption of the tissue integrity. At later stages, when collagen catabolism is occurring, there is evidence for MMP-mediated degradation of the small proportion of aggrecan remaining in the tissue, but this occurs independently of continued aggrecanase activity. Furthermore, the catabolism of link proteins by MMPs is also initiated when overt collagen degradation is evident.     

A fully coupled poroelastic reactive-transport model of cartilage

Cartilage maintains its integrity in a hostile mechanical environment. This task is made more difficult because cartilage has no blood supply, and so nutrients and growth factors need to be transported greater distances than normal to reach cells several millimetres from the cartilage surface. The chondrocytes embedded within the extracellular matrix (ECM) are essential for maintaining the mechanical integrity of the ECM, through a balance of degradation and synthesis of collagen and proteoglycans. A chondrocyte senses various chemical and mechanical signals in its local microenvironment, responding by appropriate adaption of the local ECM. Clearly a 'systems understanding' of cartilage behaviour is of critical importance in developing an integrated understanding of both normal and abnormal physiology of cartilage. In a series of papers, we have developed a reactive-transport porous-media model to investigate the coupled processes of growth factor transport, mechanical deformation and fluid flow, and in this paper, we extend the model to include biosynthesis and degradation of matrix molecules. The model is validated using three independent experimental data sets, it being found that a single set of parameters described the experimental results remarkably well. The model is then employed to make predictions about changes in proteoglycan content under a variety of conditions. This model may prove useful in predicting the behaviour of tissue engineering constructs, or predicting the outcome of repair processes in cartilage.