Biochemical analysis of normal and osteoarthritic human cartilage

Non-collagenous proteins from the articular cartilage of normal subjects and patients with degenerative joint disease were extracted sequentially. Proteoglycans and the other glycoproteins were more extractable from the osteoarthritic cartilage at lower ionic strength than those from the normal cartilage. A 50-kD protein which seems specific to osteoarthritic cartilage was identified. Three different populations of proteoglycans were purified from normal and only two from osteoarthritic cartilage. Moreover, greater amounts of albumin and fibronectin were found in the pathological cartilage. No differences were observed between link proteins from normal and osteoarthritic cartilage, nor in their molecular weight or the amounts extracted.     

Changes in the chondroitin sulfate-rich region of articular cartilage proteoglycans in experimental osteoarthritis

The chondroitin sulfate-rich region was cleaved from cartilage proteoglycans of experimental osteoarthritic canine joints to establish whether changes in this region of the molecule contribute to the well-documented increase in the chondroitin sulfate to keratan sulfate ratio in osteoarthritis. Experimental osteoarthritis was induced in eight dogs by severance of the right anterior cruciate ligament, the left joint serving as a control. Proteoglycans were extracted from the femoral cartilage of both joints, isolated as A1 fractions by associative density gradient centrifugation and cleaved with hydroxylamine. The chondroitin sulfate-rich region was isolated by either gel chromatography or dissociative density gradient centrifugation. The chondroitin sulfate-rich region from the proteoglycans of the experimental osteoarthritic joints was slightly larger in hydrodynamic size and had both a higher uronate/protein weight ratio and galactosamine/glucosamine molar ratio than the corresponding control. We conclude that the chondroitin sulfate-rich region of proteoglycans in articular cartilage of experimental osteoarthritic joints is larger and has more chondroitin sulfate than that of proteoglycans of normal cartilage.     

Nonenzymatic glycation of cartilage proteoglycans: an in vivo and in vitro study

In this study we have investigated whether proteoglycans (aggrecan) are modified by nonenzymatic glycation as in collagen. Purified human aggrecan from osteoarthritic and normal human knee articular cartilage was assayed for pentosidine, a cross-link formed by nonenzymatic glycation, using reverse-phase HPLC. In addition, an in vitro study was done by incubation of purified bovine nasal cartilage aggrecan with ribose. Pentosidine was found in all the purified human aggrecan samples. 2-3% of the total articular cartilage pentosidine was found in aggrecan. Purified link protein also contained penosidine. The in vitro study led to pentosidine formation, but did not appear to increase the molecular size of the aggrecan suggesting that pentosidine was creating intramolecular cross-links. Similar amounts of glycation were found in osteoarthritic and normal cartilage. Like collagen, aggrecan and link proteins are crosslinked by nonenzymatic glycation in normal and osteoarthritic cartilage. Crosslinking could be reproduced, in vitro, by incubating aggrecan with ribose. This revised version was published online in August 2006 with corrections to the Cover Date.     

Dibutyryl cyclic AMP affects hyaluronate synthesis and macromolecular organization in normal adult articular cartilage in vitro

When normal adult dog articular cartilage was cultured in the presence of dibutyryl cyclic AMP a higher proportion than normal of newly synthesized 35S-labeled glycosaminoglycans was released from the tissue into the culture medium, although their net synthesis was not affected. In conjunction with this release of sulfated glycosaminoglycans, 24 times more [3H]glucosamine-labeled hyaluronic acid was released from the cartilage into the medium, and net hyaluronate synthesis was enhanced 3-fold. Virtually all of the newly synthesized hyaluronic acid in the medium was associated with proteoglycans. The proteoglycans in the medium of the dibutyryl cyclic AMP treated cultures were normal in hydrodynamic size and interacted normally with hyaluronic acid to form large aggregates. These results suggest that the increase in hyaluronate synthesis caused by dibutyryl cyclic AMP mayt have destabilized the interaction of proteoglycans with the collagen meshwork of the cartilage. The changes seen in normal adult articular cartilage after incubation with dibutyryl cyclic AMP, therefore, are similar to those which are observed in cartilage of osteoarthritic joints.     

Alterations in the mechanical properties of the human chondrocyte pericellular matrix with osteoarthritis

In articular cartilage, chondrocytes are surrounded by a pericellular matrix (PCM), which together with the chondrocyte have been termed the "chondron." While the precise function of the PCM is not know there has been considerable speculation that it plays a role in regulating the biomechanical environment of the chondrocyte. In this study, we measured the Young's modulus of the PCM from normal and osteoarthritic cartilage using the micropipette aspiration technique, coupled with a newly developed axisymmetric elastic layered half-space model of the experimental configuration. Viable, intact chondrons were extracted from human articular cartilage using a new microaspiration-based isolation technique. In normal cartilage, the Young's modulus of the PCM was similar in chondrons isolated from the surface zone (68.9 +/- 18.9 kPa) as compared to the middle and deep layers (62.0 +/- 30.5 kPa). However, the mean Young's modulus of the PCM (pooled for the two zones) was significantly decreased in osteoarthritic cartilage (66.5 +/- 23.3 kPa versus 41.3 +/- 21.1 kPa, p < 0.001). In combination with previous theoretical models of cell-matrix interactions in cartilage, these findings suggest that the PCM has an important influence on the stress-strain environment of the chondrocyte that potentially varies with depth from the cartilage surface. Furthermore, the significant loss of PCM stiffness that was observed in osteoarthritic cartilage may affect the magnitude and distribution of biomechanical signals perceived by the chondrocytes.     

Alterations in the Young's modulus and volumetric properties of chondrocytes isolated from normal and osteoarthritic human cartilage

The mechanical environment of the chondrocyte is an important factor that influences the maintenance of the articular cartilage extracellular matrix. Previous studies have utilized theoretical models of chondrocytes within articular cartilage to predict the stress-strain and fluid flow environments around the cell, but little is currently known regarding the cellular properties which are required for implementation of these models. The objectives of this study were to characterize the mechanical behavior of primary human chondrocytes and to determine the Young's modulus of chondrocytes from non-osteoarthritic ('normal') and osteoarthritic cartilage. A second goal was to quantify changes in the volume of isolated chondrocytes in response to mechanical deformation. The micropipette aspiration technique was used to measure the deformation of a single chondrocyte into a glass micropipette in response to a prescribed pressure. The results of this study indicate that the human chondrocyte behaves as a viscoelastic solid. No differences were found between the Young's moduli of normal (0.65+/-0.63 kPa, n = 44) and osteoarthritic chondrocytes (0.67+/-0.86 kPa, n = 69, p = 0.93). A significant difference in cell volume was observed immediately and 600 s after complete aspiration of the cell into the pipette (p < 0.001), and the magnitude of this volume change between normal (11+/-11%, n = 40) and osteoarthritic (20+/-11%, n = 41) chondroctyes was significantly different at both time points (p < 0.002). This finding suggests that chondrocytes from osteoarthritic cartilage may have altered volume regulation capabilities in response to mechanical deformation. The mechanical and volumetric properties determined in this study will be of use in analytical and finite element models of chondrocyte-matrix interactions in order to better predict the mechanical environment of the cell in vivo.     

Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues

Introduction

The small leucine-rich proteoglycans (SLRPs) modulate tissue organization, cellular proliferation, matrix adhesion, growth factor and cytokine responses, and sterically protect the surface of collagen type I and II fibrils from proteolysis. Catabolism of SLRPs has important consequences for the integrity of articular cartilage and meniscus by interfering with their tissue homeostatic functions.

Methods

SLRPs were dissociatively extracted from articular cartilage from total knee and hip replacements, menisci from total knee replacements, macroscopically normal and fibrillated knee articular cartilage from mature age-matched donors, and normal young articular cartilage. The tissue extracts were digested with chondroitinase ABC and keratanase-I before identification of SLRP core protein species by Western blotting using antibodies to the carboxyl-termini of the SLRPs.

Results

Multiple core-protein species were detected for all of the SLRPs (except fibromodulin) in the degenerate osteoarthritic articular cartilage and menisci. Fibromodulin had markedly less fragments detected with the carboxyl-terminal antibody compared with other SLRPs. There were fewer SLRP catabolites in osteoarthritic hip than in knee articular cartilage. Fragmentation of all SLRPs in normal age-matched, nonfibrillated knee articular cartilage was less than in fibrillated articular cartilage from the same knee joint or total knee replacement articular cartilage specimens of similar age. There was little fragmentation of SLRPs in normal control knee articular cartilage. Only decorin exhibited a consistent increase in fragmentation in menisci in association with osteoarthritis. There were no fragments of decorin, biglycan, lumican, or keratocan that were unique to any tissue. A single fibromodulin fragment was detected in osteoarthritic articular cartilage but not meniscus. All SLRPs showed a modest age-related increase in fragmentation in knee articular and meniscal cartilage but not in other tissues.

Conclusion

Enhanced fragmentation of SLRPs is evident in degenerate articular cartilage and meniscus. Specific decorin and fibromodulin core protein fragments in degenerate meniscus and/or human articular cartilage may be of value as biomarkers of disease. Once the enzymes responsible for their generation have been identified, further research may identify them as therapeutic targets.     

Proteoglycans synthesized by chondrocytes of human nonarthritic and osteoarthritic cartilage

Chondrocyte cultures were developed from the cell outgrowths of explanted human nonarthritic and osteoarthritic human cartilage. Two significant differences in sulfated proteoglycan synthesis were demonstrated between the chondrocytes obtained in this manner. With 35SO4 to measure newly synthesized proteoglycan, we found that chondrocytes derived from osteoarthritic cartilage secreted significantly less (P less than 0.05) high density proteoglycan into the culture medium than did chondrocytes from nonarthritic cartilage after 20 hr of radiolabeling. This reduced amount of high density proteoglycan was sustained when chondrocytes were maintained in unlabeled culture medium ("chase" medium). In addition, the osteoarthritic chondrocytes secreted an increased amount of low density proteoglycan when compared with their nonarthritic counterparts. The elution profile of secreted high density proteoglycan isolated from the osteoarthritic chondrocyte culture medium was assessed by gel filtration on Sepharose CL-2B and revealed the presence of two proteoglycan subpopulations (Kav, 0.25, 0.58), whereas only one proteoglycan series (Kav, 0.37) was seen in the high density fraction of nonarthritic chondrocyte culture medium. Similar gel filtration profiles were also obtained when chondrocytes were maintained in chase medium. The results of this study demonstrated that stable differences in proteoglycan synthesis, but not in intracellular processing, exist between nonarthritic and osteoarthritic chondrocytes. The findings are noteworthy in that these differences were not previously apparent when organ-cultured cartilage was used to assess putative alterations in proteoglycans between the two groups.     

Expression patterns of Notch receptors and their ligands in human osteoarthritic and healthy articular cartilage

Notch pathway plays a pivotal role in cell fate determination. There is much interest surrounding its therapeutic potential, in osteoarthritis, but the expression profile of Notch-related molecules, as well as their relation with cartilage pathological parameters, remains unclear. The purpose of our study is to analyze the expression pattern of Notch family members, type II and type I collagen, in normal (healthy) and osteoarthritic human knee cartilage. Osteoarthritic cartilages were obtained from 3 patients undergoing a total knee replacement. Macroscopically normal cartilage was dissected from 3 human knees at the time of autopsy or surgery. Immunohistochemical staining was performed using Notch1,2,3 and 4, Delta, Jagged, type II collagen and type I collagen antibodies. In healthy cartilage, type II collagen was abundantly expressed while type I was absent. This latter increased proportionally to the osteoarthritic grade. Type II collagen expression remained intense in osteoarthritic cartilage. In healthy cartilage as well as in cartilage with minor lesions, Notch family member's proteins were not or just weakly expressed at the surface and in the cells. However, Notch molecules were over-expressed in osteoarthritic cartilage compared to healthy one. This expression pattern was different according to the cartilage zone and the severity of OA. Our data suggest that Notch signaling is activated in osteoarthritic cartilage, compared to healthy cartilage, with a much more abundant expression in the most damaged areas.     

Comparative analysis of collagens solubilized from human foetal, and normal and osteoarthritic adult articular cartilage, with emphasis on type VI collagen

The different collagen types were extracted sequentially, by 4 M guanidinium chloride and pepsin, from human foetal and normal and osteoarthritic adult articular cartilage. They were characterized by electrophoresis and immunoblotting. Most of the collagenous proteins present in articular cartilage from young human foetuses were solubilized: almost 40% of the total collagen was extracted in the native form with 4 M guanidinium chloride. Type VI collagen was detected in this fraction as high-molecular-mass chains (185-220 kDa) and a low-molecular-mass chain (140 kDa). Type II, IX and XI collagens were also present, but were extracted more extensively by pepsin digestion. Comparative analysis of normal and osteoarthritic cartilage from adults reveals some major differences: an increase in the solubility of the collagen and modifications of soluble collagen types in osteoarthritic cartilage. Furthermore, type VI collagen was present at a higher concentration in guanidinium chloride extracts of osteoarthritic cartilage than those of normal tissue. This finding was corroborated by electron microscopic observations of the same samples: abundant (100 nm) periodic fibrils were observed in the disorganized pericellular capsule of cloned cells in osteoarthritic cartilage. In normal tissues the pericellular zone was more compact and contained only a few such banded fibrils. The differences in the collagen types solubilized from normal and osteoarthritic cartilage, although corresponding to a minor proportion of the total collagen, demonstrate that important modifications in chondrocyte metabolism and in the collagenous network do occur in degenerated cartilage.     

Passive loss of proteoglycan from articular cartilage explants

The addition of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 0.25 mM benzamidine hydrochloride, 6.25 mM EDTA, 12.5 mM 6-aminohexanoic acid and 2 mM iodoacetic acid) to explant cultures of adult bovine articular cartilage inhibits proteoglycan synthesis as well as the loss of the macromolecule from the tissue. Those proteoglycans lost to the medium of explant cultures treated with proteinase inhibitors were either aggregates or monomers with functional hyaluronic acid-binding regions, whereas proteoglycans lost from metabolically active tissue also included a population of monomers that were unable to aggregate with hyaluronate. Analysis of the core protein from proteoglycans lost into the medium of inhibitor-treated cultures showed the same size distribution as the core proteins of proteoglycans present in the extracellular matrix of metabolically active cultures. The core proteins of proteoglycans appearing in the medium of metabolically active cultures showed that proteolytic cleavage of these macromolecules occurred as a result of their loss from the tissue. Explant cultures of articular cartilage maintained in medium with proteinase inhibitors were used to investigate the passive loss of proteoglycan from the tissue. The rate of passive loss of proteoglycan from the tissue was dependent on surface area, but no difference in the proportion of proteoglycan aggregate to monomer appearing in the medium was observed. Furthermore, proteoglycans were lost at the same rate from the articular and cut surfaces of cartilage. Proteoglycan aggregates and monomer were lost from articular cartilage over a period of time, which indicates that proteoglycans are free to move through the extracellular matrix of cartilage. The movement of proteoglycans out of the tissue was shown to be temperature dependent, but was different from the change of the viscosity of water with temperature, which indicates that the loss of proteoglycan was not solely due to diffusion. The activation energy for the loss of proteoglycans from articular cartilage was found to be similar to the binding energies for electrostatic and hydrogen bonds.     

Effects of lysosomal enzymes on the type of collagen synthesized by bovine articular cartilage

Bovine articular cartilage normally synthesizes a collagen containing three identical α-chains. After pre-incubation with rat liver lysosomal enzymes, it begins to synthesize significant amounts of the more ubiquitous collagen of the (α₁)₂α₂ type. Since lysosomes are increased in osteoarthritis, it is possible that the abnormal biosynthetic patterns exhibited by cells in areas of degeneration are caused by such enzymes.Articular cartilage is an avascular tissue with very low cell density, composed primarily of extracellular substances such as collagen, proteoglycans, and glycoproteins. The structural integrity of this tissue depends on the relative proportion, nature, and structural organization of these components. Until recently, the destruction of cartilage seen in osteoarthritis was considered to result from a “wear and tear” process. This concept is not substantiated by recent ultrastructural and biochemical findings. Cellular activity in the involved areas leads to enlarged clones of chondrocytes containing increased numbers of intracellular organelles reflecting synthetic and secretory activity (1). There is an inverse correlation between the severity of the degenerative changes and the glycosaminoglycan content of the tissue (2–7). On the other hand, radioactive sulfate incorporation increases in osteoarthritis, an indication of the attempts made by the cells involved to repair the lesion (2). The nature of the proteoglycans synthesized under these conditions (less keratan sulfate and more chondroitin-4-sulfate) reflect the behaviour of immature chondroblasts (3–8). Lysosomal proteases have been associated with the degradation of the matrix (9–12). Cathepsin-D and a neutral protease which degrade proteoglycans at pH 5.0 and 7.0 respectively are considerably increased in early osteoarthritic lesions (13–15). Although the collagen content of cartilage does not change in osteoarthritis, qualitative differences may exist. Recently, we have shown that whereas normal human cartilage synthesizes only cartilage type collagen or (α₁-Type II)₃, osteoarthritic cartilage synthesizes in addition significant amounts of (α₁)₂α₂ collagen (skin type) (16). Articular cartilage collagen is quite different from other ubiquitous forms of mammalian collagens. In addition to containing three identical α-chains, it has four to five times more hydroxylysine and glycosidically associated carbohydrate than collagen from other tissues (17). It is quite possible that the abnormal collagen deposited by the cells at the site of degeneration may give rise to a mechanically weaker structure and lead to a loss of cartilage. While attempting to elucidate the mechanism underlying this abnormal metabolic pattern, it became apparent that lysosomal enzymes can alter the function of normal cartilage cells causing them to synthesize non-specific collagen molecules.     

Gene expression profiling of serum- and interleukin-1 beta-stimulated primary human adult articular chondrocytes--a molecular analysis based on chondrocytes isolated from one donor

In order to understand the cellular disease mechanisms of osteoarthritic cartilage degeneration it is of primary importance to understand both the anabolic and the catabolic processes going on in parallel in the diseased tissue. In this study, we have applied cDNA-array technology (Clontech) to study gene expression patterns of primary human normal adult articular chondrocytes isolated from one donor cultured under anabolic (serum) and catabolic (IL-1beta) conditions. Significant differences between the different in vitro cultures tested were detected. Overall, serum and IL-1beta significantly altered gene expression levels of 102 and 79 genes, respectively. IL-1beta stimulated the matrix metalloproteinases-1, -3, and -13 as well as members of its intracellular signaling cascade, whereas serum increased the expression of many cartilage matrix genes. Comparative gene expression analysis with previously published in vivo data (normal and osteoarthritic cartilage) showed significant differences of all in vitro stimulations compared to the changes detected in osteoarthritic cartilage in vivo. This investigation allowed us to characterize gene expression profiles of two classical anabolic and catabolic stimuli of human adult articular chondrocytes in vitro. No in vitro model appeared to be adequate to study overall gene expression alterations in osteoarthritic cartilage. Serum stimulated in vitro cultures largely reflected the results that were only consistent with the anabolic activation seen in osteoarthritic chondrocytes. In contrast, IL-1beta did not appear to be a good model for mimicking catabolic gene alterations in degenerating chondrocytes.     

Proteoglycans of the intervertebral disc. Homology of structure with laryngeal proteoglycans.

The structure of the proteoglycans from normal pig nucleus pulposus and relatively normal human annulus fibrosus and nucleus pulposus was investigated in detail and the results were compared with the current structural model of proteoglycans of hyaline cartilage. Like proteoglycans of cartilage, those of intervertebral disc contain keratan sulphate and chondroitin sulphate attached to a protein core; they are able to aggregate to hyaluronic acid; the protein core likewise has three regions, one lacking glycosaminoglycans, another rich in keratan sulphate and a third region rich in chondroitin sulphate. However, disc proteoglycans contain more keratan sulphate and protein and less chondroitin sulphate and are also considerably smaller than cartilage proteoglycans. In proteoglycans of human discs, these differences appeared to be due principally to a shorter region of the core protein bearing the chondroitin sulphate chains, whereas in proteoglycans of pig discs their smaller size and relatively low uronic acid content were due to shorter chondroitin sulphate chains. There were subtle differences between proteoglycans from the nucleus and annulus of human discs. In the latter a higher proportion of proteoglycans was capable of binding to hyaluronate.     

The "instantaneous" deformation of cartilage: effects of collagen fiber orientation and osmotic stress

The present study was undertaken with two objectives in view. The first was to distinguish between the "instantaneous" deformation and creep of articular cartilage when subjected to a step loading in unconfined compression. This was done by observing changes in the specimen's diameter rather than its thickness. The second objective was to investigate experimentally the anisotropic behaviour of cartilage in a compressive loading mode, corresponding to the physiological situation. An apparatus was thus developed and constructed which enabled us to follow the "instantaneous" changes of the surface area of the sample as the latter was being loaded in unconfined compression. Specimens of human articular cartilage from normal femoral heads and condyles were tested. Full thickness specimens were tested with and without the underlying bone, as well as partial thickness specimens, characterizing the different zones of cartilage. Solutions of different ionic strength were used to vary the osmotic stress and specimens covering a considerable range of proteoglycan concentrations were selected. The effects of hydration and proteoglycan removal on the "instantaneous" deformation were also studied. The "instantaneous" deformation was found to be of a strongly anisotropic nature in all zones. The deformation was always smaller along the Indian-ink prick pattern than at 90 degrees to it, and this effect was most pronounced in the superficial zone of cartilage. The results reveal an analogy with the tensile properties of cartilage and indicate that the collagen network is mainly responsible for controlling the "instantaneous" deformation. The proteoglycans play an indirect role by modulating the stiffness of the collagen network through their osmotic pressure.     

Direct measurement of osmotic pressure of glycosaminoglycan solutions by membrane osmometry at room temperature

Articular cartilage is a hydrated soft tissue composed of negatively charged proteoglycans fixed within a collagen matrix. This charge gradient causes the tissue to imbibe water and swell, creating a net osmotic pressure that enhances the tissue's ability to bear load. In this study we designed and utilized an apparatus for directly measuring the osmotic pressure of chondroitin sulfate, the primary glycosaminoglycan found in articular cartilage, in solution with varying bathing ionic strength (0.015 M, 0.15 M, 0.5 M, 1 M, and 2 M NaCl) at room temperature. The osmotic pressure (pi) was found to increase nonlinearly with increasing chondroitin sulfate concentration and decreasing NaCl ionic bath environment. Above 1 M NaCl, pi changes negligibly with further increases in salt concentration, suggesting that Donnan osmotic pressure is negligible above this threshold, and the resulting pressure is attributed to configurational entropy. Results of the current study were also used to estimate the contribution of osmotic pressure to the stiffness of cartilage based on theoretical and experimental considerations. Our findings indicate that the osmotic pressure resulting from configurational entropy is much smaller in cartilage (based on an earlier study on bovine articular cartilage) than in free solution. The rate of change of osmotic pressure with compressive strain is found to contribute approximately one-third of the compressive modulus (H(A)(eff)) of cartilage (Pi approximately H(A)(eff)/3), with the balance contributed by the intrinsic structural modulus of the solid matrix (i.e., H(A) approximately 2H(A)(eff)/3). A strong dependence of this intrinsic modulus on salt concentration was found; therefore, it appears that proteoglycans contribute structurally to the magnitude of H(A), in a manner independent of osmotic pressure.     

Replacement of proteoglycans in embryonic chick cartilage in organ culture after treatment with testicular hyaluronidase

Explants of cartilage from tibiae of 11-12 days chick embryos were grown in organ culture. To one group hyaluronidase was added to the medium during the first 2 days of culture; the treated tissue was then cultured in medium without enzyme for a further 4 days. Control explants grown in hyaluronidase-free medium for 6 days grew rapidly in size and the total hexosamine content more than doubled during this time. After exposure to hyaluronidase, much of the hexosamine was lost from treated cartilage and appeared in the culture medium, but it was mostly replaced in the tissue during the subsequent recovery period. Analysis of cartilage and medium showed that net synthesis of hexosamine increased greatly in treated cartilage. The proteoglycans were extracted by two procedures from control and treated cartilage after 2, 4 and 6 days in culture. The hydrodynamic sizes of the purified proteoglycans were compared by gel chromatography and the composition of the gel-chromatographic fractions was determined. The proteoglycans from controls did not change during culture, but after exposure to hyaluronidase the proteoglycans from treated cartilage were of much smaller size and lower chondroitin sulphate content. During recovery, even though new proteoglycans were formed, they were nevertheless of smaller size and lower chondroitin sulphate content than control proteoglycans. They gradually became more like control proteoglycans during recovery from treatment, but even after 4 days they were not yet the same. After 2 days of treatment with the enzyme, the chondroitin sulphate in the cartilage was of shorter chain length than in controls but during recovery after 4 and 6 days in culture, the chain lengths in control and treated cartilage were similar. It is concluded that the proteoglycans formed in embryo cartilage in response to their depletion by enzyme treatment contained fewer chondroitin sulphate chains attached to the protein moiety of proteoglycans. This may have resulted from a failure under stress to glycosylate the protein moiety to the usual extent; alternatively the synthesis of normal proteoglycans of low chondroitin sulphate content may have increased, thus changing the proteoglycan population.