Osteogenic protein-1 is most effective in stimulating nucleus pulposus and annulus fibrosus cells to repair their matrix after chondroitinase ABC-induced in vitro chemonucleolysis.

BACKGROUND CONTEXT: Chondroitinase ABC (C-ABC) is used in chemonucleolysis to degrade, with great specificity, the chondroitin sulfate and dermatan sulfate chains of proteoglycans (PGs). A recent study showed that osteogenic protein-1 (OP-1) is very effective in stimulating the production and formation of the extracellular matrix by rabbit intervertebral disc cells. PURPOSE: To test the hypothesis that the repair of the extracellular matrix of the intervertebral disc after chemonucleolysis by C-ABC can be stimulated by exposure to a low dose of a growth factor, OP-1. STUDY DESIGN: An alginate bead cell culture system was used to monitor the effects of OP-1 on the repair of damaged matrices after in vitro chemonucleolysis with C-ABC. METHODS: Rabbit nucleus pulposus (NP) or annulus fibrosus (AF) cells cultured for 2 weeks in alginate gel were briefly exposed to low concentrations of C-ABC and then cultured in the presence or absence of OP-1. The control group was cultured without enzyme treatment for the same period in the absence of OP-1. At each time point, the contents of DNA and proteoglycan accumulation and proteoglycan synthesis were measured. RESULTS: NP or AF cells cultured in alginate beads, which were digested with C-ABC and then treated with OP-1, recover PG content more rapidly than those cultured in the absence of OP-1. The major contributor to the superior matrix repair in the cells treated with OP-1 was an up-regulation of proteoglycan synthesis. CONCLUSIONS: OP-1 was effective in stimulating matrix repair by NP and AF cells after their matrices were nearly totally depleted of sulfated glycosaminoglycans. The use of OP-1 after chemonucleolysis might help the disc to regain biomechanical strength, weakened by enzyme digestion, by stimulating matrix metabolism.     

Alterations in glycosaminoglycan concentration and sulfation during chondrocyte maturation

We have used antibodies to chondroitin 4- and 6-sulfate and keratan sulfate along with Alcian blue staining of sulfated proteoglycans to investigate changes in content and sulfation within the avian growth plate. In normal chicks, chondroitin 4- and 6-sulfate content were similar in the proliferating and transitional zones but in the hypertrophic zone, chondroitin 4- and 6-sulfate were slightly lower (13% and 18%, respectively) and keratan sulfate was markedly lower (58%). Compared with the proliferative zone, Alcian blue staining of sulfated glycosaminoglycans was markedly lower in both the transitional (46%) and hypertrophic (22%) zones. In tibial dyschondroplasia, where chondrocyte maturation is arrested at the transitional zone, there was no difference in the chondroitin 4- and 6-sulfate or keratan sulfate staining between the proliferative and transitional zones, which were similar to normal birds. With Alcian blue staining there was no difference in the intensity of the staining within the proliferating zone compared with normal birds but stainign in the transitional chondrocytes was markedly higher (39%). These results suggest that in the early steps of chondrocyte maturation there may be a decrease in the degree of glycosaminoglycan sulfation without any alteration in glycosaminoglycan concentration, and that further maturation may be accompanied by a change in the nature of the proteoglycans which may also affect the level of sulfation.     

Mechanisms of proteoglycan inhibition of hydroxyapatite growth

Summary Purified bovine nasal cartilage proteoglycans (aggregate and subunit containing fractions) and to a lesser degree, chondroitin 4-sulfate of physiological size, retard seeded hydroxyapatite (HA) growthin vitro. The large hydrodynamic size and high charge density of these macromolecules are believed to be associated with the ability of proteoglycans to inhibit HA formation and growth. We now demonstrate the involvement of the negative charges of proteoglycans in this inhibition (a) by comparing the inhibitory ability of chondroitin 4-sulfate and its desulfated analog, and (b) by comparing the growth of HA seed crystals coated either with proteoglycan aggregates or chondroitin 4-sulfate to that of uncoated crystals. In the desulfation experiments, desulfated chondroitin sulfate was a less efficient HA growth inhibitor than untreated, undesulfated chondroitin sulfate of similar molecular size. Dextran sulfate showed higher inhibitory effectiveness than unchanged neutral dextran. Both experiments suggest that sulfate groups play an important role in the regulation of mineral deposition by proteoglycans. In the coating experiment, precoating of HA seed crystals with proteoglycan aggregates decreased the amount of HA precipitated as a function of time, suggesting proteoglcans may block the active nucleating sites on HA surface and slow down the growth process. Chondroitin 4-sulfate has a similar but weaker coating effect. Neural dextran, having little affinity for HA, had no effect.     

Chondroitinase-resistant sulfated glycosaminoglycans synthesized by cartilages of chick embryos and of newborn chickens and rats.

Biosynthesis of chondroitinase-resistant glycosaminoglycans as minor components was studied in the cartilages of chick embryos and of newborn chickens and rats. Sternal and knee cartilages were labeled in vitro with 35SO42-, and then 35S-labeled glycosaminoglycans were analyzed. In rats up to 2 weeks old, only one glycosaminoglycan could be detected as heparan sulfate. In the chick embryos and the newborn chickens, however, keratan sulfate as well as heparan sulfate could be detected. As chondroitinase-sensitive glycosaminoglycans, large amounts of both chrondroitin 4- and 6-sulfates were synthesized in the chick cartilage, but the synthesis of chondroitin 6-sulfate could scarcely be seen in the rat cartilage. The results seem to indicate that the biosynthesis of keratan sulfate has some relation to that of chondroitin 6-sulfate.     

Chondroitinase-resistant sulfated glycosaminoglycans synthesized by cartilages of chick embryos and of newborn chickens and rats

Summary Biosynthesis of chondroitinase-resistant glycosaminoglycans as minor components was studied in the cartilages of chick embryos and of newborn chickens and rats. Sternal and knee cartilages were labeled in vitro with³⁵SO ₄ ²⁻ , and then³⁵S-labeled glycosaminoglycans were analyzed. In rats up to 2 weeks old, only one glycosaminoglycan could be detected as heparan sulfate. In the chick embryos and the newborn chickens, however, keratan sulfate as well as heparan sulfate could be detected. As chondroitinase-sensitive glycosaminoglycans, large amounts of both chondroitin 4- and 6-sulfates were synthesized in the chick cartilage, but the synthesis of chondroitin 6-sulfate could scarcely be seen in the rat cartilage. The results seem to indicate that the biosynthesis of keratan sulfate has some relation to that of chondroitin 6-sulfate.     

Structural changes in the large proteoglycan, aggrecan, in different zones of the ovine growth plate

The large cartilage proteoglycan, aggrecan, was found to vary throughout the ovine physis corresponding to the maturational state of the resident chondrocytes. Two populations of proteoglycan monomer were observed in articular, epiphyseal, and in the resting zone of growth plate cartilage. These proteoglycans contained chondroitin sulfate glycosaminoglycan chains sulfated predominantly in the 4 position along with lesser amounts of chondroitin-6-sulfate and keratan sulfate. In the proliferative zone of the growth plate, chondrocytes synthesize one population of proteoglycan monomer which was significantly larger than monomer populations in articular, epiphyseal, or resting zone and this size increase could be attributed to an increase in its constituent chondroitin sulfate side chains. As these chondrocytes progress through their life cycle they continue to modify the structural characteristics of the aggrecan molecule they synthesize. Thus, in the hypertrophic region of the growth plate, the proteoglycan monomer is larger again than in the proliferative region. Variation in sulfation pattern on aggrecan chondroitin sulfate side chains is also observed in the hypertrophic region with an increasing proportion of unsulfated residues present, which may play a role in the initiation of mineralization. In addition, increasing amounts of the carbohydrate sequence recognized by monoclonal antibody 7-D-4 are observed in the hypertrophic zone. Received: 7 July, 1995 / Accepted: 17 June 1996     

Altered synthesis and intracellular transport of proteoglycans by cyst-derived cells from human polycystic kidneys.

Employing in vitro pulse-chase techniques, we investigated the de novo synthesis and the kinetics of intracellular transport and extracellular matrix incorporation of proteoglycans (PG) by normal human renal epithelial cell and by epithelial cells isolated from cysts of autosomal dominant kidneys (ADPKD). Cell monolayers were pulsed either with (3H)leucine for 15 min and chased for seven intervals between 15 and 270 min or with (35S)sulfate for 150 min and chased for a single interval of 120 min. Total proteins and PG were isolated from cell, media, and matrix fractions and characterized by Sepharose CL-6B and DEAE-Sephacel chromatographies. ADPKD and NK cells synthesized comparable amounts of total proteins; however, the de novo synthesis of PG by ADPKD cells was significantly reduced. ADPKD versus NK cells exhibited a substantial delay in the cellular transport and extracellular release of de novo synthesized PG, indicating an impairment at the level of the Golgi complex and/or secretory vacuoles. PG synthesized by ADPKD versus NK cells had decreased charge density characteristics, probably due to a posttranslational defect in the sulfation of the PG glycosaminoglycan chains. ADPKD versus NK cells synthesized PG of higher molecular weight and had an increased proportion of chondroitin sulfate PG versus heparan sulfate PG. Collectively, these findings suggest a defect in the synthesis and intracellular transport of sulfated PG in human ADPKD cells.     

Proteoglycans synthesized during the cartilage to bone transition in developing chick embryos

Osteogenesis of the long bone involves a series of cellular and molecular events culminating in the fabrication of a mineralized matrix of the long bone. These studies focus upon the chemical identity and morphology of newly synthesized proteoglycans formed during the cartilage to bone transition in embryonic chick tibia development. As early as the onset of major ossification events (day 13; Hamburger-Hamilton Stage 39), synthesis of a small, highly 4-sulfated chondroitin sulfate proteoglycan was detected in the shaft of the tibia. The characterization of the chondroitin sulfate-rich proteoglycans from cartilage, hypertrophic cartilage, bone marrow, and new bone lead to the suggestion that these molecules can be used as distinctive markers for the cellular phenotypes involved in the cartilage to bone transition.     

Glycosaminoglycan accumulation in primary culture of rabbit intervertebral disc cells

STUDY DESIGN: With the heterogeneity of the intervertebral disc as the focus, intervertebral discs from normal young rabbits were separated into nucleus pulposus (NP), inner anulus fibrosus (IAF), and outer anulus fibrosus (OAF) zones. Disc cells from each zone were isolated and propagated under monolayer and within agarose gel culture. The metabolism of these cultured disc cells was examined in terms of glycosaminoglycan (GAG) accumulation. OBJECTIVES: The object was to study the metabolism of disc cells derived from each zone and characterize them on the basis of GAG accumulation and composition. SUMMARY OF BACKGROUND DATA: It has been shown that three-dimensional culture systems, such as within-agarose gels or in alginate beads, permit long-term maintenance of the articular chondrocyte phenotype in vitro. However, little has been reported on how the metabolism of intervertebral disc cells, especially GAG accumulation, is affected by different culture conditions. METHODS: Cells from each zone were subjected to monolayer or three-dimensional culture for up to 12 days. GAG accumulation in the different culture systems was analyzed using chemical, histologic, and immunohistologic methods. Differences of GAG and DNA content among NP, IAF, and OAF cells were statistically evaluated by analysis of variance. The data of keratin sulfate content in three-dimensional culture were compared with that in monolayer culture using nonparametric Mann-Whitney U test. RESULTS: Monolayer culture revealed that increases in GAG content were significantly higher in IAF cells than in OAF cells. However, in three-dimensional culture GAG content was similar in the two groups. AF cells in three-dimensional cultures showed immunohistochemical localization of chondroitin sulfate and keratan sulfate, suggesting the existence of pericellular matrix. High performance liquid chromatography confirmed the expression of keratan sulfate in cultured cells. CONCLUSIONS: GAG accumulation in cultures of cells from different zones of the intervertebral disc varied according to the culture conditions used. The importance of choosing the appropriate culture systems to meet the objectives of a study should be emphasized.     

Phenotypic characteristics of rabbit intervertebral disc cells. Comparison with cartilage cells from the same animals.

STUDY DESIGN: Intervertebral disc cells were extracted from the surrounding matrix, and their metabolic activities and phenotypes were studied. OBJECTIVES: To compare the metabolic activities and phenotypes of cell populations extracted from the intervertebral discs of young rabbits with those of articular and growth plate chondrocytes from the same animals. SUMMARY OF BACKGROUND DATA: The phenotype of intervertebral disc cells has been poorly studied and still is debated. METHODS: The intervertebral discs as well as articular and vertebral growth plate cartilage of rabbits were digested enzymatically. The morphology of freshly isolated cells was examined. Their contents of collagen II and X mRNAs were determined by Northern blot analysis, and their sulfation activity by 35S-sulfate incorporation as chondrocytic markers. Cells were cultured at high density or low density and grown in primary culture. The stability of their phenotype was monitored by evaluating the collagen I and II mRNA ratio. The proteoglycans newly synthesized by the cells also were quantified, and their elution profile analyzed on Sepharose 2B columns. RESULTS: The anulus fibrosus cells were morphologically undistinguishable from articular chondrocytes. The nucleus pulposus contained mainly large vacuolated cells and a few smaller cells. All freshly extracted cells expressed different levels of collagen II mRNA. Anulus fibrosus and nucleus pulposus cells contained, respectively, 22% and 8% of collagen II mRNA compared with that found in articular or growth plate chondrocytes from the same animal. Only growth plate chondrocytes expressed collagen X. When anulus fibrosus cells were incubated for 48 hours at high density, they had collagen II mRNA contents similar to those of articular and growth plate chondrocytes, but synthesized five to six times fewer sulfated proteoglycans. When seeded at low density, anulus fibrosus cells divided more slowly than articular chondrocytes and incorporated four times fewer 35S-sulfate into proteoglycans. Their collagen II mRNA content was 2.75-fold lower than that of chondrocytes, and the procollagen alpha 1II/alpha 1I mRNA ratio was 3.1 for anulus fibrosus cells and 7 for chondrocytes. No collagen X mRNA was detected. When incubated for 48 hours at high density, the nucleus pulposus giant cells had four times less collagen II mRNA content than cartilage cells but synthesized the same amounts of sulfated proteoglycans. They did not divide during 21 days in culture and still contained collagen II mRNA but no collagen X mRNA. CONCLUSIONS: Findings showed that intervertebral disc cells all express cartilage-specific matrix proteins with quantitative differences, depending on their anatomic situation. It is suggested that anulus fibrosus cells are chondrocytic cells at a different stage of differentiation than articular and growth plate chondrocytes. The phenotype of nucleus pulposus cells still is unclear. They could be chondrocytic or notochordal. A definitive answer to this important question requires differentiating markers of notochordal cells.     

Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 2. Characterization of proteoglycans

The effect of ascorbate on the proteoglycans synthesized by rabbit articular chondrocytes was studied in first- and third-passage cultures for 12 and 26 days total duration, respectively. L-Ascorbate (0.2 mM) was added daily to half of the flasks after attachment of the cells. The cultures were labeled with Na2[35S]O4 or [14C]-glucosamine and [3H]-proline. Proteoglycans were isolated from the media and pericellular matrices by dissociative extraction and associative density gradient centrifugation. There was a large decline in the amount of proteoglycan synthesized between early and late cultures. Ascorbate increased the DNA content, amount of radiosulfate incorporated into glycosaminoglycans per microgram of DNA, and the proportion of labeled proteoglycan in the pericellular fraction of both short- and long-term cultures. The proteoglycans of the media and matrices of all cultures, with and without ascorbate, eluted as aggregates under associative column chromatographic conditions. The proteoglycans of 26-day cultures exhibited a higher degree of polydispersity in size than those of the short-term culture and contained small amounts of keratan (2-5%) and dermatan sulfate (4-8%) as assessed by keratanase and chondroitinase digestions, respectively. The effect of ascorbate, therefore, was to increase the amount of proteoglycan formed and to direct it into matrix deposition rather than to alter its quality.     

Structure of chondroitin sulfate on aggrecan isolated from bovine tibial and costochondral growth plates

The structure of chondroitin sulfate on aggrecan isolated from the rib and proximal tibial growth plates of bovine fetuses was investigated, and the previously reported increase in the hydrodynamic size of chondroitin sulfate chains between the reserve and hypertrophic zones of the rib was confirmed in the tibial growth plate. Superose 6 gel chromatography, calibrated for chondroitin sulfate chain length by monosaccharide analysis, showed that the average molecular mass of chondroitin sulfate in the reserve and maturing zones of both growth plates was 21,600 and 30,400, respectively. Determination by capillary zone electrophoresis of the disaccharide composition of chains following chondroitinase digestion showed that ΔDi-0S, ΔDi-4S, and ΔDi-6S together accounted for more than 98% of the disaccharides in the digests from all zones of both growth plates; Δdisulfated and Δtrisulfated disaccharides were not detected. Furthermore, this analysis revealed a gradient in chondroitin sulfate composition from the reserve to the hypertrophic zone, characterized by a marked increase in the content of ΔDi-6S (from about 32% to about 52%) and a marked decrease in the content of ΔDi-4S (from about 53% to about 35%). Moreover, this altered pattern of sulfation was detected on chains of all sizes in the hypertrophic zone, suggesting that a proportion of the reserve zone aggrecan might be removed and replaced with aggrecan rich in chondroitin-6-sulfate synthesized during the proliferative and maturation stages of the resident chondrocytes. These data are discussed in relation to the biosynthetic mechanisms that control chondroitin sulfate chain length and sulfation on aggrecan and their modification during chondrocyte proliferation, maturation, and hypertrophy in the growth plate.     

Effects of cell density on proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel

The effects of cell density on the proliferation and chondroitin sulfate synthesis of chondrocytes embedded in Atelocollagen gel were examined. Chondrocytes of 21 10-week-old Japanese white rabbits isolated by collagenase digestion were embedded in Atelocollagen gel and cultured in Dulbecco's modified Eagles medium at cell densities of 2 x 105 cells/ml (105 group), 2 x 106 cells/ml (106 group), and 2 x 107 cells/ml (107 group) for 4 weeks. Chondrocytes in the 105 group gradually proliferated more than the other two groups. In contrast, most chondrocytes in the 107 group showed increased capability to produce chondroitin 6-sulfate. Cartilage-like tissue was produced from high-density cultures (107 cells/ml), although a decrease in cell number was seen. Even in three-dimensional cultures, the proliferation and chondroitin sulfate synthesis of chondrocytes were influenced by the cell density. These results are informative for the clinical application of chondrocyte transplantation in three-dimensional cultures for cartilage repair.     

Immunohistochemical localization of proteoglycans in interstitial elements of human bladder cancer

The immunohistochemical localization of glycosaminoglycan side chains and core protein of proteoglycan was observed, using monoclonal antibodies, on 8 specimens of non-tumor bladder tissues and 26 specimens of bladder tumors obtained from total cystectomy or transurethral resection of bladder tumor (TUR-Bt). In non-tumor tissues of human bladder examined, the surface of urothelial epithelium consisted of heparan sulfate as revealed with antibody HepSS-1, and submucosal interstitial elements consisted mainly of small proteoglycan having chondroitin 4-sulfate side chains as revealed with antibodies 6B6 and 9A2, respectively. On the other hand, in bladder tumors examined, the interstitial fibrous elements, the so-called "specific stroma" in cancer cell nests, consisted mainly of large proteoglycans having chondroitin 4-sulfate or 6-sulfate side chains as revealed with antibodies 2B1, 9A2 and 3B3, respectively. Antibodies 2B1 and 3B3 are considered to be useful to demonstrate the involvement by invasive growth of bladder tumor cells.     

Newly synthesized proteoglycans in pseudoachondroplasia

In an attempt to elucidate the biochemical defect in pseudoachondroplasia, proteoglycan metabolism was investigated in cartilage from a patient with the dominant form of this condition. Iliac-crest cartilage was radioactively labeled with ³⁵S-sulfate and the newly synthesized proteoglycans examined for their hydrodynamic size and glycosaminoglycan composition. The banding pattern of the purified proteoglycans was analyzed by gel-electrophoresis using large pore polyacrylamide-agarose. We found a normal chain-length of glycosaminoglycans and a normal ratio of chondroitin-6-sulfate to chondroitin-4-sulfate. The proteoglycans were not enriched in keratan sulfate. Gel electrophoretic analysis of the proteoglycans disclosed a banding pattern comparable to that of two normal controls. In contrast to the findings of other authors no differences between the proteoglycans of pseudoachondroplastic and normal cartilage were detected.     

Effects of high molecular weight hyaluronan on the distribution and movement of proteoglycan around chondrocytes cultured in alginate beads

OBJECTIVE: To evaluate the effects of high molecular weight hyaluronan (HA) on the distribution and movement of proteoglycan (PG) formed around rabbit chondrocytes cultured in alginate beads. DESIGN: Rooster comb-derived HA (MW 8x10(5) Da) was co-polymerized in alginate gel to study the direct effects of extrinsic HA on chondrocytes. PG metabolism of rabbit chondrocytes cultured in alginate beads was examined by measuring the incorporation of [(35)S]sulfate into glycosaminoglycan in two distinct regions, the cells with their cell-associated matrix (CM) and the further-removed matrix (FRM). Immunohistochemical analysis was performed using monoclonal antibodies against chondroitin sulfate and keratan sulfate. Autoradiography using degenerated cartilage tissue from the rabbit osteoarthritis (OA) model was performed to discover the effect of HA on the distribution of newly-synthesized PG in the cartilage tissue. RESULTS: The incorporation of [(35)S]sulfate into newly-synthesized PG in the cells with CM decreased with the addition of 0.125-1.0 mg/ml HA, while the incorporation in the FRM increased. These effects of HA on the distribution of newly-synthesized PG were the same either in chondrocytes with CM or chondrocytes without CM. Immunohistochemical analysis showed that staining of PG in the CM was decreased and staining in the FRM was increased in the HA treated group compared to the control group. Autoradiography using degenerated cartilage tissue from the rabbit OA model indicated that [(35)S]-labeled macromolecules showed a more diffuse distribution in the HA treated group compared with the control group. CONCLUSION: These results indicate that extrinsic HA could affect the movement of newly-synthesized PG from the CM to the FRM in both alginate beads and cartilage tissue.     

Immunohistochemical study on the relationship between cancer cell growth and stroma changes of gastric carcinoma

To elucidate the stromal characteristics in gastric carcinoma, the localization of chondroitin sulfate proteoglycans (PGs), fibronectin (FN), and type IV collagen was observed by immunohistochemical methods in 46 surgically resected stomachs including 41 carcinomas, 2 adenomas, and 3 ulcers. Chondroitin 4-sulfate PG revealed by 9A2 antibody and FN were distributed abundantly in the stroma of gastric carcinoma. On the other hand chondroitin 6-sulfate PG revealed by 3B3 antibody and type IV collagen were located more broadly in the stroma of undifferentiated carcinoma, especially scirrhous carcinoma than differentiated carcinoma. Type IV collagen was ultrastructurally localized around smooth muscle cells, myofibroblasts, and capillaries. And Bromodeoxyuridine was labelled in the stromal cells including endothelial cells and smooth muscle cells whose proliferative activities were suggested. In addition to carcinoma, muscularis mucosae of adenoma and ulcer were stained by 3B3 antibody. In gastric carcinoma, extracellular matrix was more strikingly distributed compared with noncarcinomatous region and in particular, stromal changes may be promoted by cancer cells in scirrhous carcinoma. These results suggest that specific stromal changes in gastric carcinoma may be related with the histologic types and growth patterns.     

Matrix replenishment by intervertebral disc cells after chemonucleolysis in vitro with chondroitinase ABC and chymopapain

BACKGROUND CONTEXT: One of the advantages of chemonucleolysis for the treatment of a herniated intervertebral disc is the potential for the disc to self-repair. It has been suggested that the enzymes used for chemonucleolysis differentially affect the potential of the disc cells to promote repair. PURPOSE: To test the ability of nucleus pulposus and anulus fibrosus cells to repair the extracellular matrix degraded in vitro by either chondroitinase ABC or chymopapain. STUDY DESIGN: An alginate cell culture system was used to monitor the progress of matrix repair after chemonucleolysis in vitro. METHODS: Rabbit nucleus pulposus or anulus fibrosus cells precultured for 10 days in alginate gel were briefly exposed to low concentrations of chondroitinase ABC or chymopapain and then returned to normal culture conditions for up to 4 weeks. At each time point, the contents of DNA and matrix macromolecules and proteoglycan synthesis were measured. RESULTS: The DNA content of enzyme-treated alginate beads during the following 4 weeks of culture was higher in the chondroitinase ABC group than in the chymopapain group (NP, p<.01, and AF, p<.05). The content of proteoglycan in beads containing nucleus pulposus and anulus fibrosus cells in the chondroitinase ABC group was higher than that in the chymopapain group (NP and AF, p<.001). The rate of proteoglycan synthesis and the content of collagen did not, however, differ between those two groups. CONCLUSIONS: Intervertebral disc cells exposed to chondroitinase ABC reestablish a matrix richer in proteoglycan than cells exposed to chymopapain. This may be because of differences in the substrate spectrum of each enzyme. Although these results cannot be translated directly to the in vivo situation, they suggest the possibility that cells in discs subjected to chondroitinase ABC-induced chemonucleolysis retain a greater ability to replenish their extracellular matrix with proteoglycans than cells in discs exposed to chymopapain.