Synthesis of Cartilage Matrix by In Vitro. III. Effects of Ascorbate Mammalian Chondrocytes

Chondrocytes isolated from bovine articular cartilage were plated at high density and grown in the presence or absence of ascorbate. Collagen and proteoglycans, the major matrix macromolecules synthesized by these cells, were isolated at times during the course of the culture period and characterized. In both control and ascorbate-treated cultures, type II collagen and cartilage proteoglycans accumulated in the cell-associated matrix. Control cells secreted proteoglycans and type II collagen into the medium, whereas with time in culture, ascorbate-treated cells secreted an increasing proportion of types I and III collagens into the medium. The ascorbate-treated cells did not incorporate type I collagen into the cell-associated matrix, but continued to accumulate type II collagen in this compartment. Upon removal of ascorbate, the cells ceased to synthesize type I collagen. Morphological examination of ascorbate-treated and control chondrocyte culture revealed that both collagen and proteoglycans were deposited into the extracellular matrix. The ascorbate-treated cells accumulated a more extensive matrix that was rich in collagen fibrils and ruthenium red-positive proteoglycans. This study demonstrated that although ascorbate facilitates the formation of an extracellular matrix in chondrocyte cultures, it can also cause a reversible alteration in the phenotypic expression of those cells in vitro. Embryonic chick chondrocytes easily modulate or "dedifferentiate" in culture (19). They tend to lose their characteristic extracellular matrix, become motile, and assume a spindle shape similar to that of fibroblasts (18). These fibroblastlike chick chondrocytes synthesize predominantly hyaluronic acid and small amounts of chondroitin sulfate-containing proteoglycans. The shift from a polygonal to a spindle-shaped chondrocyte is also accompanied by a change in synthesis from the cartilage-specific type II collagen to the typical interstitial type I collagen ( 19, 32). Thus, the switching of collagen gene expression is accompanied by alterations in both cell morphology and matrix biosynthesis. The present study was undertaken to examine the effects of ascorbate on long-term chondrocyte cultures. We used bovine articular chondrocytes, cultured under conditions that have been shown to preserve the phenotype of these cells for long periods of time in vitro (13, 14). In the absence of ascorbate, these chondrocytes synthesize and accumulate a metachromatic matrix (14) that contains type II collagen, as well as monomeric and aggregated proteoglycan species characteristic of the parent cartilage (13). In the presence of ascorbate, the extracellular matrix showed significantly increased amounts of collagen and ruthenium red-positive proteoglycans. Biochemical data indicate that ascorbate-treated chondrocytes synthesize collagens of types I, II, and III. Type II collagen was incorporated into the matrix, and collagens of types | and III were released into the culture medium. Type I collagen synthesis ceased when ascorbate is removed from the medium. Proteoglycan synthesis was stimulated by ascorbate, yet the proteoglycan hydrodynamic size was not altered and remained identical to that of the parent cartilage. MATERIALS AND METHODS Materials used in this study and their sources were as follows: PD-10 Sepharose CL-2B (Pharmacia Fine Chemicals, Piscataway, N J); ultrapure guanidine HC1 (GdnHCl) (Research Plus Lab, Denville, N J); cesium chloride (CsC1) (Beckman Instruments, Inc., Palo Alto, CA); pepsin (Millipore Corp., Bedford, MA); [3H]proline 2,3,4,5aH; 90 Ci/mmol; ICN Biochemicals, lrvine, CA); Na235SO~ (840 mCi/mmol), Aquasol II, and NCS (New England Nuclear, Boston, MA); 2,5diphenyloxazole and 1,4-bis[2-(5-phenoxazoyl)]benzene (Research Products International, Elk Grove, IL); 6-aminohexanoic acid and phenylmethyisuifonyl fluoride (Sigma Chemical Co., St. Louis, MO); benzamidine hydrochloride, glucaronolactone, DMSO, and XRP-l x-ray film (Eastman Kodak Co., RochTHE JOURNAL OF CELL BIOLOGY • VOLUME 99 DECEMBER 1984 1960-1969 1960 © The Rockefeller University Press • 0021-9525/84/12/1960/10 $1.00 on O cber 9, 2017 jcb.rress.org D ow nladed fom ester, NY); cyanogen bromide (Aldrich Chemical Co.,, Milwaukee, WI); purified collagenase (Form lid (Advance Biofaetors Co., Lynbrook, NY); hyaluronidase (Worthington Biochemical Corp., Freehold, N J); all other chemicals were reagent grade (Fisher Scientific Co., Lynbrook, NY). Assonatively extracted Swarm rat chondrosarcoma proteoglycan aggregate and monomer were generous gifts from Dr. J. Kimura, Rush Medical College, Chicago, IL; monomeric pig dermal fibroblast prote~aglycans from Dr. J. Gregory, Rockefeller University, New York; and purified high-molecular-weight rooster comb hyaluronic acid from Dr. A. Balazs, Columbia University, New York. The GdnHO solutions used for the isolation and characterization of proteoglycans were buffered with 0.05 M sodium acetate to pH 6.2. They routinely contained the following proteina~ inhibitors at the concentrations indicated: 0.01 M 6-aminohexanoic acid, 0.01 M Na2EDTA, 0.005 M benzamidine hydrochloride, and 0.001 M phenylmethylsnlfonyl fluoride. The 0.5 and 4 M GdnHCI solutions are referred to as the associative and dissociative extraction solutions, respectively. When proteoglycans were isolated from medium, equal volumes of a double concentration of the associative or dissociative solutions were added. Proteoglycan aggregates (Al) and monomers (AID1) from bovine nasal septum were prepared by CsCl density gradient centrifogation, according to the method of Sajdera and Hascall (26) and used as cartier proteoglycans. Bovine types I and II carrier collagens were prepared from calf skin and nasal septum cartilage, respectively, by mild pepsin digestion, acid extraction, and salt precipitation (22). Type V collagen was extracted from 14-d-old chick embryos, and purified by differential salt precipitation (31). Isolation and Culture Characteristics of Chondrocgtes: Procedures for the isolation and characterization of bovine articular chondrocytes have been described in detail elsewhere (13). Cells were plated at high density (2 x l05 cells/cm 2) in 35or 60-mm culture dishes, 75-cm 2 flasks, or in roller bottles, and were grown for various periods before labeling or fixation. The growth medium was Ham's F-12, supplemented with 10% fetal bovine serum, 25 mM HEPES (pH 7.2), 50 ug/ml gentamycin, and 50 ug/ml amphoteflon B. In the experimental series, ascorbate was added at a concentration of 50 vg/ml. Cultures were refed with fresh medium every 48 h. Morphology: Chondrocytes were grown for 14 d in roller bottles or on Thermanox coverslips that had been placed into the bottoms of 8-well Multiplate dishes (Lux Scientific, Inc., Thousand Oaks, CA). Cells were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, dehydrated, and embedded in EPOn, as described in detail elsewhere (14). Some cultures were exposed to 0. 1% ruthenium red during Pre7 and postfixation. Sections were cut on a LKB 8810A ultramicrotome (LKB Instruments, Inc., Rockville, MD) with glass or diamond knives. Thick sections were stained with toluidine blue, thin sections with uranyl acetate followed by lead citrate. Thin sections were examined in a Philips EM-301 electron microscope (Philips Electronic Instruments, Inc., Mahwah, NJ). Determination of 35S-labeled Proteoglycans Synthesized in Culture: The formation of3SS-labeled proteoglycan aggregated in vitro was investigated, using 35-mm dish cultures that were pulse labeled with Na2~5SO4 (20 #Ci/ml for 15 h) on days 5 and 14 of incubation. The medium and the associatively extracted (4°C, 4 h) matrix proteoglycans were frozen at -70"C for further analysis. The remaining matrix proteoglycans were dissociatively extracted (4°C, 2 h). The unextractable residue was digested with papain (100 ug/ml, in 0.05 M sodium acetate buffer, pH 5.8, containing 20 mM L-Cysteine and 5 mM disodium EDTA (60°C, 24 li). The papain digest contained freed 35S-labeled glycosaminoglycans, which were counted for 35S. Proteoglycans were analyzed from medium fraction after addition of an equal volume of 8 M GdnHCl (4°C, 4 h). In order to separate 3~S incorporated into macromolecules from incorporated 35S in the medium and matrix extracts, 250-#1 aiiquots of each sample were applied to PD-10 columns (5.5 x I 1.8 cm), and equilibrated and eluted with 4 M GdnHCl (12). Unlabeled proteoglycan monomer (AID1; 1.5 mg/ml) from bovine nasal septum was added to all sample solutions to serve as carrier in subsequent analyses and chromatographic separations. 1-ml fractions were collected in scintillation vials, mixed with 0.9 ml of 70% ethanol and 14 ml of Aquasol II, then counted in a Packard model 3333 scintillation counter (Packard Instrument Co., Downers Grove, IL). The excluded volume (Vo), which contained 35S-labeled macromolecules, was used to determine net proteoglycan synthesis. Determination of Proteoglycan Monomer Size Synthesized by Chondrocytes In Vitro: Dish cultures were pulse labeled with Na235SO4 for 15 h on days 5 and 14. After dissociative extraction, the hydrodynamic size of 35S-labeled proteoglycan monomers was determined by molecular sieve chromatography. Portions of the pooled Vo fractions from PD-10 columns were chromatographed on a Sepharose CL-2B column (120 x 0.6 cm), equilibrated in 4 M GdnHC1. The sample was eluted in 4 M GdnHC1 and collected in 0.5-ml fractions. Each fraction was counted as described in the previous section. The column was precalibrated with a variety of macromolecules: (a) purified high-molecular-weight hyaluronic acid; (b) proteoglycan monomers (AID1) from bovine nasal septum and bovine articular cartilage; (c) Swarm rat chondrosarcoma proteoglycan monomer (A1D1); (d) ftbroblast proteoglycan monomer from pig dermis; and (e) glucaronolaetone. Free 35SO4 was added to all samples in order to determine total column volume (V0. Macromolecnles used in the calibration of the column were monitored for uronic acid after fractionation, according to the technique of Bitter and Muir (4). Proteoglycan aggregates of the associatively extracted cell-assonated matrix were examined, utilizing a separate CL-2B column and 0.5 M GdnHCI as a eluent. Fractions were collected and the radioactivity was determined as above. Labeling of Collagen Synthesized by Chondrocyte Cultures: Chondrocytes grown in flasks or roller bottles were exposed for 14 days to medium containing 5 ~mCi/ml [3H]proline. Labeled media were collected every 48 h and frozen for subsequent analyses. The long-term labeled (14 d) cell layers were scraped from the dishes or bottles and suspended in cold 0.5 M acetic acid solution (pH adjusted to 2.5 with 0.I N HCI). In some experiments, [3H]proline (5 ~Ci/ml) was administered to previously unlabeled cultures for a 12-h period. The media were removed and the cell layers treated as outlined above. In pulseor long-term labeled, ascorbate-treated cultures, fresh ascorbate was added at each feeding. Isolation of Collagen from Cell Cultures: Media from labeling experiments were adjusted to 30% saturation with ammonium sulfate and kept at 4°C for 18 h. The precipitated collagen was collected by centflfugation at 10,000 g for 30 rain, then dissolved in 0.5 M acetic acid. After removal of undissolved material by ceutrifugation, the acetic acid solution was brought to 100 #g/ml pepsin and incubated at 4°C for 18 h. The digest was adjusted to 2 M NaCI and kept at 4"(2 for 18 h in order to reprecipitate all collagen types (22). The salt-precipitated media collagens were collected by centrifugation, dissolved in 5 ml of 0.5 M acetic acid, dialyzed against the same solution for 48 h at 4°C, and then lyophilized. Cell layers from shortand long-term labeling experiments were extracted with 0.5 M acetic acid containing 100 pg/ml pepsin at 4°C for 48 h. The cell layer residue was further extracted with 50 mM Tris-HCl buffer, pH 7.5, 1 M NaCI for 24 h at 4°C. The extractions were combined and neutralized, and collagen was selectively precipitated from solution with a final 4.4 M NaCI concentration. After centrifugation, the precipitated collagens were resuspended in 5 ml of 0.5 M acetic acid, dialyzed against the same solution, and lyophilized. Determination of Total Protein and Collagen: Chondrocytes, grown in the presence or absence of ascorbate for 5 d, were pulse-labeled with 5 pCi/ml [3H]proline for 18 h. Total protein and collagen in the medium and cell layer were determined separately for each of three dishes, according to a modification of the "bacterial collagenas¢ digestion procedure" of Peterkofsky (25). Trichloroacetic acid pellets from the media and cell-assonated matrix were pretreated with bovine testicular hyaluronidase (50 pg/ml in 0.5 M phosphate buffer, pH 5.5, 0.1 M NaCI) for 1 h at 37°C. The hyaluronidase digests were trichloroacetic acid precipitated, then subjected to the collagenase digestion procedure of Peterkofsky (25). Collagenase digestion was started with an excess of enzyme, and additional enzyme was added half way through the incubation to insure complete digestion. SDS Gel Electrophoresis: The procedures of Laemmli (15) were followed, using either 11 x 0.5-cm tube gels or 1.5-mm-thick slab gels of 6% polyacrylamide. Collagen samples were dissolved in 6 M urea containing 1% SDS + 1% mercaptoethanol, and were heat denatured at 60°C for I0 rain. After electrophoresis, gels were stained with Coomassie Blue, destained, and scanned in an ISCO gel scanner (ISCO Inc., Lincoln, NE) at 580 nm. Tube gels were sliced into l-mm sections on a Miclde gel slicer, dissolved in NCS tissue solubilizer at 60°C for 1 h, and then counted in a Packard model 3333 scintillation counter. After destaining the slab gels and after indicating the positions of the echains, gels were prepared for fluorography, according to the techniques of Bonner and Laskey (6). Dried gels were exposed to XRP-1 film for a period of 1-2 wk at -70°C. For quantitation of collagen types synthesized over various time periods in culture, x-ray fluorograms were placed over gels, and the regions on the gels corresponding to the dark bands on the x-ray fluorograms film were excised for scintillation counting. The gel slices were solubilized in NCS at 60°C for 1 h, and then counted in a liquid scintillation counter.