Characterization of adult bovine articular chondrocytes in culture

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proved to be more polar on thin-layer chromatography than arachidonic acid. Consequently, we suspect that the chemotactic lipid is a hydroperoxy acid or, possibly, a mixture of positional isomers of arachidonic acid hydroperoxides. Final identification, of course, must await analysis by gas chromatography and mass spectrometry. Arachidonic acid is a ubiquitous constituent of cell membrane phospholipids and can be made accessible by the action of phospholipases to a variety of chemical transformations in response to membrane pertur-

bations. Phagocytic leukocytes (particularly PMN) generate abundant amounts of oxygen-derived free radicals and singlet oxygen upon stimulation of their plasma membrane’ and have been demonstrated previously to be capable of producing oxygenation products of arachidonic acid and other lipid peroxides.‘,’ Consequently, under conditions that exist at most foci of inflammation, it is quite possible that potent biologically active lipids may be generated from arachidonic acid by mechanisms involving free radicals.

REFERENCES

I. Perez HD, Goldstein IM. Weksler BB. Generation of a biologically active lipid from arachidonic acid by exposure to a superoxide-generating system. Agents Actions (in press). 2. Perez HD, Weksler BB, Goldstein IM. Generation of a chemotactic lipid from arachidonic acid by exposure to a superoxide-generating system. Inflammation (in press). 3. Goetzl EJ, Sun FF. Generation of unique monohydroxy-eicosatetraenoic acids from arachidonic acid by human neutrophils. J Exp Med 1979; 150:406-l 4. 4. Kellogg EW Ill, Fridovich I. Superoxide, hydrogen peroxide and singlet oxygen in lipid peroxidation by a xanthine oxidase system. J Biol Chem 1975; 250:8812217. 5. Zigmond SH, Hirsch JG. Leukocyte locomotion and chemotaxis: New methods for evaluation and demonstration

Characterization

of a cell-derived chemotactic

factor. J Exp Med

1973;

137:387-410. 6. Weksler BB, Marcus AJ, JalTe E. Synthesis of prostaglandin I, (prostacyclin) by cultured human and bovine endothelial cells. Proc Natl Acad Sci USA 1977; 74:392226. 7. Goldstein IM, Roos D, Kaplan HB, Weissmann G. Complement and immunoglobulins stimulate superoxide production by human leukocytes independently of phagocytosis. J Clin Invest 1975; 56: I 155-63. 8. Goldstein IM, Malmsten CL, Kindahl H. Thromboxane generation by human peripheral blood polymorphonuclear leukocytes. J Exp Med 1978; 148:787-92.

of adult bovine articular

chondrocytes

in culture

By Klaus E. Kuettner, Robert K. Schenk, Jon Daniel, Vincent A. Memoli, Nancy C. Wrobel, Gary S. Gall, and Bendicht U. Pauli; Rush Medical College, and University of Illinois Dental School, Chicago, Illinois

A

RTICULAR CHONDROCYTES may undergo a variety of phenotypic alterations in culture. They may lose their characteristic morphology, become fibroblastoid, and may synthesize noncartilaginous macromolecules, such as type I collagen. Conditions are described for the isolation of articular chondrocytes and the maintenance of cartilage phenotype in vitro. MATERIALS AND METHODS Articular cartilage slices were obtained from IS-mo-old bovine metacarpophalangeal joints. They underwent sequential digestion in 1% pronase followed by 0.4% collagenase in F- I2 media in the presence of 5% calf serum.’ After filtration (90 pm Nitex), 2 x IO5 cells/cu cm were plated in either 35-mm tissue culture dishes or roller bottles in F-l 2 media, supplemented with antibiotics and 10% fetal bovine serum.* Chondrocytes fixed in buffered glutaraldehyde containing 0.1% ruthenium red (RR), were examined by light and transmission electron microscopy. Collagen type determination of ‘H-proline-labeled proteins isolated from cultures were performed by electrophoretic and CNBr peptide analysis.

Biosynthesis of proteoglycans was measured by j5S0, incorporation into macromolecules extracted under dissociative conditions (excluded from P-IO gels). Proteoglycan monomer size of “SO,-labeled macromolecules was determined by Sepharose 2BCL chromatography under dissociative conditions. The amount of proteoglycan extractable as aggregate was evaluated by associative extration of “SO,labeled cultures followed by sequential CsCI, density gradient ultrafiltration and chromatography of the fraction with density > I .70 on Sepharose 2BCL under associative condisions. Wet and dry weight determinations were used as an estimate of total water content of the cell-associated matrix.

RESULTS AND DISCUSSION Isolated chondrocytes prior to culture were typically rounded with scant territorial matrix, which Supported in part by NIH Grant AM-09132 and in part by NIH Grants CA-21566, CA-25034, and the Council for Tobacco Research-USA, Inc.. Grant 1206. o 1981 by Grune & Stratton, Inc. 0049-0172/81/1005~051/$01.00/0

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102

0

1

Fig. 1. Chondrocytes grown in roller bottles form typical streaks. which consist of multiple cell layers and abundant cartilage-like matrix. (Magnification x880.)

could be removed by mild trypsinization. From the start of the culture, chondrocytes were separated from each other by a rapidly synthesized RR-positive territorial matrix and some extraterritorial matrix. Multilayers of chondrocytes were surrounded by a distinct territorial matrix. These cells were embedded in an abundant extraterritorial matrix composed of a network of collagen fibrils with an approximate periodicity of 670 A. RR-positive globules were abundant and often associated with thin electron-dense filaments suggestive of proteoglycan aggregates, or were associated with collagen hbrils. With progression in culture, disseminated cartilaginous nodules were visible in the culture dishes. These nodules appeared as

2A 1

long streaks (50 x I .5 mm) in the roller bottles (Fig. I). Ultrastructurally, these nodules and streaks appeared almost identical to cartilage in vivo. Electrofiuorographs of collagen that was extracted from ‘H-proline-labeled cultures after mild pepsin digestion showed I band in the position of the 01 1 chain. An a2 chain could not be detected, despite slicing of the gels for scintillation counting. Cyanogen bromide peptide analysis confirmed that the major radioactive peptides comigrated with unlabeled peptides obtained from type II collagen. Type I collagen was not detectable in these cultures, even after a culture period of 1 mo. In these long-term cultures, however, bands tentatively identified as type

2B 2

3

4

1

2

3

4

-a 1

da 2

Fig. 2. (A) Seven percent slab gel electrofiuorographs of media collagen from chondrocyte cultures labeled with ‘f-f-proline on days 2(l). 4f21. 713). and lOf4) of incubation. Positions of la and a2 chains of type I collagen are indicated. f8J Seven percent slab gel electrofluorographs of reduced media collagen from chondrocyte cultures labeled on days 411). 7f2). 12(3), and 14f4) of incubation.

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OSTEOARTHRITISSYMPOSIUM

AB collagen and type 111 collagen appeared as previously described by other? (Fig. 2). The 35S0, proteoglycan synthesis was demonstrable from the start of the culture and remained relatively constant for approximately 2 wk, after which time it slowly decreased in both cell-associated matrix and media. The percentage of unextractable proteoglycan in the cell-associated layer in the dishes was low and relatively constant throughout the 4 wk culture period. The majority of the synthesized “SO,-labeled proteoglycan monomers had a K,, value identical to that obtained from bovine articular cartilage in shortterm organ culture and that isolated from fresh tissues. Monomer size remained relatively constant throughout the course of the culture, with a slight decrease in older cultures (>2l days; Fig. 3). Proteoglycan aggregate was extracted from both culture dishes and roller bottle cultures, under associative conditions. Approximately 90% of 35S04labeled proteoglycan was extractable from the cellassociated matrix using associative solvent. This value is in contrast to that obtained for roller bottle cultures where only approximately 50% of 35S0, proteoglycan could be extracted associatively. The water content of cell-associated matrix was less than that of the roller culture (91.4% versus 99.2%). These differences suggest that the roller culture cell-associated matrix may have a greater degree of organization than that grown in standard tissue culture dishes. These data indicate that articular chondrocytes grown in mass roller cultures are capable of synthesizing a phenotypically stable, tissue-like matrix in vitro. Current studies are aimed at characterizing the role of physical forces and nonstructural matrix components in the synthesis and organization of the cartilaginous matrix.

REFERENCES

I. Memoli VA, Pauli BU, Daniel J, Schenk RK, Kuettner KE. Isolated chondrocytes produce cartilage-like matrix in vitro. J Cell Biol 1980;87:E956 2. Memoli VA, Schenk RK. Pauli BU, et al. Maintenance

,

Fig. 3. The ‘%01 proteoglycan monomer size was determined by Sepharose 2BCL chromatography under dissociative conditions. (Al Short-term cartilage organ culture; (B) chondrocyte culture, day 7: (Cl chondrocyte culture, day 21. Note: K, values of monomeric proteoglycan of A and B are identical to each other and to those from fresh cartilage (data not shown). These in vitro K, values remain constant until approximately day 18, after which time a slight decrease of the monomer occurs (Cl. The smaller peak probably represents breakdown products of the monomer due to the prolonged pulse (15 hr). This K, value is slightly larger than that obtained for proteoglycans synthesized by fibroblasts.

of bovine articular chondrocyte phenotype in vitro. Tram Orthop Res Sot 198 1 (in press). 3. Benya PD, Padilla SR, Nimni ME. Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture. Cell I978;15: 1313-2 1.