Age-related changes in rabbit articular chondrocytes

Age-related changes in rabbit articular chondrocytes

Mechanisms of Aging and Development, 37 (1986) 231 - 240 231 Elsevier Scientific Publishers Ireland Ltd. AGE-RELATED CHANGES IN RABBIT ARTICULAR CH...

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Mechanisms of Aging and Development, 37 (1986) 231 - 240

231

Elsevier Scientific Publishers Ireland Ltd.

AGE-RELATED CHANGES IN RABBIT ARTICULAR CHONDROCYTES

JOCELYNE DOMINICE, CAROLE LEVASSEUR, SUZANNE LARNO, XAVIER RONOT and MONIQUE ADOLPHE Laboratoire de Pharmacologie Cellulaire de l'Ecole Pratique des ttautes Etudes. Institut Biomedical des Cordeliers, 15 rue de I'Ecole de M~decine. 75006 Paris {FranceJ

(Received June 14th, 1986) (Revision received October 15th, 1986)

SUMMARY Cell culture techniques have been used extensively in the study of the aging process at the cellular level. The "senescent" articular chondrocyte seems to be a good model to examine the responses to aging in .osteoarthritis, one of the most frequent diseases of old age. Thus/n vitro chondrocyte "senescence", established by weekly subculture was characterized by a declining proliferation rate during late passages, from a rapid growth rate in early subculture to a complete loss of the proliferation capacity after 8 -+ 1 passages. Flow cytometric analysis show a time course decrease in the fraction S and G2 + M during the subculture, and a concomittant enhancement in protein content related to the increase of cell size. The immunocytochemistry assays revealed an appearance of a rigid cytoarchitecture with an increase in the number, and organization, of three cytoskeletal components: actin, tubulin and vimentin. The cultured chondrocytes therefore undergo in vitro aging analogous to that described for diploid fibroblasts, and could constitute a cellular model for pharmacological and toxicological assays.

K e y words: Aging; Cultured chondrocytes; Growth kinetics; Flow cytomet ry; Cytoskele-

ton INTRODUCTION Diploid human fibroblasts can be kept in culture for prolonged periods of time under standardized quantitative cell culture conditions, but they do have a definitive and ultimately limited mitotic life span in vitro. This limited life span has been interpreted as a manifestation of aging at the cellular level [1-2]. This has been largely confirmed, not only for human fibroblasts but also for a variety of other vertebrate cell types [3]. Recently Evans and Georgescu [4] showed that cells derived from articular cartilage. 0047-6374/87/$03.50 Printed and Published in Ireland

© 1987 Elsevier Scientific Publishers Ireland Ltd.

232 upon subsequent subculture, underwent in vitro senescence in a manner analogous to that described for several other types of diploid cells. The authors essentially defined morphological modifications, after studying dog, human and rabbit chondrocytes from different aged donors. The model using rabbit articular chondrocytes studied in our laboratory which is similar to that of Evans and Georgescu could be suitable for studying the mechanism of aging of chondrocytes in vitro, and its possible correlation with a frequent disease of old-age: osteoarthritis. Our contribution has been to determine, using chondrocytes of various ages from the first to the seventh passage, various parameters which could be modified during in vitro aging: cell proliferaton capacity, cell size, flow cytometric analysis of cell cycle and protein content, and cytoskeleton organization using monoclonal antibodies to actin, tubulin and vimentin. MATERIALS AND METIIODS Cell culture Articular cartilage was removed from the shoulder and knee joints of I 2-month-old Fauve de Bourgogne rabbits. The chondrocytes were enzymatically released from cartilage slices by the technique of Green [5], which gives a pure population of chondrocytes. Isolated cells were then cultured in HAM F 12 medium supplemented with 10% (v/v) fetal calf serum (FCS, IBF), penicillin 10 IU/ml and streptomycin 10/ag/ml (SPECIA), and maintained at 37°C in an atmosphere of 5% CO2 in air. When monolayers reached confluence, cells were trypsinized and removed. Chondrocytes were inoculated into 25 cm 2 flasks (2.5 × 106 cells/flask) for cellular size measurement and flow cytometric analysis, and 35-mm diameter Petri dishes (7 X 104 cells/dish) for studying cell growth. The study of the cytoskeleton was performed on cells grown on glass cover slips that were placed in 35-mm diameter Petri dishes. The cultures were subcultured weekly in 75-cm 2 flasks with a constant seeding density of 1.5 X 10 6 cells in 15 ml of medium. The medium was changed halfway through the week-long incubation. At the fourth and at the seventh passages, chondrocytes were distributed and the same experiments performed as described above for the first passage. Cellular measurements Cells from Petri dishes were detached by incubation with 1 ml of phosphate buffered saline (PBS) (GIBCO) containing 0.1% (v/v) trypsin and 0.02% (v/v) EDTA. The number of cells was determined with a hemocytometer. Cell volumes were determined with an electronic particule sizer (Coulter Counter, Coultronics) interfaced with a pulse height analyser (Coulter Channel Analyser, Coultronics) after diluting the cell suspension with isoton cell-counting medium. Polystyrene beads of different diameter were used as a standard. Flow cytometric analysis Cells (seeded at density of 5 × 104/ml into 25-cm 2 plastic flasks) were harvested by

233 trypsinization after 2 days in culture. Suspension of isolated cells were fixed in 70% (v/v) ethanol, treated with ribonuclease (1 mg/ml, Sigma), and stained with propidium iodide (0.05 mg/ml, Sigma) as described by Crissman and Steinkamp [6]. The relative cellular DNA content was measured using about 50 000 cells with a Cytofluorograf FC 200/4800 A (Ortho Instruments). The relative percentage of cells in GO/I, S and G2 + M in DNA distribution histrograms were estimated according to the peak-reflect method [7]. For analysis of cellular protein, stock solutions (1 mg/mt) of fluorescein isothiocyanate, isomer 1 (Polysciences, Ltd., Northampton, U.K.)were prepared in absolute ethanol immediately before use, and a subsequent dilution was made in PBS. Ethanol-fixed cells were stained for protein at room temperature in fluorescein isothiocyanate solution (1/ag/ml). Flow cytometric analysis was performed within I h after staining [8].

Immunofluorescence localization of cytoskeleton Indirect immunofluorescence was performed on cell growth on glass cover slips and subsequently acetone-fixed at -20°C for 5 min. The cover slips were air dried and incubated with mouse monoclonal anti-actin lgM (Amersham), mouse monoclonal anti-atubulin IgG1 (Amersham), or anti-vimentin lgG (Sanbio), in a humidified atmosphere at 37°C for 45 min and washed thoroughly with PBS. They were then incubated with fluorescein-labeled sheep anti-mouse lgG (Amersham) for actin and tubulin, or fluoresceinlabeled goat lgG anti-mouse (Nordic Immunology) for vimentin, for 45 min at 37°C, washed thoroughly in several changes.of PBS and mounted on glass slides for microscopic observation. RESULTS

Cell measurements Cell count was performed daily in each group (first, fourth and seventh passages). Representative growth curves are presented in Fig. 1 and show the changes in cell density during 4-day periods. The chondrocytes multiplied rapidly during early passages, but entered a stage of declining growth rate in later subcultures. In all passages, cells multiplied exponentially from day 1 to day 3, and the slope was gradually affected. Thus, the cell cultures at the late passage had significantly longer cell population doubling times: 16.25 h at the first passage versus 22.75 h at the fourth and 28.5 h at the seventh. The chondrocytes were unable to grow after the ninth (+1) subculture (data not shown). It was found that the average volume of cells increased by 36% after the first passage (2510 -+ 131 ~tm3) to the fourth passage (3425 -+ 280/am3). Flow cytometric analysis Flow cytometric analysis of DNA showed a progression of cells in the GO/l, S and G2 + M phases, following subculture (Fig. 2). The first passage population was actively multiplying after 2 days of culture with 43% of cells in S and G2 + M. After the fourth and seventh passages in culture the S and G2 + M phases were gradually affected: 33% then 25%. In parallel, the proportion of cells in GO/I phase was enriched. This assay confirmed the decrease in proliferative capacity observed by cell numeration.

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After 3 days in culture, flow cytometric analysis o f protein demonstrated a high protein content in the late passage population as compared with the early passage population value (Fig. 3). This resulted in an enhancement o f 37% in the protein content during subculture and was comparable with the increase of 36% in cell size.

Immunofluorescence localization of cytoskeleton The immunofluorescence microscopy revealed an unusually organized configuration of the cytoskeleton in the "senescent" cells. In cells at the first passage, actin microfilaments formed a cytoplasmic network with a somewhat granulous aspect (Fig. 4A). When the actin fibers seen in Fig. 4B were observed as large actin cables, cells reached

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Fig. 4. Cytoskeleton organization in chondrocyte culture at different passages: Distribution of actin fibers: (~,) early passase, (B) late passage. Distribution of tubulin fibers: (C) early passage, (D) late passage. Distribution of vimentin fibers: (E) early passage, (F) late passage. Magnification: × 2270.

239 later stages of their lifespan. The cytoplasm of this enlarged chondrocytes was commonly observed to contain numerous actin fibers in ordered arrangements. In early passage the orientation of the actin fibers was random, with the subculture actin fibers running parallel to the long axis of the cells. Observation of the chondrocytes by indirect immunofluorescence microscopy using monoclonal anti-tubulin antibodies revealed an extensive microtubular system radiating from the centriolar region to the cell periphery (Fig. 4C). In large senescent chondrocytes the density of tubulin fibers was increased, and appeared to be locally ordered (Fig. 4D). A similar parallel arrangement of intermediate filaments was also observed using fluorescent monoclonal antibodies against these structures which were seen as large bundles radiating from the perinuclear region (more pronounced at early passage) into the peripheral cytoplasm (Fig. 4E,F). Vimentin containing intermediate ffdaments also appear to be increased in abundance and were tightly packed and ordered in the peripheral cytoplasm. Microscopic examination also revealed that, according to the age, the cell size distribution became heterogeneous, with numerous enlarged cells. DISCUSSION This study shows that the rabbit articular chondrocyte, under tissue culture conditions, has a replicative lifespan similar, although less pronounced, to that of cells of other origins [2,3,9,10]. The proliferative capacity of chondrocytes showed a great decrease in the later passages, it seems that less cells are capable of entering division and those that initiate their cycle have a prolonged generation time. The prolongation of the generation time seems to be due to an increase in the duration of the GoGI phase of the cell cycle. In addition, previous studies [11] comparing cell cultures derived from young and old rabbits clearly indicate that statistically significant differences in growth kinetics can be observed in vitro as a function of the donor's age. Thus the decrease in the proliferative capacity of cultured chondrocytes is also observed in cartilage from old animals. Another feature of increase in cell size, which is common to/n vitro aging in many species analysed [3,12,13,14], is also observed in the old chondrocyte. A parallel increase in protein content was also found. These studies also provide data on the age-related changes in cytoskeleton organization. With increasing number of passages the density of fdaments of different biochemical nature, tubulin, actin or vimentin, increased in the cytoplasm of the enlarged ceils. Wang and Gundersen [15] recently described an increased organization of cytoskeleton accompanying the aging of human fibroblasts in vitro. Moreover, van Gansen et al. [16] described a modification of actin patterns according to the age of mouse fibroblasts and found an unbalanced ratio between soluble and insoluble actin with an increase of total actin in the declining terminal cultures. Modifications of vimentin filaments have also been observed in cultured endothelial cells after repeated subculture [ 17]. Thus the loss of proliferative potential by chondrocytes in culture is accompanied by the maintenance of a highly organized state of the cytoskeleton in senescent cells. The enhancement of the cytofilaments and their rigid architecture in senescent cells should be

240 regarded as an i n d i c a t i o n o f the progressive i n c a p a c i t y o f these enlarged ceils to divide a n d so to m a i n t a i n a high replicative capacity. T h u s t h e decline o f t h e division p o t e n t i a l o f c u l t u r e d c h o n d r o c y t e s a c c o m p a n i e d by various changes o f several p a r a m e t e r s could be c o n s i d e r e d as a m a n i f e s t a t i o n o f cartilage aging. O n t h e o t h e r h a n d , a c c o r d i n g to Evans a n d G e o r g e s c u [4] such a m o d e l could also r e p r o d u c e c e r t a i n aspects o f a r t h r o s i s disease w h i c h is o f t e n related to o l d age. F u r t h e r m o r e , t h e main i n t e r e s t o f this in vitro m o d e l s h o u l d be 1o p e r m i t the s t u d y o f t h e effects o f various drugs p o t e n t i a l l y active o n c a r t i l a g i n o u s tissue to slow d o w n or to accelerate the aging process. A('KNOWLED(;EM I:NTS

Dr. M. Wolfelsperger is g r a t e f u l l y a c k n o w l e d g e d for p r o v i d i n g p h o t o m i c r o g r a p h s . RI' FI'RENCES 1 L. Haytlick and P.S. Moorhead, t h e serial cultivation of human diploid cell strains. Exp. ('ell Res. 25 (1961) 5 8 5 - 6 2 1 . 2 L. Hayflick, The limited in vitro lifetime of human diploid cell strains. E.xp. ('ell R~,s.. 3 7 t 1 9 6 5 ) 614 -636. 3 V.J. Cristofalo, Handbook o tCell Biology o.t Aging, CRC Press Inc. Boca Raton, I.lorida. 1985. 4 C.H. Evans and H.I. Georgescu, Obse:cvations on the senescence of cells derived from articular cartilage. Mech. Ageing Def.. 22 (1983) 179 .- 191. 5 W.T. Green, Behaviour of articular chondrocytes in cell culture. LTin. Orthop.. 75 ( 1971 ) 248 261). 6 H.A. Crissman and J.A. Steinkamp, Rapid simultaneous measurement of DNA, protein and cell volume in single cells from large mammelian cell population. J. Cell Biol., 59 (1973) 766-771. 7 B. Barlogie and B. Drewinko, D.A. Johnston, T. Buchner, W.It. |tauss and E.J. I'teirich, Pulse cytometric analysis of synchronized cells in vitro. Cancer Res., 36 (1976) 1176 1181. 8 lEA. Crissman and J.A. Steinkamp, Rapid one step staining procedures for analysis of cellular DNA and protein by single and dual laser flo~, cytometry. Cytometrv. 3 (1982) 84 9 0 . 9 Y. le Guilly, M. Simon. P. Lenoir and M. Bourel, Longterm culture of human adult liver cells: morphological changes related It) in vitro senescence and effect of donor's age on gro~ th potent ial. Gerontologia, 19 (1973) 303- 313. 10 E.L. Schneider and J.R. Smith, The relationship of in vitrt, studies to in vivo human aging. Int. Rev. CytoL. 69 (1981) 261--270. 11 M. Adolphe, X. Ronot, P. Jaffray0 C. Hecquel,J. l.ontagne and P. Lechat. Effect of donor',~ age ,m the growth kinetics of rabbit articular chondrocytes in culture. Mech. Ageing Dev.. 23 (1983) 191-198. 12 P. Polgar, L. Taylor and L. Brown, Plasma membrane as~bciated metabolic parameters and tile aging of human diploid fibroblasts. Mech. Ageing Dev.. 7 (1978) 151 - 160. 13 G. Leutert, Morphological aging changes in human articular cartilage. Mech. AgeingDer. 14 t 1980) 469-475. 14 J.W.I.M. Simons, The use of frequency distributions of cell diameters to characteri.,'c cell populations in tissue culture. Exp. Cell Res., 45 ( 1967 ) 336- 350. 15 E. Wang and D. Gundersen, Increased organization of cytoskeleton accompanying the aging ol human fibroblasts in vitro. Exp. CellRes., 154 (1984) 191 202. 16 P. van Gansen, A. Pays and L. Malherbe, Actin content and organization of microfilament~ tn primary cultures of mouse embryonic fibroblasts (in vitro ageing). Biol. Cell. 54 (19851 251 260. 17 M. Hormia, E. Linder, V.P. Lehto, T. Vartio, R.A. Badley and !. Virtanen. Vimentin filamem, m cultured endothelial ceils form butyrate-sensitive juxtanuclear masses at'tcr repeated suhcuhur¢. Exp. CelIRes.. 138 (1982) 159-166.