- Email: [email protected]
Observations on the senescence of cells derived from articular cartilage
Mechanisms of Ageing and Development, 22 (1983) 179-191 Elsevier Scientific Publishers Ireland Ltd.
179
OBSERVATIONS ON THE SENESCENCE OF CELLS DERIVED FROM ARTICULAR CARTILAGE
C H R I S T O P H E R H. E V A N S and H E L G A I. G E O R G E S C U Department of Orthopaedic Surgery, 986 Scai[e Hall, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 (U.S.A.) (Received October 15th, 1982) (Revision received February 3rd, 1983)
SUMMARY In the experiments described here, we have sought to determine whether primary cultures of cells derived from articular cartilage will, upon subsequent subculture, undergo in vitro senescence in a manner analogous to that described for several other types of diploid cell. Using cells from the articular cartilage of rabbits, dogs and man, we have established that the population doubling capacity of cultures of these cells is directly related to the specific lifespan of the donor organism. Furthermore, the doubling capacity of the initial cultures of lapine articular chondrocytes is inversely related to the age of the donor rabbit. By these criteria, serially passaged primary cultures of cells derived from articular cartilage appear, a priori, to be a valid system for studies of cellular ageing. Monolayer cultures of lapine chondrocytes appear to "dedifferentiate" after several passages. However, the same cells can be grown as clones, under which conditions they appear to retain better their differentiated properties. Even under these circumstances, lapine articular chondrocytes have a limited capacity for growth, which can be calculated to approximate to the same average n u m b e r of cell divisions as undergone by monolayer cultures. Lapine chondrocytes frequently transform into established lines of fibroblastic cells. Transformation of canine chondrocytes was more rare, while human chondrocytes have not been observed to transform. This suggests that resistance to transformation is somehow related to lifespan. In addition to furthering our understanding of cellular ageing, studies of the senescence of articular chondrocytes could provide new insights into the aetiology of primary osteoarthritis.
K e y words: Chondrocyte; Cartilage; Cellular senescence; Cell division; Osteoarthritis (XI47-6374/83/$3.(11) Printed and Published in Ireland
© 1983 Elsevier Scientific Publishers Ireland Ltd.
180 INTRODUCTION The concept of ageing at the cellular level is now c o m m o n currency a m o n g gerontologists. Most support the view of Hayflick [1], that normal diploid cells have a finite mitotic capacity, reflecting the cellular ageing which imposes a m a x i m u m species' lifespan. Hayflick's original system of cellular senescence in vitro used h u m a n fibroblasts. Fibroblasts derived from human embryonic tissue undergo 5 0 _ 10 population doublings in culture before losing completely their ability to divide. Cellular age is conveniently measured as the n u m b e r of population doublings that the culture has achieved. T h r e e lines of evidence suggest that the finite iifespan of cultured diploid fibroblasts is in some way connected with actual cellular ageing. Firstly, experiments have shown a clear correlation between the lifespan of the donor species and the n u m b e r of doublings that a population of its fibroblasts will undergo [2]. Secondly, there may be an inverse correlation between the age of the donor animal and the n u m b e r of doublings that populations of its fibroblasts will achieve in culture [1,3-5]. Lastly, several of the changes found in last passage cells in vitro also occur in old cells in vivo [2]. Apart from obvious practical advantages, this system permits certain aspects of cellular senescence to be studied under standard conditions in isolation from the confusion of secondary, extrinsic factors which operate in the complexity of the whole organism. Most investigations of in vitro senescence have for reasons of convenience employed primary cultures of dermal or lung fibroblasts. While much important information has emerged from such investigations, fibroblasts are not without their drawbacks as a model for cellular ageing. The term "fibroblast'" is an imprecise one based largely on morphological criteria. Yet not all ~'fibroblasts'" are equivalent, and it is difficult to relate fibroblasts grown in culture with their precursors in situ. A related disadvantage is the lack of suitable, specific, markers for fibroblasts which would facilitate such comparisons. Inasmuch as ageing and differentiation are interrelated p h e n o m e n a , the lack of detailed information concerning the differentiation of fibroblasts either in vivo or in vitro is unfortunate. Another qualification is the observation that senescent changes in old organisms are more prominent in populations of non-dividing differentiated cells than in cells, such as fibroblasts, which divide [6,7]. Articular chondrocytes have several potential advantages as an alternative type of cell with which to study cellular ageing in vivo and in vitro. They constitute a population of differentiated cells which are exclusive to articular cartilage, an isolated, aneural, alymphatic and avascular connective tissue. As no other type of cefi normally occurs in articular cartilage, articular chondrocytes may be cultured without fear of contamination by other types of cell (although there is a possibility of cellular heterogeneity among populations of chondrocytes [8]). These properties facilitate comparisons between chondrocytes grown in culture and their in vivo counterparts. Furthermore, articular chondrocytcs typically
181
produce a characteristic "type IV" proteoglycan [9] and type II collagen [10], which is found only in articular cartilage and perhaps, transiently, during the development of the eye [11]. Loss of differentiated status is associated with the production of altered types of proteoglycan and collagen [9,12,13]. Unlike marly other highly differentiated types of cell, chondrocytes are readily cultured. Depending on the culture conditions, there is a variable loss of differentiated function during the in vitro cultivation of chondrocytes [12-15]. Chondrocytes rarely, if ever, divide in the articular cartilage of adult rabbits under nonpathological conditions [161. This circumstance may promote their cellular senescence [6,7]. Indeed, ultrastructural studies of articular cartilage from a variety of vertebrates, including man, rodents and newts, have demonstrated marked senescent changes in the chondrocytes of older animals [17-20]. Physiological changes in the chondrocytes of ageing animals are indicated by altered enzyme activities [21] and rates of [3H]uridine incorporation [22]. In the work reported here, we have tried to establish whether cultures of articular chondrocytes undergo cellular senescence in the "classical" in vitro manner exemplified by fibroblasts, and to what extent this might be applied legitimately to the study of ageing in chondrocytes. Previous investigations of this type appear to be limited to those of Mayne et al. [23], who reported a finite in vitro lifespan for cultures of embryonic chick chondrocytes. In addition to furthering our understanding of cellular gerontology, the cellular ageing of chondrocytes merits attention as a possible perpetrator of one of the most troublesome conditions of old age-osteoarthritis [24].
MATERIALS AND METHODS
Materials
Ham's F-12 medium, foetal bovine serum and the trypsin used for detaching cells were purchased from GIBCO, Grand Island, New York; sterile plastic culture vessels (25-cm 2) and pipettes were from Fisher Scientific; New Zealand white rabbits aged 4 weeks, 6 months and retired "breeders" approximately 3.5 years' old, specific pathogen-free, were from Hilltop Lab Animals, Scottdale, PA. Articular cartilage was removed from the femoral heads of young adult dogs, which were kindly given to use by the surgical research laboratory of the University of Pittsburgh School of Medicine. Human articular cartilage was obtained from fresh autospy material at the University of Pittsburgh Health Center. Collagenase and trypsin used in releasing chondrocytes from the articular cartilage were purchased from Worthington Biochemicals, Freehold, N J, and all other biochemicals from the Sigma Chemical Company, St. Louis, MO. Cell culture
Articular chondrocytes were liberated from the articular cartilage of rabbits,
182 dogs and humans by partial digestion of the matrix with trypsin and collagenase (0.2% w/v each), as described by Green [25]. Monolayer cultures were established by seeding l0 s cells into 25-cm 2 culture vessels, with 4 m l of Ham's F-12 medium supplemented by 10% (v/v) foetal bovine serum, 100 units/ml penicillin and 100 )xg/mi streptomycin. Cultures were incubated at 37°C in an atmosphere of 95% air and 5% CO2. When confluent, cells were detached with trypsin and reseeded at a 1 : 2 or 1:4 split ratio. Lapine articular chondrocytes were also cultured as clones [25]. When seeded sparsely (10 3 cells per 25-cm 2 vessel), the chondrocytes attach to the bottom of the culture vessel as single isolated cells. Upon incubation in Ham's F-12 medium with 10% foetal bovine serum at 37°C, these individual, freshly isolated chondrocytes grow slowly into small clones of around 1000 cells, each derived from the originally seeded cell. Under these conditions, they retain many of their differentiated functions and secrete a cartilaginous matrix [14,15,25]. RESULTS Primary cultures of freshly isolated lapine and human chondrocytes formed monolayers of flattened, polygonal cells (Fig. 1). Those from the articular cartilage of dogs were much smaller than those from the other two sources, and differed in morphology.
(a) Fig. I. Early passage monolayer cultures of articular chondrocytes from (a) rabbit, x I00; (b) dog, x320; (c)human, xl0(}.
183
Cc)
Fig. 2. Lapine articular chondrocytes.
Passage 3.
x 100.
On successive subculture, chondrocytes deviated from this regular morphology. With lapine chondrocytes, this alteration occurred after only two or three population doublings, producing cells of the type shown in Fig. 2. Although these cells no longer resembled their freshly isolated precursors, they did not present the aspect of typical fibroblasts. Late passage derivatives of the original monolayer of lapine chondrocytes adopted a variety of bizarre morphologies (Fig. 3) similar to those seen in late passage human and murine fibroblasts [1,26]. Canine chondrocytes retained their original form better than did the chondrocytes of rabbits. Even in late passage (Fig. 3) there was some retention of the original morphology. The original populations of lapine, canine and human articular chondrocytes had in vitro doubling capacities related to their respective specific maximum lifespans. When the data of Mayne et al. [23] are included (Table I), there is an impressive demonstration of the connection between lifespan and doubling capacity of the original population of articular chondrocytes. Populations of articular chondrocytes from baby rabbits routinely undergo more doublings in vitro than do those from young adult rabbits (Table II). Even though the difference is quite small (8-10 doublings vs. 11-12 doublings), it has proved reproducible in five separate experiments. Populations of articular chondrocytes from 3.5year-old rabbits underwent only 3-4 doublings in culture. Lapine chondrocytes in clonal culture behaved in the manner described by
×
~r
×
c~r ~r
0
186
TABLE I RELATIONSHIP BETWEEN POPULATION DOUBLING CAPACITY OF CULTURES OF CHONDROCYTES AND AGEING
A him a l
Maximum lifespan (years)
Population doublings
Rabbit (adult) Adult dog Chick a Young adult human
6 18 30 100
8-10 19 30 35-40
aData of Mayne et al. [231.
i
.
Fig. 4. Lapine articular chondrocytes grown in clonal culture [14,15,251 (x 100) showing production of metachromatically (dark) staining matrix. Toluidine blue stain.
187 TABLE II RELATIONSHIP BETWEEN POPULATION DOUBLING CAPACITY OF CULTURES OF LAPINE CHONDROCYTES AND DONOR AGE
Age of r a b b i t
Populationdoublings
4 weeks 6 months 3.5 years
11-12 8-10 3-4
G r e e n [14,15,25]. T h e y r e t a i n e d t h e i r f l a t t e n e d , p o l y g o n a l s h a p e a n d s e c r e t e d l a r g e a m o u n t s of m e t a c h r o m a t i c a l l y staining m a t r i x (Fig. 4). C l o n e s of this t y p e c o n t a i n e d a b o u t 1000 cells. W h e n such cells w e r e r e l e a s e d f r o m t h e i r m a t r i x by c o l l a g e n a s e a n d trypsin, a n d r e c u l t i v a t e d as m o n o l a y e r s , t h e y s e l d o m d i v i d e d for
iiiii
Fig. 5. 25-cm2 Falcon flasks with matrix-producing clones of lapine articular chondrocytes. Left: first passage clones seeded at (from top to bottom) 103, 104 and 103 chondrocytes per flask. Right: second passage clones. No clones were able to grow to a third passage (not shown).
188 m o r e than one passage. Furthermore, when recultivated as clones, very few of the cells were still able to divide and produce "second generation" clones (Fig. 5). None of the cells contained in these "second generation" clones were capable of further growth, either as monolayers or clones. Ageing cultures of lapine chondrocytes frequently transformed into established lines of apparently unlimited mitotic potential. Transformation was typically accompanied by a switch to a morphology that was unequivocally fibroblastic. It is interesting to note that cultures which failed to transform resisted this conversion to a completely fibroblastic form, although, as described above, their morphologies were markedly different from those of their precursors. Whereas eight out of twelve initial isolates of lapine chondrocytes eventually transformed, only three out of seven cultures of canine chondrocytes did so, while none of our three lines of cells derived from human articular cartilage have yet been observed to transform. DISCUSSION Justification for the use of serially passaged, diploid fibroblasts as in in vitro model of cellular senescence rests largely upon the dependence of population doubling capacity on specific lifespan and donor age. By these criteria of justification, the use of serially passaged, diploid cells derived from articular cartilage should be afforded similar status (Tables 1 and I1). Two objections could be raised to this contention: firstly, that chondrocytes do not normally divide in vivo [16] and thus have no bearing on cellular senescence in vitro; and, secondly, that chondrocytes dedifferentiate in culture so that we are observing the senescence of a fibroblastic precursor, not true chondrocytes. If cellular senescence is indeed a function of the n u m b e r of divisions a cell has undergone, then the first objection is a valid one. But it has been proposed [6,7,26] that cellular ageing is a post-mitotic process both in vivo and in vitro. From this perspective, the objection is inapplicable. The advantages offered by chondrocytes, described in the Introduction, will be valuable in the further work necessary to test such matters. The second putative objection raises a n u m b e r of complicated issues. Differentiation, so far as articular chondrocytes are concerned, is usually monitored as the production of type II collagen; accessory criteria include the synthesis of type IV proteoglycan [9], cartilaginous matrix material [15], and cellular morphology. By these criteria, lapine [13] and chick [23] articular chondrocytes dedlfferentiate quite quickly in culture. However, this cannot be held responsible for the limited division potential of lapine chondrocytes. Using the culture system of clones, developed by Green [14,15,251, we have grown lapine chondrocytes under conditions where they retain many of their differentiated properties. A simple calculation reveals that the original chondrocyte must have divided on average about 9-10 times to produce a clone of around 10(10 cells. This is not to claim that each individual daughter cell has divided a specific number of times. It
189 is quite likely, as occurs with monolayers of ageing fibroblasts, that many cells divide much more than this, while others do not divide at all, to give this average figure. When harvested from their matrix and reseeded, either as clones or monolayers, these cells rarely divide for more than one passage. Thus, even though the chondrocytes of such clones appear to have resisted overt dedifferentiation, their in vitro lifespan is still around ari average of ten generations. Hence, dedifferentiation cannot be a trivial explanation for the in vitro senescence of articular chondrocytes. The interrelationship between ageing and differentiation is not a simple one. Loss of differentiated function may itself be a result of the ageing process, which could explain the apparent similarity between certain features of senescent and immature tissue. Ultrastructural studies by Weiss [27] and Silberberg et al. [28] have revealed the presence of "fibroblastic" and "atypical, oblong'" cells in the articular cartilage of older animals. Of significance is the suggestion by Silberberg et al. [28] that these morphologically aberrant cells may represent the oldest chondrocytes in the joint. With respect to this, it is interesting to note that, whereas lapine articular chondrocytes easily lose their original morphology in vitro, canine and human chondrocytes showed greater morphological stability. Thus the ability to maintain differentiated properties, such as cellular morphology, may reflect the specific lifespan. A related observation is that, in the three mammals studied here, the case of spontaneous transformation into established lines was also related to lifespan. Thus articular chondrocytes from the rabbit transform readily, while those from dogs do so less readily and human chondrocytes not at all. The morphological changes which occur in late passage chondrocytes are typical of ageing cells both in vivo and in vitro. The results reported here are of interest for additional reasons. Primary osteoarthritis is a common, severely debilitating disease of old age. Its marked correlation with age is a prima facie reason for considering the possible involvement of ageing in its genesis. The earliest detectable changes in preosteoarthritic cartilage occur in the articular cartilage. As this is an isolated tissue, dependent on its articular chondrocytes for maintenance of its functional integrity, it is vulnerable to the effects of aberrant chondrocytic metabolism such as might result from ageing. An indication that commonality may exist between osteoarthritis and the ageing of chondrocytes in vivo and in vitro is the occurrence of abnormal types of collagen in osteoarthritic cartilage [29], hypertrophying chondrocytes at the base of the epiphyseal growth plate [30] and in late passage cultures of chondrocytes [23]. ACKNOWLEDGEMENTS This work was supported, in part, by a grant from the Western Pennsylvania Chapter of the Arthritis Foundation. We thank Dr. W.T. Green for criticism and discussion,
190 REFERENCES 1 L. Hayflick, The limited in vitro lifespan of human diploid cell strains. Exp. Cell Res., 37 (1965) 614-636. 2 L. Hayflick, The cellular basis for biological aging. In C.E. Finch and L. Hayflick (eds.), Handbook of the Biology of Aging, Van Nostrand Reinhold Co., New York, 1977, pp. 159-186. 3 S. Goldstein, J.W. Littlefield and J.S. Soeldner, Diabetes mellitus and aging: diminished plating etficiency of cultured human fibroblasts. Proc. Natl. Acad. Sci. U.S.A., 64 (1969) 15_5--160. 4 G.M. Martin, C.A. Sprague and C,J. Epstein, Replicative lifespan of cultivated human cells. Effects of donor's age, tissue and genotype. Lab. Invest., 23 (197{)) 86-92. 5 S. Goldstein, E.J. Moerman, J.S. Soeldner, R.E. Gleason and D.M. Barnett, Chronological and physiological age affect replcative lifespan of fibroblasts from diabetic, prediabatic and normal donors. Science, 199 (1978) 781-782. 6 C.H. Evans. On the ageing of organisms and their cells. IVied. Hypoth., 5 (1979) 53-66. 7 C.H. Evans, Is the G0(A)-state time-independent? J. Theor. Biol.. 79 (1979) 259-262. 8 S. Trippel, M. Ehrlich, L. Lippiello and H. Mankin, Metabolic activity of isolated chondrocyte populations. Trans. Orthop. Res. Soc., 3 (1977) 76. 9 M. Okayama, M. Pacifici and H. Holtzer, Difference among sulphated proteoglycans synthesised in nonchondrogenic cells, presumptive chondroblasts and chondroblasts. Proc. Natl. Acad. Sci U.S.A., 73 (1976) 3224-3228. 10 E.J. Miller, Isolation and characterisation of a collagen from chick cartilage containing three identical a chains. Biochemistry, 10 (1971) 1652-1658. 11 T.F. Linsenmayer and C.D, Little, Embryonic neural retina collagen: In vitro synthesis of high molecular weight forms of type II plus a new genetic type. Proc. Natl. Acad. Sci. U.S,A., 75 (1078) 3235-3239. 12 K. Madsen and S. Lohmander, Production of cartilage-type proteoglycans in cultures of chondrocytes from elastic cartilage. Arch. Biochem. Biophys., 196 (1979) 192-198. 13 P.D. Benya, S.R. Padilla and M.E. Nimni, The progeny of rabbit articular chondrocytes synthesize collagen types 1 and Ill and type 1 trimer, but not type 11, Verification by cyanogen bromide peptide analysis. Biochemistry, 16 (1977) 865-872. 14 W.T. Green, Behavior of articular chondrocytes in cell culture. Clin. Orthop., 75 (1971) 248-260. 15 W.T. Green and R.J, Ferguson, Histochemical and electron microscopic comparison of tissue produced by rabbit articular chondrocytes in vivo and in vitro. Arthritis Rheum., 18 (1075) 273-280. 16 H. Mankin, Mitosis in articular cartilage of immature rabbits. Clin. Orthop., 34 (1964) 170--176. 17 J.C. Jeanny and M. Gontcharoff, l~tude en microscopie 61ectronique et par cytoph,otometrie /a balayage de la structure et la distribution de la chromatine dans les noyaux des cellules cartilageneuse de la Triticus cristatus au cours de la s6nescence. Biol Cell., ,t2 (1978) 233 244. 18 C.H. Barnett, W. Cochrane and A.J. Palfrey, Age changes in articular cartilage of rabbits. Ann. Rheum. Dis., 22 (1963) 389-40{I. 19 R. Silberberg, M. Hasler and P.A. Lesker, Aging of the shoulder joint of guinea pigs. Electron microscopic and quantitative histochemical aspects. J. Gerontol., 28 (1973) 18-36. 20 R. Silberberg, Ultrastructure of articular cartilage in health and disease, Clin, Orthop., 57 (196~) 233-257. 21 R. Silberberg and P. Lesker, Enzyme activity in aging articular cartilage. Experientia, 27 (19711 133-135. 22 l.J. Singh and E.A. Tonna, Autoradiographic evaluation of 3bl-uridine utilization by ageing mouse cartilage. Gerontology, 12 (1972) 41-48. 23 R. Mayne, M.S. Vail, P.M. Mayne and E.J. Miller, Changes in type of collagen synthesised as clones of chick chondrocytes grow and eventually lose division capacity. Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 1674-1678. 24 C.H, Evans, H.I. Georgescu and R.A. Mazzocchi, Does cellular ageing of chondrocytes engender primary osteoarthritis? Trans. Orthop. Res. Soc., 6 (1981) 153. 25 W.T. Green, Articular cartilage repair. Behavior of rabbit chondrocytes during tissue culture and subsequent allografting. Clin. Orthop., 124 (1977) 237-250.
191 26 C.H. Evans, P. Van Gansen and I. Rassen, Transcription in early and late passage embryonic mouse fibroblasts in primary culture: a correlated biochemical, autoradiographical and ultrastructural study. Biol. Cell., 33 (1978) 117-128. 27 C. Weiss, Ultrastructural characteristics of osteoarthritis. Fed. Proc. 32 (1975) 1459-1466. 28 R. Silberberg, M. Silberberg and D. Feir, Life cycle of articular cartilage cells: an electron microscopic study of the hip joint of the mouse. A m . J. Anat., 114 (1964) 17-22. 29 M. Nimni and K. Deshmukh, Differences in collagen metabolism between normal and osteoarthritic human articular cartilage. Science, 181 (1973) 751-752. 30 S. Gey, P.K. Muller, C. Lemmer, K. Remberger, K. Matzen and K. Kuhn, Immunohistological study on collagen in cartilage-bone metamorphosis and degenerative osteoarthritis. Klin. Wochenschr., 54 (1976) 969-976.
179
OBSERVATIONS ON THE SENESCENCE OF CELLS DERIVED FROM ARTICULAR CARTILAGE
C H R I S T O P H E R H. E V A N S and H E L G A I. G E O R G E S C U Department of Orthopaedic Surgery, 986 Scai[e Hall, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 (U.S.A.) (Received October 15th, 1982) (Revision received February 3rd, 1983)
SUMMARY In the experiments described here, we have sought to determine whether primary cultures of cells derived from articular cartilage will, upon subsequent subculture, undergo in vitro senescence in a manner analogous to that described for several other types of diploid cell. Using cells from the articular cartilage of rabbits, dogs and man, we have established that the population doubling capacity of cultures of these cells is directly related to the specific lifespan of the donor organism. Furthermore, the doubling capacity of the initial cultures of lapine articular chondrocytes is inversely related to the age of the donor rabbit. By these criteria, serially passaged primary cultures of cells derived from articular cartilage appear, a priori, to be a valid system for studies of cellular ageing. Monolayer cultures of lapine chondrocytes appear to "dedifferentiate" after several passages. However, the same cells can be grown as clones, under which conditions they appear to retain better their differentiated properties. Even under these circumstances, lapine articular chondrocytes have a limited capacity for growth, which can be calculated to approximate to the same average n u m b e r of cell divisions as undergone by monolayer cultures. Lapine chondrocytes frequently transform into established lines of fibroblastic cells. Transformation of canine chondrocytes was more rare, while human chondrocytes have not been observed to transform. This suggests that resistance to transformation is somehow related to lifespan. In addition to furthering our understanding of cellular ageing, studies of the senescence of articular chondrocytes could provide new insights into the aetiology of primary osteoarthritis.
K e y words: Chondrocyte; Cartilage; Cellular senescence; Cell division; Osteoarthritis (XI47-6374/83/$3.(11) Printed and Published in Ireland
© 1983 Elsevier Scientific Publishers Ireland Ltd.
180 INTRODUCTION The concept of ageing at the cellular level is now c o m m o n currency a m o n g gerontologists. Most support the view of Hayflick [1], that normal diploid cells have a finite mitotic capacity, reflecting the cellular ageing which imposes a m a x i m u m species' lifespan. Hayflick's original system of cellular senescence in vitro used h u m a n fibroblasts. Fibroblasts derived from human embryonic tissue undergo 5 0 _ 10 population doublings in culture before losing completely their ability to divide. Cellular age is conveniently measured as the n u m b e r of population doublings that the culture has achieved. T h r e e lines of evidence suggest that the finite iifespan of cultured diploid fibroblasts is in some way connected with actual cellular ageing. Firstly, experiments have shown a clear correlation between the lifespan of the donor species and the n u m b e r of doublings that a population of its fibroblasts will undergo [2]. Secondly, there may be an inverse correlation between the age of the donor animal and the n u m b e r of doublings that populations of its fibroblasts will achieve in culture [1,3-5]. Lastly, several of the changes found in last passage cells in vitro also occur in old cells in vivo [2]. Apart from obvious practical advantages, this system permits certain aspects of cellular senescence to be studied under standard conditions in isolation from the confusion of secondary, extrinsic factors which operate in the complexity of the whole organism. Most investigations of in vitro senescence have for reasons of convenience employed primary cultures of dermal or lung fibroblasts. While much important information has emerged from such investigations, fibroblasts are not without their drawbacks as a model for cellular ageing. The term "fibroblast'" is an imprecise one based largely on morphological criteria. Yet not all ~'fibroblasts'" are equivalent, and it is difficult to relate fibroblasts grown in culture with their precursors in situ. A related disadvantage is the lack of suitable, specific, markers for fibroblasts which would facilitate such comparisons. Inasmuch as ageing and differentiation are interrelated p h e n o m e n a , the lack of detailed information concerning the differentiation of fibroblasts either in vivo or in vitro is unfortunate. Another qualification is the observation that senescent changes in old organisms are more prominent in populations of non-dividing differentiated cells than in cells, such as fibroblasts, which divide [6,7]. Articular chondrocytes have several potential advantages as an alternative type of cell with which to study cellular ageing in vivo and in vitro. They constitute a population of differentiated cells which are exclusive to articular cartilage, an isolated, aneural, alymphatic and avascular connective tissue. As no other type of cefi normally occurs in articular cartilage, articular chondrocytes may be cultured without fear of contamination by other types of cell (although there is a possibility of cellular heterogeneity among populations of chondrocytes [8]). These properties facilitate comparisons between chondrocytes grown in culture and their in vivo counterparts. Furthermore, articular chondrocytcs typically
181
produce a characteristic "type IV" proteoglycan [9] and type II collagen [10], which is found only in articular cartilage and perhaps, transiently, during the development of the eye [11]. Loss of differentiated status is associated with the production of altered types of proteoglycan and collagen [9,12,13]. Unlike marly other highly differentiated types of cell, chondrocytes are readily cultured. Depending on the culture conditions, there is a variable loss of differentiated function during the in vitro cultivation of chondrocytes [12-15]. Chondrocytes rarely, if ever, divide in the articular cartilage of adult rabbits under nonpathological conditions [161. This circumstance may promote their cellular senescence [6,7]. Indeed, ultrastructural studies of articular cartilage from a variety of vertebrates, including man, rodents and newts, have demonstrated marked senescent changes in the chondrocytes of older animals [17-20]. Physiological changes in the chondrocytes of ageing animals are indicated by altered enzyme activities [21] and rates of [3H]uridine incorporation [22]. In the work reported here, we have tried to establish whether cultures of articular chondrocytes undergo cellular senescence in the "classical" in vitro manner exemplified by fibroblasts, and to what extent this might be applied legitimately to the study of ageing in chondrocytes. Previous investigations of this type appear to be limited to those of Mayne et al. [23], who reported a finite in vitro lifespan for cultures of embryonic chick chondrocytes. In addition to furthering our understanding of cellular gerontology, the cellular ageing of chondrocytes merits attention as a possible perpetrator of one of the most troublesome conditions of old age-osteoarthritis [24].
MATERIALS AND METHODS
Materials
Ham's F-12 medium, foetal bovine serum and the trypsin used for detaching cells were purchased from GIBCO, Grand Island, New York; sterile plastic culture vessels (25-cm 2) and pipettes were from Fisher Scientific; New Zealand white rabbits aged 4 weeks, 6 months and retired "breeders" approximately 3.5 years' old, specific pathogen-free, were from Hilltop Lab Animals, Scottdale, PA. Articular cartilage was removed from the femoral heads of young adult dogs, which were kindly given to use by the surgical research laboratory of the University of Pittsburgh School of Medicine. Human articular cartilage was obtained from fresh autospy material at the University of Pittsburgh Health Center. Collagenase and trypsin used in releasing chondrocytes from the articular cartilage were purchased from Worthington Biochemicals, Freehold, N J, and all other biochemicals from the Sigma Chemical Company, St. Louis, MO. Cell culture
Articular chondrocytes were liberated from the articular cartilage of rabbits,
182 dogs and humans by partial digestion of the matrix with trypsin and collagenase (0.2% w/v each), as described by Green [25]. Monolayer cultures were established by seeding l0 s cells into 25-cm 2 culture vessels, with 4 m l of Ham's F-12 medium supplemented by 10% (v/v) foetal bovine serum, 100 units/ml penicillin and 100 )xg/mi streptomycin. Cultures were incubated at 37°C in an atmosphere of 95% air and 5% CO2. When confluent, cells were detached with trypsin and reseeded at a 1 : 2 or 1:4 split ratio. Lapine articular chondrocytes were also cultured as clones [25]. When seeded sparsely (10 3 cells per 25-cm 2 vessel), the chondrocytes attach to the bottom of the culture vessel as single isolated cells. Upon incubation in Ham's F-12 medium with 10% foetal bovine serum at 37°C, these individual, freshly isolated chondrocytes grow slowly into small clones of around 1000 cells, each derived from the originally seeded cell. Under these conditions, they retain many of their differentiated functions and secrete a cartilaginous matrix [14,15,25]. RESULTS Primary cultures of freshly isolated lapine and human chondrocytes formed monolayers of flattened, polygonal cells (Fig. 1). Those from the articular cartilage of dogs were much smaller than those from the other two sources, and differed in morphology.
(a) Fig. I. Early passage monolayer cultures of articular chondrocytes from (a) rabbit, x I00; (b) dog, x320; (c)human, xl0(}.
183
Cc)
Fig. 2. Lapine articular chondrocytes.
Passage 3.
x 100.
On successive subculture, chondrocytes deviated from this regular morphology. With lapine chondrocytes, this alteration occurred after only two or three population doublings, producing cells of the type shown in Fig. 2. Although these cells no longer resembled their freshly isolated precursors, they did not present the aspect of typical fibroblasts. Late passage derivatives of the original monolayer of lapine chondrocytes adopted a variety of bizarre morphologies (Fig. 3) similar to those seen in late passage human and murine fibroblasts [1,26]. Canine chondrocytes retained their original form better than did the chondrocytes of rabbits. Even in late passage (Fig. 3) there was some retention of the original morphology. The original populations of lapine, canine and human articular chondrocytes had in vitro doubling capacities related to their respective specific maximum lifespans. When the data of Mayne et al. [23] are included (Table I), there is an impressive demonstration of the connection between lifespan and doubling capacity of the original population of articular chondrocytes. Populations of articular chondrocytes from baby rabbits routinely undergo more doublings in vitro than do those from young adult rabbits (Table II). Even though the difference is quite small (8-10 doublings vs. 11-12 doublings), it has proved reproducible in five separate experiments. Populations of articular chondrocytes from 3.5year-old rabbits underwent only 3-4 doublings in culture. Lapine chondrocytes in clonal culture behaved in the manner described by
×
~r
×
c~r ~r
0
186
TABLE I RELATIONSHIP BETWEEN POPULATION DOUBLING CAPACITY OF CULTURES OF CHONDROCYTES AND AGEING
A him a l
Maximum lifespan (years)
Population doublings
Rabbit (adult) Adult dog Chick a Young adult human
6 18 30 100
8-10 19 30 35-40
aData of Mayne et al. [231.
i
.
Fig. 4. Lapine articular chondrocytes grown in clonal culture [14,15,251 (x 100) showing production of metachromatically (dark) staining matrix. Toluidine blue stain.
187 TABLE II RELATIONSHIP BETWEEN POPULATION DOUBLING CAPACITY OF CULTURES OF LAPINE CHONDROCYTES AND DONOR AGE
Age of r a b b i t
Populationdoublings
4 weeks 6 months 3.5 years
11-12 8-10 3-4
G r e e n [14,15,25]. T h e y r e t a i n e d t h e i r f l a t t e n e d , p o l y g o n a l s h a p e a n d s e c r e t e d l a r g e a m o u n t s of m e t a c h r o m a t i c a l l y staining m a t r i x (Fig. 4). C l o n e s of this t y p e c o n t a i n e d a b o u t 1000 cells. W h e n such cells w e r e r e l e a s e d f r o m t h e i r m a t r i x by c o l l a g e n a s e a n d trypsin, a n d r e c u l t i v a t e d as m o n o l a y e r s , t h e y s e l d o m d i v i d e d for
iiiii
Fig. 5. 25-cm2 Falcon flasks with matrix-producing clones of lapine articular chondrocytes. Left: first passage clones seeded at (from top to bottom) 103, 104 and 103 chondrocytes per flask. Right: second passage clones. No clones were able to grow to a third passage (not shown).
188 m o r e than one passage. Furthermore, when recultivated as clones, very few of the cells were still able to divide and produce "second generation" clones (Fig. 5). None of the cells contained in these "second generation" clones were capable of further growth, either as monolayers or clones. Ageing cultures of lapine chondrocytes frequently transformed into established lines of apparently unlimited mitotic potential. Transformation was typically accompanied by a switch to a morphology that was unequivocally fibroblastic. It is interesting to note that cultures which failed to transform resisted this conversion to a completely fibroblastic form, although, as described above, their morphologies were markedly different from those of their precursors. Whereas eight out of twelve initial isolates of lapine chondrocytes eventually transformed, only three out of seven cultures of canine chondrocytes did so, while none of our three lines of cells derived from human articular cartilage have yet been observed to transform. DISCUSSION Justification for the use of serially passaged, diploid fibroblasts as in in vitro model of cellular senescence rests largely upon the dependence of population doubling capacity on specific lifespan and donor age. By these criteria of justification, the use of serially passaged, diploid cells derived from articular cartilage should be afforded similar status (Tables 1 and I1). Two objections could be raised to this contention: firstly, that chondrocytes do not normally divide in vivo [16] and thus have no bearing on cellular senescence in vitro; and, secondly, that chondrocytes dedifferentiate in culture so that we are observing the senescence of a fibroblastic precursor, not true chondrocytes. If cellular senescence is indeed a function of the n u m b e r of divisions a cell has undergone, then the first objection is a valid one. But it has been proposed [6,7,26] that cellular ageing is a post-mitotic process both in vivo and in vitro. From this perspective, the objection is inapplicable. The advantages offered by chondrocytes, described in the Introduction, will be valuable in the further work necessary to test such matters. The second putative objection raises a n u m b e r of complicated issues. Differentiation, so far as articular chondrocytes are concerned, is usually monitored as the production of type II collagen; accessory criteria include the synthesis of type IV proteoglycan [9], cartilaginous matrix material [15], and cellular morphology. By these criteria, lapine [13] and chick [23] articular chondrocytes dedlfferentiate quite quickly in culture. However, this cannot be held responsible for the limited division potential of lapine chondrocytes. Using the culture system of clones, developed by Green [14,15,251, we have grown lapine chondrocytes under conditions where they retain many of their differentiated properties. A simple calculation reveals that the original chondrocyte must have divided on average about 9-10 times to produce a clone of around 10(10 cells. This is not to claim that each individual daughter cell has divided a specific number of times. It
189 is quite likely, as occurs with monolayers of ageing fibroblasts, that many cells divide much more than this, while others do not divide at all, to give this average figure. When harvested from their matrix and reseeded, either as clones or monolayers, these cells rarely divide for more than one passage. Thus, even though the chondrocytes of such clones appear to have resisted overt dedifferentiation, their in vitro lifespan is still around ari average of ten generations. Hence, dedifferentiation cannot be a trivial explanation for the in vitro senescence of articular chondrocytes. The interrelationship between ageing and differentiation is not a simple one. Loss of differentiated function may itself be a result of the ageing process, which could explain the apparent similarity between certain features of senescent and immature tissue. Ultrastructural studies by Weiss [27] and Silberberg et al. [28] have revealed the presence of "fibroblastic" and "atypical, oblong'" cells in the articular cartilage of older animals. Of significance is the suggestion by Silberberg et al. [28] that these morphologically aberrant cells may represent the oldest chondrocytes in the joint. With respect to this, it is interesting to note that, whereas lapine articular chondrocytes easily lose their original morphology in vitro, canine and human chondrocytes showed greater morphological stability. Thus the ability to maintain differentiated properties, such as cellular morphology, may reflect the specific lifespan. A related observation is that, in the three mammals studied here, the case of spontaneous transformation into established lines was also related to lifespan. Thus articular chondrocytes from the rabbit transform readily, while those from dogs do so less readily and human chondrocytes not at all. The morphological changes which occur in late passage chondrocytes are typical of ageing cells both in vivo and in vitro. The results reported here are of interest for additional reasons. Primary osteoarthritis is a common, severely debilitating disease of old age. Its marked correlation with age is a prima facie reason for considering the possible involvement of ageing in its genesis. The earliest detectable changes in preosteoarthritic cartilage occur in the articular cartilage. As this is an isolated tissue, dependent on its articular chondrocytes for maintenance of its functional integrity, it is vulnerable to the effects of aberrant chondrocytic metabolism such as might result from ageing. An indication that commonality may exist between osteoarthritis and the ageing of chondrocytes in vivo and in vitro is the occurrence of abnormal types of collagen in osteoarthritic cartilage [29], hypertrophying chondrocytes at the base of the epiphyseal growth plate [30] and in late passage cultures of chondrocytes [23]. ACKNOWLEDGEMENTS This work was supported, in part, by a grant from the Western Pennsylvania Chapter of the Arthritis Foundation. We thank Dr. W.T. Green for criticism and discussion,
190 REFERENCES 1 L. Hayflick, The limited in vitro lifespan of human diploid cell strains. Exp. Cell Res., 37 (1965) 614-636. 2 L. Hayflick, The cellular basis for biological aging. In C.E. Finch and L. Hayflick (eds.), Handbook of the Biology of Aging, Van Nostrand Reinhold Co., New York, 1977, pp. 159-186. 3 S. Goldstein, J.W. Littlefield and J.S. Soeldner, Diabetes mellitus and aging: diminished plating etficiency of cultured human fibroblasts. Proc. Natl. Acad. Sci. U.S.A., 64 (1969) 15_5--160. 4 G.M. Martin, C.A. Sprague and C,J. Epstein, Replicative lifespan of cultivated human cells. Effects of donor's age, tissue and genotype. Lab. Invest., 23 (197{)) 86-92. 5 S. Goldstein, E.J. Moerman, J.S. Soeldner, R.E. Gleason and D.M. Barnett, Chronological and physiological age affect replcative lifespan of fibroblasts from diabetic, prediabatic and normal donors. Science, 199 (1978) 781-782. 6 C.H. Evans. On the ageing of organisms and their cells. IVied. Hypoth., 5 (1979) 53-66. 7 C.H. Evans, Is the G0(A)-state time-independent? J. Theor. Biol.. 79 (1979) 259-262. 8 S. Trippel, M. Ehrlich, L. Lippiello and H. Mankin, Metabolic activity of isolated chondrocyte populations. Trans. Orthop. Res. Soc., 3 (1977) 76. 9 M. Okayama, M. Pacifici and H. Holtzer, Difference among sulphated proteoglycans synthesised in nonchondrogenic cells, presumptive chondroblasts and chondroblasts. Proc. Natl. Acad. Sci U.S.A., 73 (1976) 3224-3228. 10 E.J. Miller, Isolation and characterisation of a collagen from chick cartilage containing three identical a chains. Biochemistry, 10 (1971) 1652-1658. 11 T.F. Linsenmayer and C.D, Little, Embryonic neural retina collagen: In vitro synthesis of high molecular weight forms of type II plus a new genetic type. Proc. Natl. Acad. Sci. U.S,A., 75 (1078) 3235-3239. 12 K. Madsen and S. Lohmander, Production of cartilage-type proteoglycans in cultures of chondrocytes from elastic cartilage. Arch. Biochem. Biophys., 196 (1979) 192-198. 13 P.D. Benya, S.R. Padilla and M.E. Nimni, The progeny of rabbit articular chondrocytes synthesize collagen types 1 and Ill and type 1 trimer, but not type 11, Verification by cyanogen bromide peptide analysis. Biochemistry, 16 (1977) 865-872. 14 W.T. Green, Behavior of articular chondrocytes in cell culture. Clin. Orthop., 75 (1971) 248-260. 15 W.T. Green and R.J, Ferguson, Histochemical and electron microscopic comparison of tissue produced by rabbit articular chondrocytes in vivo and in vitro. Arthritis Rheum., 18 (1075) 273-280. 16 H. Mankin, Mitosis in articular cartilage of immature rabbits. Clin. Orthop., 34 (1964) 170--176. 17 J.C. Jeanny and M. Gontcharoff, l~tude en microscopie 61ectronique et par cytoph,otometrie /a balayage de la structure et la distribution de la chromatine dans les noyaux des cellules cartilageneuse de la Triticus cristatus au cours de la s6nescence. Biol Cell., ,t2 (1978) 233 244. 18 C.H. Barnett, W. Cochrane and A.J. Palfrey, Age changes in articular cartilage of rabbits. Ann. Rheum. Dis., 22 (1963) 389-40{I. 19 R. Silberberg, M. Hasler and P.A. Lesker, Aging of the shoulder joint of guinea pigs. Electron microscopic and quantitative histochemical aspects. J. Gerontol., 28 (1973) 18-36. 20 R. Silberberg, Ultrastructure of articular cartilage in health and disease, Clin, Orthop., 57 (196~) 233-257. 21 R. Silberberg and P. Lesker, Enzyme activity in aging articular cartilage. Experientia, 27 (19711 133-135. 22 l.J. Singh and E.A. Tonna, Autoradiographic evaluation of 3bl-uridine utilization by ageing mouse cartilage. Gerontology, 12 (1972) 41-48. 23 R. Mayne, M.S. Vail, P.M. Mayne and E.J. Miller, Changes in type of collagen synthesised as clones of chick chondrocytes grow and eventually lose division capacity. Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 1674-1678. 24 C.H, Evans, H.I. Georgescu and R.A. Mazzocchi, Does cellular ageing of chondrocytes engender primary osteoarthritis? Trans. Orthop. Res. Soc., 6 (1981) 153. 25 W.T. Green, Articular cartilage repair. Behavior of rabbit chondrocytes during tissue culture and subsequent allografting. Clin. Orthop., 124 (1977) 237-250.
191 26 C.H. Evans, P. Van Gansen and I. Rassen, Transcription in early and late passage embryonic mouse fibroblasts in primary culture: a correlated biochemical, autoradiographical and ultrastructural study. Biol. Cell., 33 (1978) 117-128. 27 C. Weiss, Ultrastructural characteristics of osteoarthritis. Fed. Proc. 32 (1975) 1459-1466. 28 R. Silberberg, M. Silberberg and D. Feir, Life cycle of articular cartilage cells: an electron microscopic study of the hip joint of the mouse. A m . J. Anat., 114 (1964) 17-22. 29 M. Nimni and K. Deshmukh, Differences in collagen metabolism between normal and osteoarthritic human articular cartilage. Science, 181 (1973) 751-752. 30 S. Gey, P.K. Muller, C. Lemmer, K. Remberger, K. Matzen and K. Kuhn, Immunohistological study on collagen in cartilage-bone metamorphosis and degenerative osteoarthritis. Klin. Wochenschr., 54 (1976) 969-976.