Calcif. Tiss. Res. 19, 179--187 (1975) 9 by Springer-Verlag 1975
Osteogenesis by Chondrocytes from Growth Cartilage of Rat Rib u
S h i m o m u r a , T. u
a n d F. Suzuki
Department of Biochemistry, Osaka University, Dental School, Osaka Received March 6, accepted July 18, 1975 Chondrocytes were isolated from growth cartilage and resting cartilage of rat rib and cultivated in vitro. The cultivated chondrocytes were placed in Millipore diffusion chambers, which were then implanted into the abdominal cavities of rats for several weeks and prepared for histological analysis. The results indicate that growth cartilage cells have a remarkable osteogenic potential, even after cultivation in vitro, whereas resting cartilage cells show no osteogenic activity. However, growth cartilage cells alone do not form new bone but require the participation of certain host cells to initiate osteogenic differentiation. Key words: Osteogenesis - - Chondrocytes - - Growth cartilage.
Introduction The process of replacing cartilage b y bone is k n o w n as e n d o c h o n d r a l ossification. D u r i n g t h e first stages of conversion into bone, e p i p h y s e a l cartilage cells increase t h e i r r a t e of proliferation, enlarge, a n d become h y p e r t r o p h i c . I t has been a s s u m e d in t h e p a s t t h a t t h e h y p e r t r o p h i c cells u n d e r g o d e g e n e r a t i v e changes a n d d e a t h [1, 2, 3, 8, 17, 22]. However, H o l t r o p , on t h e basis of e x p e r i m e n t a l t r a n s p l a n t a t i o n of g r o w t h cartilage labeled w i t h 3 H - t h y m i d i n e [10, 11, 12], suggested t h a t some of t h e cells survive a n d r e d i f f e r e n t i a t e in t h e m e t a p h y s i s to c o n t r i b u t e to ossification. S h i m o m u r a a n d R a y h a v e confirmed her findings t h a t h y p e r t r o p h i c c h o n d r o c y t e s can survive a n d t r a n s f o r m into bone-forming cells [19 ]. R e c e n t i n v e s t i g a t i o n s using electron m i c r o s c o p y h a v e led to opposing interpret a t i o n s [3, 13, 14, 20], a n d c o n t r o v e r s y persists concerning t h e m e c h a n i s m of e n d o c h o n d r a l ossification. I n t h e p r e s e n t s t u d y , h o n d r o c y t e s i s o l a t e d from g r o w t h cartilage of r a t rib were c u l t i v a t e d in vitro a n d t r a n s p l a n t e d to i n v e s t i g a t e t h e m e c h a n i s m of endoc h o n d r a l ossification.
Materials and Methods The costochondral junction was removed with aseptic technique from the ribs of 10 young Sprague-Dawley rats, weighing 100-120 g. After removing its surrounding soft tissue, growth cartilages (GC) were cut off and chopped up with a scalpel. They were incubated twice with 4 ml of 0.1% EDTA in Ca ~+- and Mg2+-free, balanced salt solution for 20 min at 37 ~ Then the material was digested successively with 4 ml of 0.2% trypsin for 1 h and with 4 ml of 0.2% collagenase for 3 h at 37 ~ The resulting cell suspension was filtered through a nylon sieve of 45 ~m pore size. The filtrate was centrifuged and the pellet was washed with Ham's F-12 medium (Nissui Pharmaceutical Co.) supplemented with 10% fetal calf serum (GIBCO) and 50 ~g/ml of ascorbic acid [18]. Chondroeytes from resting cartilage (RC) were obtained from the same rib in a similar manner. For reprints: Fujio Suzuki, Ph. D., Department of Biochemistry, Osaka University, Dental School, 32, Joancho, Kitaku, Osaka 530, Japan.
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Fig. 1. GC cells (chondrocytes from growth cartilage), cultivated for 1 week. The cells are polygonal and the accumulated extracellular material is refractile. W h e n stained with toluidine blue, the latter shows metachromasia. Phase contrast, • 100
Inocula of 3-5 • 10~ cells/5 ml were cultivated in 50 mm Petri dishes (Falcon) for 1 week at 37 ~ in a gas-flow, humidified incubator (5% C0 z in air). After 1 week, the cells were detached from the dishes by t r e a t m e n t with a mixture of 0.2% trypsin and 0.2% collagenase (1:2, v/v) for 1 h at 37 ~ and washed and resuspended in the complete medium [4]. Experiment 1. The chondrocytes obtained were then placed in diffusion chambers made with Millipore HA-membranes, 100 ~m thick and 0.45 ~m pore size, a t a cell density of 5 • 104 1 • 10~ cells/0.2 ml/ehamber. As controls, pieces of intact GC tissue were placed in similar chambers. The chondroeytes or GC tissue sealed in the chambers were transplanted intraperitoneally as isografts into male Sprague-Dawley rats, weighing 100-120 g. The animals were killed 2 a n d 6 weeks after the operation and the implanted chambers were removed. Experiment 2. The chambers containing pieces of GC tissue or cultivated cartilage cells were implanted into the abdominal cavities of rats, as described in Experiment 1. Two weeks later, they were removed from the host rats, the filters punctured with a 21 gauge syringe needle, a n d each chamber returned to the abdominal cavity of the same r a t where it was left for another 4 weeks. All specimens were fixed in 10% neutral formalin and decalcified in 10% EDTA. They were t h e n dehydrated, embedded in paraffin and sectioned at 5 ~m thickness. Sections were stained with hematoxylin a n d eosin. Host rats which developed infection or died during the experimental period were eliminated from the results.
Results Cell Culture T h e y i e l d of c h o n d r o c y t e s f r o m GC t i s s u e of cells. T h e y i e l d of R C cells w a s o n l y o n e - f o u r t h of G C cells, w h e n g r o w n i n H a m ' s F - 1 2 m e d i u m calf s e r u m in vitro w e r e f o u n d t o b e p o l y g o n a l
10 r a t s w a s 6.0 • 106-8.0 • 108 t h a t of G C cells. supplemented with 10% fetal and epithelial-like. The tissue
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Fig. 2. RC cells (chondrocytes from resting cartilage), cultivated for 1 week. The cells are fibroblastic and not polygonal, showing less intense metachromasia when stained with toluidine blue. Phase contrast, • 100
exhibited properties of well-differentiated cartilage [15], including the formation of a refractile matrix and metachromatic staining with toluidin blue, as shown in Fig. 1. On the other hand, RC cells grown under tile same conditions as GC cells were not polygonal but spindle-shaped like fibroblasts and showed less intense metachromasia, as shown in Fig. 2. The details of the characteristics of these cells will be published in another paper.
Transplantation I t has been shown by Lacroix [17], Gillette [6] and Holtrop [10, 11, 12] that transplanted GC forms new bone on the metaphyseal side within 2 weeks after operation, although no new bone was observed with transplanted RC. These results were confirmed by Shimomura and R a y (not published)i I n the present study, intact pieces of GC or chondrocytes cultivated in vitro were placed in diffusion chambers constructed with Millipore membranes and transplanted intraperitoneally into rats.
a) Results o/Experiment 1. In the chambers the control pieces of GC showed remarkable outgrowth along the inner surface of the membrane, with no bone formation occurring throughout the period from 2 to 6 weeks after transplantation. When chambers containing cultivated RC or GC cells were removed 2 weeks after implantation, they were filled with cells and matrix, so that their contents were no longer fluid. No new bone formed in 3 chambers with pieces of GC, 5 chambers with RC cells and 5 chambers with GC cells. The chambers of cultivated RC cells contained disorganized masses of chondroeytes which had poorly proliferated even after 6 weeks, although they appeared 2 Calcif. Tiss. Res.
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Fig. 3. Cultivated GC cells in a Millipore chamber, 6 weeks after transplantation into the abdominal cavity of rat, showing high proliferation rate and the cartilage cells occasionally appear to be realigned into column-like structures. Hematoxylin and eosin, X 200 Table 1. Results of experiment la Materials
Number of chambers implanted
Number of chambers with newly-formed bone (after 6 weeks)
Piece of GC tissue Cultivated RC cells Cultivated GC cells
6 8 8
0 0 1b
GC ~ Growth cartilage, RC ~ Resting cartilage. a See text for details. The results of transplantation for two weeks are not tabulated. b The filter of the chamber had a tiny fracture.
to be viable. I n contrast, c u l t i v a t e d GC cells proliferated r a p i d l y a n d sometimes appeared to be realigned into a column-like s t r u c t u r e w i t h i n 6 weeks (Fig. 3). This m a y indicate the d e v e l o p m e n t of GC cells toward c n d o c h o n d r a l ossification. However, no new bone formed in 21 chambers, even 6 weeks after t r a n s p l a n t a t i o n , as shown i n Table 1, b u t new bone formed i n only one c h a m b e r c o n t a i n i n g cultiv a t e d GC cells. The filter of one c h a m b e r of c u l t i v a t e d GC cells (6 weeks) h a d a t i n y fracture which p e r m i t t e d the host cells to stream into the c h a m b e r a n d produce new bone (Fig. 4). This newly formed bone c o n t a i n e d a large marrow c a v i t y a n d typical cartilage tissue was f o u n d persisting i n its vicinity. This finding suggested t h a t some u n k n o w n t y p e of host cell e n h a n c e d the osteogenic p o t e n t i a l of the chondrocytes. E x p e r i m e n t 2 was carried out to confirm this possibility.
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Fig. 4. Cultivated GC ceils in a ruptured Millipore chamber, 6 weeks after transplantation into the abdominal cavity of rat, forming a new bone which contains a large marrow cavity. Persistent cartilage islands are seen in its vicinity. Hematoxylin and eosin, • 100
Fig. 5. Cultivated RC cells in a punctured Millipore chamber, 6 weeks after transplantation into the abdominal cavity of rat, do not form new bone. Pairs of cartilage cells are occasionally seen. Hematoxylin and eosin, • 200
b) Results o/ Experiment 2. W h e n the chambers with needle holes were removed, the c u l t i v a t e d RC cells in the chambers resembled the original resting cartilage tissue, pairs of cells being seen occasionally (Fig. 5). No n e w bone f o r m a t i o n was observed, although the invasion b y the host cells was evident. On
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Fig. 6. (a) Cultivated GC cells in a punctured Millipore chamber 6 weeks after transplantation into the abdominal cavity of rat, forming a large a m o u n t of new bone containing a m~rrow cavity. A needle hole is seen in the lower Ieft. The connective tissue cells Crom t h e host stream into the chamber accompanying with vascular buds. The distance between two filters is increased, suggesting an increased volume of contents. Hematoxylin a n d eesin, • 40 (b) A higher magnification o5 Fig. 6a. Hematoxylin and eosin, • 100
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Table 2. Results of Experiment 2 a Materials
Number of chambers implanted
Number of chambers with newly formed bone
Piece of GC tissue Cultivated RC cells Cultivated GC cells
6 7 6
5b 0 6
GC = Growth cartilage, RC ~ Resting cartilage. a See text, for details. b One case failed to form new bone. The reason is not clear.
the other hand, the pieces of GC or cultivated GC cells showed advanced stages of endoehondral ossification with connective tissue cells accompanied by vascular buds streaming into the chamber from the host tissue through the needle holes in 11 out of 12 chambers (Fig. 6a and 6b). The summary of results is shown in Table 2. Discussion Early work, based principally on embryonic chick cartilage, showed that chondrocytes proliferating in vitro rapidly lose their cartilagenous properties and resemble fibroblasts [15]. However, several recent reports indicate that chondroid expression persists under certain conditions [5, 7, 9]. Utilizing the technique presented in this paper, homogenous "differentiated" cells [15], i.e., polygonal cells producing a metachromatic extracellular material has become available from GC tissue. The introduction of this culture system may enable us to study the role of the hypertrophic cells and the mechanism of endochondral ossification. The fate of hypertrophic cells in the growth cartilage remains controversial. I t has been assumed by many investigators that the last hypertrophic cell in the cell column of growth cartilage usually dies [1, 2, 3, 8, 17, 21, 22] as a result of interrupted nutrition caused by provisional calcification of the intracellular matrix. Also, it has been postulated that bone is laid down by osteogenic precursor cells derived from the walls of proliferating sinusoid vessels [22] or some other undifferentiated cell source [17, 23, 24, 25]. Thus it has been a predominant idea since Lacroix [17] that the osteogenic cells arc formed through an induction mechanism, whatever the responding cells arc. However, Holtrop [10, 11, 12 ] and Shimomura and R a y [19 ] maintain that the hypertrophic chondroeytes can survive and transform into osteoblasts and osteocytes. Support is given by Kuhlman [16] from his biochemical study and by Holtrop [13, 14] and Silberman eta/. [20] with their electron microscopic findings. Our results as described in this paper indicate that GC cells have a remarkable osteogenie potential, even after cultivation in vitro, whereas RC cells showed no osteogenic activity during the experiment. However, GC cells alone did not form new bone but required the participation of certain host cells to initiate osteogenic activity. There remains to be considered in more detail the role of
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h y p e r t r o p h i c c h o n d r o c y t e s a n d u n k n o w n r e c i p i e n t cells in e n d o c h o n d r a l ossific a t i o n ; in o t h e r words, t h e f u n d a m e n t a l m e c h a n i s m of b o n e i n d u c t i o n , if one exists. Acknowledgement. The authors wish to thank Professor Y. Takeda, Osaka University, for his support and continued interest in this work. They are very grateful to Dr. M. Sato, Osaka University, and Professor T. S. Okada, Kyoto University, for valuable advice on the ceil culture techniques. Their special thanks go to Professors K. Kawakatsu and T. Miyazaki, Osaka University, for their encouragement. Finally, thanks are also due to Mrs. E. Ichihara and Z. Roda for assistance in the preparation of this manuscript.
References 1. Bentley, G., Greer, R. B.: The fate of chondrocytes in cndochondral ossification in the rabbit. J. Bone Jt. Surg. B 52, 571-577 (1970) 2. Bloom, W., Fawcett, D. W.: A textbook of histology, 9th ed., p. 212 262. PhiladelphiaLondon-Toronto: W. B. Saunders Co. 1968 3. Brighton, C. T., Sugioka, Y., Hunt, R. : Cytoplasmic structures of epiphyseal plate chondrocytes; Quantitative evaluation using electron micrographs of rat costochondral junctions with special reference to the fate of hypertrophic cells. J. Bone Jr. Surg. A 55, 771-784 (1973) 4. Cahn, R . D . , Coon, H. G., Cahn, M.B.: Growth of differentiated cells: Cell culture and cloning techniques. In: Methods in developmental biology, (Wilt, F., Wessells, N. K., eds.), p. 493-530. New York: Thomas Crowell 1967 5. Coon, H . G . : Clonal stability and phenotypic expression of chick cartilage cells. Proc. nat. Acad. Sci. (Wash.) 55, 66-73 (1966) 6. Gillette, R., Mardfin, D., Schour, I.: Osteogenesis in subcutaneous rib transplants between normal and ia rats. Amer. J. Anat. 99, 447-471 (1956) 7 Green, W.T., Jr. : Behavior of articular chondrocytes in cell culture. Clin. Orthop. 75, 248-260 (1971) 8. Ham, A. W.: Histology, 5th ed., p. 373-450. Philadelphia and Montreal: J. B. Lippincott Co. 1965 9. Ham, R. G., Sattler, G. L.: Clonal growth of differentiated rabbit cartilage cells. J. cell. Physiol. 72, 109-114 (1968) 10. Holtrop, M. E. : The origin of bone cells in endochondral ossification. In: Calcified tissues, (Fleisch, H., ed.), p. 32 36. Berlin-Heidelberg-New York: Springer 1966 11. Holtrop, M. E. : The potencies of the epiphyseal cartilage in endochondral ossification. Proc. kon. ned. Akad. Wet. Ser. C 70, 21-28 (1967) 12. Holtrop, M. E. : Factors influencing the growth rate in endochondral ossification. Proc. kon. ned. Akad. Wet. Ser. C 79, 29 38 (1967) 13. Holtrop, M. E. : The ultrastructure of the epiphyseal plate. I. The flattened chondrocytes. Calcif. Tiss. Res. 9, 131-139 (1972) 14. Holtrop, M. E. : The ultrastructure of the epiphyseal plate. II. The hypertrophic chondrocytes. Calcif. Tiss. Res. 9, 140-151 (1972) 15. Holtzer, H., Abbott, J., Lash, J., Holtzer, S.: The loss of phenotypic traits by differentiated cells in vitro. I. Dedifferentiation of cartilage cells. Proc. nat. Acad. Sci. (Wash.) 46, 1533-1542 (1960) 16. Kuhlman, R . E . , McNamee, M . J . : The biochemical importance of the hypertrophic cartilage cell area to enchondral bone formation. J. Bone Jt. Surg. A 52, 1025-1033 (1970) 17. Laeroix, P.: The organization of bones, p. 1-235. London: J. A. Churchill 1951 18. Levenson, G . E . : The effect of ascorbic acid on monolayer cultures of three types of chondrocytes. Exp. Cell Res. 55, 225-228 (1969) 19. Shimomura, Y., Ray, R. D. : The fate of the hypertrophic cells in the growth cart;lage I. Transplantation of the growth cartilage. Cent. Jap. J. orthop, traumat. Surg. 16, 726-728 (1973)
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20. Silberman, M., Frommer, J. : Ultrastructure of developing cartilage in the mandibular condyle of the mouse. Acta anat. (Basel) 90, 330-346 (1974) 21. Sissons, H. A. : The growth of bone. In: Biochemistry and physiology of bone (Bourne, G. H., ed.), vol. III, p. 145-180. New York: Academic Press 1971 22. Trueta, J.: The role of the vessels in osteogenesis. J. Bone Jr. Surg. B 45, 402418 (1963) 23. Urist, M. R., McLean, F. C. : Osteogenic potency and new bone formation by induction in transplants to the anterior chamber of the eye. J. Bone Jr. Surg. A 34, 443-470 (1952) 24. Urist, M. 1~. : Bone formation by autoinduction. Science I~0, 893-899 (1965) 25. Urist, M. R., Dowel1, T. A., Hay, P . H . , Strates, B. S.: Inductive substrates for bone formation. Clin. Orthop. ~9, 59-96 (1968)
Osteogenesis by Chondrocytes from Growth Cartilage of Rat Rib u
S h i m o m u r a , T. u
a n d F. Suzuki
Department of Biochemistry, Osaka University, Dental School, Osaka Received March 6, accepted July 18, 1975 Chondrocytes were isolated from growth cartilage and resting cartilage of rat rib and cultivated in vitro. The cultivated chondrocytes were placed in Millipore diffusion chambers, which were then implanted into the abdominal cavities of rats for several weeks and prepared for histological analysis. The results indicate that growth cartilage cells have a remarkable osteogenic potential, even after cultivation in vitro, whereas resting cartilage cells show no osteogenic activity. However, growth cartilage cells alone do not form new bone but require the participation of certain host cells to initiate osteogenic differentiation. Key words: Osteogenesis - - Chondrocytes - - Growth cartilage.
Introduction The process of replacing cartilage b y bone is k n o w n as e n d o c h o n d r a l ossification. D u r i n g t h e first stages of conversion into bone, e p i p h y s e a l cartilage cells increase t h e i r r a t e of proliferation, enlarge, a n d become h y p e r t r o p h i c . I t has been a s s u m e d in t h e p a s t t h a t t h e h y p e r t r o p h i c cells u n d e r g o d e g e n e r a t i v e changes a n d d e a t h [1, 2, 3, 8, 17, 22]. However, H o l t r o p , on t h e basis of e x p e r i m e n t a l t r a n s p l a n t a t i o n of g r o w t h cartilage labeled w i t h 3 H - t h y m i d i n e [10, 11, 12], suggested t h a t some of t h e cells survive a n d r e d i f f e r e n t i a t e in t h e m e t a p h y s i s to c o n t r i b u t e to ossification. S h i m o m u r a a n d R a y h a v e confirmed her findings t h a t h y p e r t r o p h i c c h o n d r o c y t e s can survive a n d t r a n s f o r m into bone-forming cells [19 ]. R e c e n t i n v e s t i g a t i o n s using electron m i c r o s c o p y h a v e led to opposing interpret a t i o n s [3, 13, 14, 20], a n d c o n t r o v e r s y persists concerning t h e m e c h a n i s m of e n d o c h o n d r a l ossification. I n t h e p r e s e n t s t u d y , h o n d r o c y t e s i s o l a t e d from g r o w t h cartilage of r a t rib were c u l t i v a t e d in vitro a n d t r a n s p l a n t e d to i n v e s t i g a t e t h e m e c h a n i s m of endoc h o n d r a l ossification.
Materials and Methods The costochondral junction was removed with aseptic technique from the ribs of 10 young Sprague-Dawley rats, weighing 100-120 g. After removing its surrounding soft tissue, growth cartilages (GC) were cut off and chopped up with a scalpel. They were incubated twice with 4 ml of 0.1% EDTA in Ca ~+- and Mg2+-free, balanced salt solution for 20 min at 37 ~ Then the material was digested successively with 4 ml of 0.2% trypsin for 1 h and with 4 ml of 0.2% collagenase for 3 h at 37 ~ The resulting cell suspension was filtered through a nylon sieve of 45 ~m pore size. The filtrate was centrifuged and the pellet was washed with Ham's F-12 medium (Nissui Pharmaceutical Co.) supplemented with 10% fetal calf serum (GIBCO) and 50 ~g/ml of ascorbic acid [18]. Chondroeytes from resting cartilage (RC) were obtained from the same rib in a similar manner. For reprints: Fujio Suzuki, Ph. D., Department of Biochemistry, Osaka University, Dental School, 32, Joancho, Kitaku, Osaka 530, Japan.
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Fig. 1. GC cells (chondrocytes from growth cartilage), cultivated for 1 week. The cells are polygonal and the accumulated extracellular material is refractile. W h e n stained with toluidine blue, the latter shows metachromasia. Phase contrast, • 100
Inocula of 3-5 • 10~ cells/5 ml were cultivated in 50 mm Petri dishes (Falcon) for 1 week at 37 ~ in a gas-flow, humidified incubator (5% C0 z in air). After 1 week, the cells were detached from the dishes by t r e a t m e n t with a mixture of 0.2% trypsin and 0.2% collagenase (1:2, v/v) for 1 h at 37 ~ and washed and resuspended in the complete medium [4]. Experiment 1. The chondrocytes obtained were then placed in diffusion chambers made with Millipore HA-membranes, 100 ~m thick and 0.45 ~m pore size, a t a cell density of 5 • 104 1 • 10~ cells/0.2 ml/ehamber. As controls, pieces of intact GC tissue were placed in similar chambers. The chondroeytes or GC tissue sealed in the chambers were transplanted intraperitoneally as isografts into male Sprague-Dawley rats, weighing 100-120 g. The animals were killed 2 a n d 6 weeks after the operation and the implanted chambers were removed. Experiment 2. The chambers containing pieces of GC tissue or cultivated cartilage cells were implanted into the abdominal cavities of rats, as described in Experiment 1. Two weeks later, they were removed from the host rats, the filters punctured with a 21 gauge syringe needle, a n d each chamber returned to the abdominal cavity of the same r a t where it was left for another 4 weeks. All specimens were fixed in 10% neutral formalin and decalcified in 10% EDTA. They were t h e n dehydrated, embedded in paraffin and sectioned at 5 ~m thickness. Sections were stained with hematoxylin a n d eosin. Host rats which developed infection or died during the experimental period were eliminated from the results.
Results Cell Culture T h e y i e l d of c h o n d r o c y t e s f r o m GC t i s s u e of cells. T h e y i e l d of R C cells w a s o n l y o n e - f o u r t h of G C cells, w h e n g r o w n i n H a m ' s F - 1 2 m e d i u m calf s e r u m in vitro w e r e f o u n d t o b e p o l y g o n a l
10 r a t s w a s 6.0 • 106-8.0 • 108 t h a t of G C cells. supplemented with 10% fetal and epithelial-like. The tissue
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Fig. 2. RC cells (chondrocytes from resting cartilage), cultivated for 1 week. The cells are fibroblastic and not polygonal, showing less intense metachromasia when stained with toluidine blue. Phase contrast, • 100
exhibited properties of well-differentiated cartilage [15], including the formation of a refractile matrix and metachromatic staining with toluidin blue, as shown in Fig. 1. On the other hand, RC cells grown under tile same conditions as GC cells were not polygonal but spindle-shaped like fibroblasts and showed less intense metachromasia, as shown in Fig. 2. The details of the characteristics of these cells will be published in another paper.
Transplantation I t has been shown by Lacroix [17], Gillette [6] and Holtrop [10, 11, 12] that transplanted GC forms new bone on the metaphyseal side within 2 weeks after operation, although no new bone was observed with transplanted RC. These results were confirmed by Shimomura and R a y (not published)i I n the present study, intact pieces of GC or chondrocytes cultivated in vitro were placed in diffusion chambers constructed with Millipore membranes and transplanted intraperitoneally into rats.
a) Results o/Experiment 1. In the chambers the control pieces of GC showed remarkable outgrowth along the inner surface of the membrane, with no bone formation occurring throughout the period from 2 to 6 weeks after transplantation. When chambers containing cultivated RC or GC cells were removed 2 weeks after implantation, they were filled with cells and matrix, so that their contents were no longer fluid. No new bone formed in 3 chambers with pieces of GC, 5 chambers with RC cells and 5 chambers with GC cells. The chambers of cultivated RC cells contained disorganized masses of chondroeytes which had poorly proliferated even after 6 weeks, although they appeared 2 Calcif. Tiss. Res.
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Fig. 3. Cultivated GC cells in a Millipore chamber, 6 weeks after transplantation into the abdominal cavity of rat, showing high proliferation rate and the cartilage cells occasionally appear to be realigned into column-like structures. Hematoxylin and eosin, X 200 Table 1. Results of experiment la Materials
Number of chambers implanted
Number of chambers with newly-formed bone (after 6 weeks)
Piece of GC tissue Cultivated RC cells Cultivated GC cells
6 8 8
0 0 1b
GC ~ Growth cartilage, RC ~ Resting cartilage. a See text for details. The results of transplantation for two weeks are not tabulated. b The filter of the chamber had a tiny fracture.
to be viable. I n contrast, c u l t i v a t e d GC cells proliferated r a p i d l y a n d sometimes appeared to be realigned into a column-like s t r u c t u r e w i t h i n 6 weeks (Fig. 3). This m a y indicate the d e v e l o p m e n t of GC cells toward c n d o c h o n d r a l ossification. However, no new bone formed in 21 chambers, even 6 weeks after t r a n s p l a n t a t i o n , as shown i n Table 1, b u t new bone formed i n only one c h a m b e r c o n t a i n i n g cultiv a t e d GC cells. The filter of one c h a m b e r of c u l t i v a t e d GC cells (6 weeks) h a d a t i n y fracture which p e r m i t t e d the host cells to stream into the c h a m b e r a n d produce new bone (Fig. 4). This newly formed bone c o n t a i n e d a large marrow c a v i t y a n d typical cartilage tissue was f o u n d persisting i n its vicinity. This finding suggested t h a t some u n k n o w n t y p e of host cell e n h a n c e d the osteogenic p o t e n t i a l of the chondrocytes. E x p e r i m e n t 2 was carried out to confirm this possibility.
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Fig. 4. Cultivated GC ceils in a ruptured Millipore chamber, 6 weeks after transplantation into the abdominal cavity of rat, forming a new bone which contains a large marrow cavity. Persistent cartilage islands are seen in its vicinity. Hematoxylin and eosin, • 100
Fig. 5. Cultivated RC cells in a punctured Millipore chamber, 6 weeks after transplantation into the abdominal cavity of rat, do not form new bone. Pairs of cartilage cells are occasionally seen. Hematoxylin and eosin, • 200
b) Results o/ Experiment 2. W h e n the chambers with needle holes were removed, the c u l t i v a t e d RC cells in the chambers resembled the original resting cartilage tissue, pairs of cells being seen occasionally (Fig. 5). No n e w bone f o r m a t i o n was observed, although the invasion b y the host cells was evident. On
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Fig. 6. (a) Cultivated GC cells in a punctured Millipore chamber 6 weeks after transplantation into the abdominal cavity of rat, forming a large a m o u n t of new bone containing a m~rrow cavity. A needle hole is seen in the lower Ieft. The connective tissue cells Crom t h e host stream into the chamber accompanying with vascular buds. The distance between two filters is increased, suggesting an increased volume of contents. Hematoxylin a n d eesin, • 40 (b) A higher magnification o5 Fig. 6a. Hematoxylin and eosin, • 100
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Table 2. Results of Experiment 2 a Materials
Number of chambers implanted
Number of chambers with newly formed bone
Piece of GC tissue Cultivated RC cells Cultivated GC cells
6 7 6
5b 0 6
GC = Growth cartilage, RC ~ Resting cartilage. a See text, for details. b One case failed to form new bone. The reason is not clear.
the other hand, the pieces of GC or cultivated GC cells showed advanced stages of endoehondral ossification with connective tissue cells accompanied by vascular buds streaming into the chamber from the host tissue through the needle holes in 11 out of 12 chambers (Fig. 6a and 6b). The summary of results is shown in Table 2. Discussion Early work, based principally on embryonic chick cartilage, showed that chondrocytes proliferating in vitro rapidly lose their cartilagenous properties and resemble fibroblasts [15]. However, several recent reports indicate that chondroid expression persists under certain conditions [5, 7, 9]. Utilizing the technique presented in this paper, homogenous "differentiated" cells [15], i.e., polygonal cells producing a metachromatic extracellular material has become available from GC tissue. The introduction of this culture system may enable us to study the role of the hypertrophic cells and the mechanism of endochondral ossification. The fate of hypertrophic cells in the growth cartilage remains controversial. I t has been assumed by many investigators that the last hypertrophic cell in the cell column of growth cartilage usually dies [1, 2, 3, 8, 17, 21, 22] as a result of interrupted nutrition caused by provisional calcification of the intracellular matrix. Also, it has been postulated that bone is laid down by osteogenic precursor cells derived from the walls of proliferating sinusoid vessels [22] or some other undifferentiated cell source [17, 23, 24, 25]. Thus it has been a predominant idea since Lacroix [17] that the osteogenic cells arc formed through an induction mechanism, whatever the responding cells arc. However, Holtrop [10, 11, 12 ] and Shimomura and R a y [19 ] maintain that the hypertrophic chondroeytes can survive and transform into osteoblasts and osteocytes. Support is given by Kuhlman [16] from his biochemical study and by Holtrop [13, 14] and Silberman eta/. [20] with their electron microscopic findings. Our results as described in this paper indicate that GC cells have a remarkable osteogenie potential, even after cultivation in vitro, whereas RC cells showed no osteogenic activity during the experiment. However, GC cells alone did not form new bone but required the participation of certain host cells to initiate osteogenic activity. There remains to be considered in more detail the role of
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h y p e r t r o p h i c c h o n d r o c y t e s a n d u n k n o w n r e c i p i e n t cells in e n d o c h o n d r a l ossific a t i o n ; in o t h e r words, t h e f u n d a m e n t a l m e c h a n i s m of b o n e i n d u c t i o n , if one exists. Acknowledgement. The authors wish to thank Professor Y. Takeda, Osaka University, for his support and continued interest in this work. They are very grateful to Dr. M. Sato, Osaka University, and Professor T. S. Okada, Kyoto University, for valuable advice on the ceil culture techniques. Their special thanks go to Professors K. Kawakatsu and T. Miyazaki, Osaka University, for their encouragement. Finally, thanks are also due to Mrs. E. Ichihara and Z. Roda for assistance in the preparation of this manuscript.
References 1. Bentley, G., Greer, R. B.: The fate of chondrocytes in cndochondral ossification in the rabbit. J. Bone Jt. Surg. B 52, 571-577 (1970) 2. Bloom, W., Fawcett, D. W.: A textbook of histology, 9th ed., p. 212 262. PhiladelphiaLondon-Toronto: W. B. Saunders Co. 1968 3. Brighton, C. T., Sugioka, Y., Hunt, R. : Cytoplasmic structures of epiphyseal plate chondrocytes; Quantitative evaluation using electron micrographs of rat costochondral junctions with special reference to the fate of hypertrophic cells. J. Bone Jr. Surg. A 55, 771-784 (1973) 4. Cahn, R . D . , Coon, H. G., Cahn, M.B.: Growth of differentiated cells: Cell culture and cloning techniques. In: Methods in developmental biology, (Wilt, F., Wessells, N. K., eds.), p. 493-530. New York: Thomas Crowell 1967 5. Coon, H . G . : Clonal stability and phenotypic expression of chick cartilage cells. Proc. nat. Acad. Sci. (Wash.) 55, 66-73 (1966) 6. Gillette, R., Mardfin, D., Schour, I.: Osteogenesis in subcutaneous rib transplants between normal and ia rats. Amer. J. Anat. 99, 447-471 (1956) 7 Green, W.T., Jr. : Behavior of articular chondrocytes in cell culture. Clin. Orthop. 75, 248-260 (1971) 8. Ham, A. W.: Histology, 5th ed., p. 373-450. Philadelphia and Montreal: J. B. Lippincott Co. 1965 9. Ham, R. G., Sattler, G. L.: Clonal growth of differentiated rabbit cartilage cells. J. cell. Physiol. 72, 109-114 (1968) 10. Holtrop, M. E. : The origin of bone cells in endochondral ossification. In: Calcified tissues, (Fleisch, H., ed.), p. 32 36. Berlin-Heidelberg-New York: Springer 1966 11. Holtrop, M. E. : The potencies of the epiphyseal cartilage in endochondral ossification. Proc. kon. ned. Akad. Wet. Ser. C 70, 21-28 (1967) 12. Holtrop, M. E. : Factors influencing the growth rate in endochondral ossification. Proc. kon. ned. Akad. Wet. Ser. C 79, 29 38 (1967) 13. Holtrop, M. E. : The ultrastructure of the epiphyseal plate. I. The flattened chondrocytes. Calcif. Tiss. Res. 9, 131-139 (1972) 14. Holtrop, M. E. : The ultrastructure of the epiphyseal plate. II. The hypertrophic chondrocytes. Calcif. Tiss. Res. 9, 140-151 (1972) 15. Holtzer, H., Abbott, J., Lash, J., Holtzer, S.: The loss of phenotypic traits by differentiated cells in vitro. I. Dedifferentiation of cartilage cells. Proc. nat. Acad. Sci. (Wash.) 46, 1533-1542 (1960) 16. Kuhlman, R . E . , McNamee, M . J . : The biochemical importance of the hypertrophic cartilage cell area to enchondral bone formation. J. Bone Jt. Surg. A 52, 1025-1033 (1970) 17. Laeroix, P.: The organization of bones, p. 1-235. London: J. A. Churchill 1951 18. Levenson, G . E . : The effect of ascorbic acid on monolayer cultures of three types of chondrocytes. Exp. Cell Res. 55, 225-228 (1969) 19. Shimomura, Y., Ray, R. D. : The fate of the hypertrophic cells in the growth cart;lage I. Transplantation of the growth cartilage. Cent. Jap. J. orthop, traumat. Surg. 16, 726-728 (1973)
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