Proc. Natl. Acad. Sci. USA Vol. 74, No. 12, pp. 5589-5592, December 1977 Cell Biology Transitions in types during matrix-induced , , and formation (immunofluorescence/) A. H. REDDI*, RENATE GAYt, STEFFEN GAYt, AND EDWARD J. MILLERt * Laboratory of Biological Structure, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20014; and t Institute of Dental Research, University of Alabama, Birmingham, Alabama 35294 Communicated by Charles Huggins, September 19, 1977

ABSTRACT The localization of types I, II, and HI cartilage matrix consists of type II molecules. The availability during bone matrix-induced sequential differentiation of car- of purified, chemically characterized collagen types has led to tilage, bone, and bone marrow was studied by specific immu- characteriza- nofluorescence. Subcutaneous transplantation of coarse pow- the production, isolation, and immunochemical ders of demineralized rat bone matrix into allogeneic recipients tion of the collagen-type specific antibodies (7). This develop- resulted in new bone formation. After a transient appearance ment, in turn, resulted in the immunofluorescent localization of polymorphonuclear leukocytes in the implant, of collagen types in tissue sections (8, 9). Such immunohistolo- appeared in close continguity to the matrix on day 3. Type III gical localization is particularly useful in studies of the distri- collagen was then localized as a fine network around the in- bution of the various types of collagen in complex tissues with vading fibroblasts. On days 4-6 smaller amounts of type I were as developing bone, where a bio- also detected around these proliferating cells. With the onset a variety of cell types sucfi of chondrogenesis, colgen was detected in the cartilage chemical approach is of limited utility. This paper presents the matrix on day 6 and persisted until the early stages of bone sequential transitions in the developmental appearance of formation. Vascular invasion of the implant was accompanied collagen types I, II, and III during matrix-induced endochon- by osteogenesis on day 10. was demonstrated dral bone formation. in the newly deposited bone matrix coating the surfaces of cartilage spicules and particles of implanted bone powder. On day 17 and thereafter, type III collagen was localized as a fi- AND METHODS brous array around nests of hematopoietic cells. MATERIALS Preparation and Transplantation of Matrix. Dehydrated Collagens and proteoglycans are the two major classes of ex- diaphyseal shafts of rat femur and tibia were pulverized in a tracellular matrix macromolecules in skeletal and dental tissues. CRC Micro Mill (Technilab Instruments, Vineland, NJ) and An analysis of changes in these matrix constituents during cell sieved to a discrete particle size of 74-420 ,m. The powders differentiation and morphogenesis may lead to a better un- were demineralized with 0.5 M HC1, extracted with water, derstanding of the biological role of these macromolecules in ethanol, and ether, and prepared as described (1). Demineral- development. Transplantation of demineralized bone matrix ized bone matrix was transplanted subcutaneously during ether from the rat diaphysis to subcutaneous sites in allogeneic re- anesthesia in male rats of Long-Evans strain, age 28-35 days cipients results in new endochondral bone formation accom- (1). There were two sites over the thorax in each rat (2). On panied by hematopoietic bone marrow differentiation in the designated days, as indicated in Table 1, the rats killed and the newly formed ossicle (1-5). The details of the sequential cascade subcutaneous button-like plaques were dissected out and frozen of cell differentiation and morphogenesis have been described on the cryostat block. elsewhere (3). In brief, on transplantation of demineralized bone Preparation of Collagen Antibodies. Antibodies were matrix a transient inflammation-like response ensues. On day produced in rabbits, guinea pigs, and rats. The antigens used 3, responding fibroblasts (mesenchymal cells) appear in the were bovine and human types I and III collagens derived from vicinity of the matrix particles, interact with the matrix, pro- , as well as bovine and human type II collagen from ar- liferate, and then emerge as on days 5 and 6. ticular cartilage. Type-specific antibodies were purified by These chrondrocytes undergo hypertrophy and, on day 9, the immunoadsorption as described (9). onset of calcification of the matrix is evident. On day 10 vas- Indirect Immunofluorescence Localization. Frozen tissue cular invasion of the calcified cartilage matrix occurs, which sections (4-6 ,m) were cut with the aid of an Ames Cryostat at results in chondrolysis and initial osteogenesis. The newly -200. All sections used for type II collagen localization were formed bone is eventually remodeled by osteoclasts and func- processed with and without hyaluronidase pretreatment in tional bone marrow develops in the ossicle by day 21. order to reduce the masking due to proteoglycan constituents. The occurrence and distribution of several genetically distinct Tissue sections were exposed to purified antibodies (about 20 collagen types is now well established (6) and it is expected that ug/ml) for 30 min at room temperature. This was followed by the cellular transitions outlined above would be accompanied rinsing three times in phosphate-buffered saline (pH 7.4) for by alterations in the type of collagen produced. Types I and III 10 min each rinse. The sections were subsequently treated with collagens usually coexist in the same tissue, such as blood vessels, fluoresceinisothiocyanate(FITC)-conjugated goat antibodies dermis, uterus, and intestinal wall. On the other hand, only type to rabbit IgG (diluted 1:20) and FITC-conjugated goat anti- I collagen occurs in bone, , , and tooth, whereas bodies to guinea pig IgG or rat IgG (Behring Werke, Marburg, Germany and Nordic, Tilburg, The Netherlands). Sections were The costs of publication of this article were defrayed in part by the then mounted as described (10). The crossreaction of bovine payment of page charges. This article must therefore be hereby marked anticollagen antibodies with human tissues (9) and rat tissues "advertdiment" in accordance with 18 U. S. C. §1734 solely to indicate (11) has been documented. Thus, the reaction of antibodies to this fact. bovine collagens with the various collagen types in normal rat 5589 Downloaded by guest on September 24, 2021 5590 Cell Biology: Reddi et al. Proc. Natl. Acad. Sci. USA 74 (1977) Table 1. Distribution of collagen types I, II, and III in matrix-induced plaques Chondrocytes and Osteoblasts and Mesenchymal cells cartilage matrix bone matrix Bone marrow Day I II III I II III I II III I II III 3 - - ++ - ______4 (+) - ++ - 5 + - +++ - - - _ _ - 6 + - +++ + _ _ _ _ 7 - - +++ - ++ - _ _ _ 8 - - ++ - +++ _ _ _ _ 9 - - + (+) +++ ------10 - - + + +- + 11 - - + +++ 14 - - - - ++ - +++ - - - - - 17 - - - - + - +++ - - (+) - + 19 ------(+) - ++ 21 ------(+) - ++ Staining pattern: -, not detectable; (+), trace; +, small amount; ++, moderate; and +++, abundant. epiphyseal growth plate is very similar to that observed in drocytes and cartilaginous matrix, as evidenced by a significant normal human eiphyseal growth plate (12). Antibodies to type reduction in staining for type II collagen (Fig. 1). These events I collagen show a specific staining with bone osteoid, whereas were accompanied by an extensive increase in type I collagen antibodies against type II react with the matrix of hyaline staining in areas of osteogenesis. This was most noticeable in cartilage and antibodies against type III collagen react with the regions adjacent to the implanted matrix, as well as on calcified reticular fiber network of bone marrow. cartilage spicules. On day 17 and thereafter, around areas of active osteogenesis, RESULTS the development of hematopoietic bone marrow in certain On transplantation of demineralized diaphyseal bone matrix cavernous spaces was evident and was characterized by the into subcutaneous sites in allogenic recipients, a sequential appearance of a fine network of type III collagen fibers among matrix-cell interaction ensued, resulting in endochondral bone the hematopoietic cells (Fig. 1). formation (1-3). The alterations in prevalence of types I, II, and III collagens during endochondral bone formation are described DISCUSSION in detail in Table 1. Bone matrix-induced endochondral bone formation is a useful On days 1 and 2, a compact conglomerate of the implanted experimental model that recapitulates several aspects of bone matrix was observed accompanied by an inflammatory re- formation in epiphyseal growth plate (1, 5, 13). In addition, the sponse, which was indicated by the presence of strands of fibrin sequential cellular transitions are similar to that observed in as well as polymorphonuclear leukocytes. No collagen staining fracture healing. In view of these correlations, the present in- was detected. vestigation of changes in collagen types I, II, and III during bone On day 3 numerous mesenchymal cells appeared in close matrix-induced endochondral bone formation is informative. proximity to the implanted particles of bone matrix. At this In this regard, immunohistological localization reveals the exact stage, a fibrous network of type III collagen appeared; type II topography of the various collagen types in the changing tissue. and type I collagens were not observed at this stage (Fig. 1). This approach is thus more precise than techniques that require However, there was a faint reaction on the part of antibodies isolation of tissue elements and biochemical characterization to type I collagen with the implanted bone powder. of the collagen. Nevertheless, the immunohistological tech- On days 4 and 5 there was a progressive increase in the ap- niques are limited by the fact that certain tissue components pearance of fibers reactive with type III specific antibodies may mask the collagenous components in a given tissue. Fur- (Table 1). At this time, there was also a limited reaction for ther, as yet uncharacterized collagenous components may be antibodies to type I collagen in certain areas. present which share similar antigenic determinants with the On days 6-7, the onset of chondrogenesis was associated with well-characterized collagen types used as antigens in the present the appearance of chrondroblasts and the initial detection of study. material reacting with antibodies to type II collagen (Fig. 1). One of the salient findings in the present study is the occur- The type III collagen network persisted around the newly rence of type III collagen around the proliferating mesenchy- formed cartilaginous tissue on day 7. There was no detectable mal cells in contact with the implanted matrix. This particular appearance of type I collagen in chrondrogenic areas. Staining phase of the sequence is thus very similar to the reported in- for type I collagen was still observed over the surface of im- crease in type III collagen in rat and human tissues during the planted bone matrix. early phases of wound healing and the formation of granulation The accumulation of type II collagen in the extracellular tissue (11, 14). It thus appears that the early mesenchymal cells matrix continued to day 11 (Fig. 1). At this time, significant that start to synthesize predominantly type III collagen may amounts of type I collagen were observed in the form of parallel serve as progenitor cells which eventually differentiate to fiber bundles indicating the regions of osteogenesis. Concom- chondroblasts. This situation is, of course, different from that itantly, there was degeneration of hypertrophied chondrocytes observed in developing chick limb bud, both in vivo (15-17) and marked reduction of type III collagen around the carti- and in vitro (18). These studies indicated that mesenchymal laginous matrix. cells destined to form cartilage initially synthesized largely type On days 12-18 there was further disappearance of chon- I collagen (15, 16, 18). Downloaded by guest on September 24, 2021 Cell Biology: Reddi et al. Proc. Natl. Acad. Sci. USA 74 (1977) 5591

Type III Type 11 Type I

Day 3

Day 7

Day 1 1

Day 1 7

FIG. 1. Cryostat sections of bone matrix-induced plaques stained with antibodies directed against (Left) type III collagen, (Middle) type II collagen, and (Right) type I collagen. (X80.) B, implanted bone matrix particle. (Day 3) A loose network of fibers reacting with antibodies to (Left) type III collagen, and the absence of reactivity with antibodies to (Middle) type II collagen. (Right) There is a faint staining of implanted particles of bone matrix with antibodies to type I collagen. (Day 7) Persistence of staining with antibodies to (Left) type III collagen around proliferating chondrocytes which stain well with antibodies to (Middle) type II collagen. (Right) The most abundant staining with antibodies to type I collagen is still observed for the implanted bone particles. (Day 11) Slight reactivity with antibodies to (Left) type III collagen remains diffused around cartilage matrix, which is abundant in fibers derived from (Middle) type II collagen. (Right) At the same time, newly synthesized bundles of type I collagen can be observed (arrows). (Day 17) Calcified cartilage matrix in which staining with antibodies to (Middle) type II collagen can be observed surrounded by newly formed osteoid tissue in which there is a great deal of reactivity with antibodies to (Right) type I collagen. (Left) Moderate staining with antibodies to type III collagen is observed in areas of active bone marrow formation.

Chondrogenesis was associated with a localization of type II as a fibrous array in hematopoetic bone marrow is not collagen. With the onset of vascularization, there was osteo- known. genesis. The occurrence of type I collagen on the surfaces of In summary, the results of this study demonstrate the intri- type II collagen-reactive cartilage spicules is reminiscent of cate cellular changes with attendant transitions in the collagen earlier observations in developing chick long (17). The phenotype during matrix-induced endochondral bone mor- significance of the subsequent localization of type III collagen phogenesis. Downloaded by guest on September 24, 2021 5592 Cell Biology: Reddi et al. Proc. Natl. Acad. Sci. USA 74 (1977)

We thank Drs. S. B. Doty, G. R. Martin, K. A. Piez, and J. D. Termine dran G. N. & Reddi, A. H. (Plenum Press, New York), pp. for reviewing the manuscript. We extend our appreciation to Miss 318-375. Noreen E. Sullivan for excellent technical assistance. This work was 8. Timpl, R., Wick, G. & Gay, S. (1977) J. Immunol., in press. supported by U.S. Public Health Service Grants DE-02670 and HL- 9. Nowack, H., Gay, S., Wick, G., Becker, U. & Timpl, R. (1977) 11310. J. Immunol. Meth. 12, 117-124. 10. Gay, S., Martin, G. R., Muller, P. K., Timpl, R. & Kuhn, K. (1976) Proc. Natl. Acad. Sci. USA 73,4037-4040. 11. Kent, G., Gay, S., Inouye, T., Bahu, R., Minick, D. T. & Popper, H. (1976) Proc. Natl. Acad. Sci. USA 73,3719-3722. 1. Reddi, A. H. & Huggins, C. B. (1972) Proc. Natl. Acad. Sci. USA 12. Gay, S., Muller, P. K., Lemmen, C., Remberger, K., Matzen, K. 69, 1601-1605. & Kuhn, K. (1976) Klin. Wochenschr. 54,969-976. 2. Reddi, A. H. & Huggins, C. B. (1975) Proc. Natl. Acad. Sci. USA 13. Nakagawa, M. & Urist, M. R. (1977) Proc. Soc. Exp. Biol. Med. 72,2212-2216. 154,568-572. 3. Reddi, A. H. & Anderson, W. A. (1976) J. Cell Biol. 69, 557- 14. Bailey, A. J., Sims, T. J., LeLous, M. & Bazin, S. (1975) Biochem. 572. Biophys. Res. Commun. 66,1160-1165. 4. Reddi, A. H. (1975) in Influences on Gene 15. von der Mark, H., von der Mark, K. & Gay, S. (1976) Dev. Biol. Expression, eds. Slavkin, H. C. & Greulich, R. C. (Academic 48,237-249. Press, New York), pp. 619-625. 16. Linsenmeyer, T. F., Toole, B. P. & Trelstad, R. L. (1973) Dev. 5. Reddi, A. H. (1976) in Biochemistry of Collagen, eds. Rama- Biol. 35, 232-239. chandran, G. N. & Reddi, A. H. (Plenum Press, New York), pp. 17. von der Mark, K., von der Mark, H. & Gay, S. (1976) Dev. Biol. 449-478. 53, 153-170. 6. Miller, E. J. (1976) Mol. Cell Biochem. 13, 165-192. 18. von der Mark, K. & von der Mark, H. (1977) J. Cell Biol. 73, 7. Timpl, R. (1976) in Biochemistry of Collagen, eds. Ramachan- 736-747. Downloaded by guest on September 24, 2021