Cell Biology and Biochemistry of Endochondral Bone Development

Coll. Res. Vol. 111981, p. 209-226

Cell Biology and Biochemistry of Endochondral Bone Development A.H.REDDI Laboratory of Biological Structure, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20205

Introduction The differentiation of mesenchym al cells to bone can occur by one of two routes. First, there is a direct development of bone from mesenchyme, as in the skulI, which is referred to as intramembranous ossification. Second, an intervening cartilage model preceedes the formation of bone from mesenchymal cells; and this occurs in the long bones and is known as endochondral bone formation. It has been emphasized that these two modes of ossification connote only the environment in which bone formation occurs and, they do not significantly differ in the kind of bone formed (Harn and Cormack, 1979). The aim of this review is to describe recent progress in the realm of the cell biology and biochemistry of endochondral bone development. It is our intention to provide only a selective survey of the area, and no attempt has been made to be comprehensive. Recent reviews of bone development include those of Hall (1978) and Harn and Cormack (1979). Additional information may be obtained from the four volume treatise on bone (Boume, 1971-1976). Cell Biology of Endochondral Bone Development The development and growth of long bones occurs via proliferation of mesenchymal cells, differentiation and hypertrophy of chondrocytes and caIcification of the cartilage matrix. The caIcified cartilage is replaced by bone in the metaphysis. ConcurrentIy, bone is remodeled to form a tubular bone that permits hematopoietic cell differentiation. This entire sequence of bone formation occurs in a continuum in the epiphyseal growth plate. In most laboratory rodents the process is initiated in utero prior to birth. In the human embryo endochondral bone development begins in the 7th to 8th week after conception (Langman, 1969). As the avian or mammalian embryo develops, mesodermal outgrowths from the trunk give rise to the limb buds, the site of future limb growth and development. The mesenchym al cells in the central core of the bud divide rapidly, resulting in the condensation of mesenchym al cells. By mechanisms which are not

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entirely elear, the central core forms chondroblasts and chondrocytes, and the periphery forms the perichondrium. The cartilage model increases in size by a combination of interstitial and appositional growth. Much of the cell division occurs in the ends while the mid-region chondrocytes enlarge. The developing vascular system penetrates the perichondrium in the central zone and heraIds a new cellular phenotype, the basophilic osteogenic cells that differentiate into osteoblasts in elose proximity to the invading endothelial cells and pericytes (Trueta, 1963). Despite its importance to osteogenesis and bone formation wc still do not understand the role played by angiogenesis. The collar of bone in the midshaft of the developing bone is now covered by a periosteum of mesenchymal cells. The central bony shaft is progressively mineralized, and the cartilaginous ends now constitute the epiphyses. The longitudinal growth of bone is mainly due to interstitial growth of chondrocytes in the epiphyseal growth plate by sequential proliferation, maturation and calcification of the cartilage. Capillaries invade the longitudinal columns of hypertrophie cartilage, and osteogenic cells in elose apposition to the cartilage spicules proliferate and differentiate into osteoblasts and secrete bone matrix. Further growth of bone occurs by appositional growth and remodeling by osteoelasts to yield the final shape and form. In order to avoid the problems presented by the task of working with sm all amounts of tissue obtainable from prenatal fetuses we have explored the potential of matrix-initiated endochondral bone development (Reddi and Huggins, 1972; 1975; Reddi and Anderson, 1976). This method also avoids the spatial and temporal difficulties presented by the growth plate. The sequential developmental changes elicited by subcutaneous implantation of demineralized collagenous bone matrix is reminiscent of endochondral bone development in the fetus and neonate (Harn and Cormack, 1979; Reddi, 1976). On subcutaneous implantation of collagenous diaphyseal bone matrix there was an instantaneous formation of a blood elot. One day after implantation the implant was in the form of a button-like planoconvex plaque and consisted of the implanted matrix and an irregular fibrin network with enmeshed polymorphonuelear leukocytes. On days 3 and 4 spindle-shaped mesenchymal cells were in the vicinity of the matrix (Fig. 1) and proliferated as assessed by 3H-thymidine incorporation and ornithine decarboxylase activity (Rath and Reddi, 1978; 1979a). The earliest chondroblasts were evident on day 5, and chondrocytes were abundant on days 7-8 (Fig.2). On day 9 calcification of the hypertrophie cartilage matrix was observed, and vascular invasion occurred (Fig.3). Several multinueleated chondroelasts were noted, and the degradation of cartilage matrix was evident (Fig.3). On days 10 and 11, basophilic osteogenic cells and osteoblasts were seen (Fig.4) in the vicinity of invading capillaries. New bone formation proceeded by appositional growth on calcified cartilage spicules and on the surface of the implanted bone matrix partieles. On days 12-18 bone remodeling occurred, resulting in selective dissolution of implanted bone matrix and the formation of an ossiele consisting essentially of the newly induced bone. During the bone remodeling phase sinusoids with littoral endothelium enlarged by confluence in the developing ossiele. The resulting stroma appeared to be a conducive microenvironment for proliferation and differentiation of extravascular colonies of hematopoietic stern cells, developing erythroid and granulocytic cells and megakaryocytes (Reddi and Huggins, 1975; Reddi and Anderson, 1976). The response to bone matrix is rather specific and requires prior demineralization

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Figs. 1-4. Photomicrographs of various development stages of matrix-induced bone formation. M, implanted collagenous matrix. X 220. Figure 1 - day 3. Mesenchymal cells in dose apposition to the implanted matrix. Figure 2 - day 7. cartilage cells in contact with implanted matrix. Figure 3 - day 9. Vascular invasion of the hypertrophie cartilage matrix. Note the multinudeate chondrodasts (arrows) in the vicinity of degenerating chondrocytes. Figure 4 - day 11. Bone formation is evident and the arrows point to a palisade of osteoblasts.

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of the matrix (Reddi, 1974; 1975; 1976). The sequential developmental events are summarized in Figure 5. Thus, this experimental model is useful to explore endochondral bone development in the postfetal rat. Origin of Bone Cells The foregoing consideration of the development of endochondral bone reveals the heterogenous nature of the cell populations in this tissue. A logical question, then, is what are the developmental lineages of the main cell types in bone, namely the bone forming osteoblast and the bone resorbing osteoelast. There is compelling evidence to suggest that osteoblasts arise from the local mesenchymal cell population and the osteoelasts are derived from blood-borne monocyticmacrophage cells (Owen, 1978; 1980). It was earlier believed that there might be a common precursor for osteoblasts and osteoelasts (Young, 1962; Friedenstein, 1976; Rasmussen and Bordier, 1973; Harn and Cormack, 1979). In an incisive study, Fischman and Hay (1962) demonstrated by 3H-thymidine autodiagraphy that the monocytes fuse to form the osteoelasts in the regenerating newt limb. Very shortly thereafter similar findings were reported using injections of charcoal into rabbits (Jee and Nolan, 1963). Additional evidence was adduced by Buring (1975) in parabiotic rats during heterotopic bone formation and by Gothlin and Ericsson (1976) in a fracture-healing model in rats by the use of 3H-thymidine autoradiography. Further evidence for the hematogenous origin of osteoelasts was derived from the quail-chick chimera system pioneered by Le Douarin (1973). This experimental model circumvents the possible pitfalls of an exogenous marker such as 3H-thymidine, as it is based on a "bui!t-in" consisting of structural differences in the interphase nuelei of two species of birds, the Japanese quai! and the chick. The quai! cells have nueleolus-associated heterochromatin that can be stained by the Feulgen cytochemical reaction for DNA, and therefore represents a useful nuelear marker. Furthermore, the chorioallantoic membrane of the host is a richly vascularized immunologically privileged site for transplantation of donor tissue. Kahn and Simmons (1975) grafted developing quaillimbs to the chick chorioallantoic membrane and coneluded that osteoblastic cells were of the quail-type and the osteoelasts were mostly of the chick-type supporting the notion that the latter cells were blood-borne. These experiments were confirmed and extended by Jotereau and Le Douarin (1978). These authors conelusively demonstrated that osteoblasts and osteocytes were derived from the limb bud mesenchyma whereas the osteoelasts arose from blood-borne hemotopoietic cell lineage. A systematic study of certain developmental anomalies may lead to a better understanding of fundamental biological principles. This certainly was the case in the study of osteopetrosis in rodents by Walker and his coworkers (Marks and Walker, 1976). A characteristic feature of this condition is the defect in bone resorption and remodeling leading to formation of dense bone. This defect was corrected by establishing cross-circulation by parabiosis between an osteopetrotic animal and anormal litter-mate. Additional proof that it was a cellular defect of the macrophage-osteoelast was provided by the curing of osteopetrosis by injection of a suspension of bone marrow or spleen cells into irradiated recipients. These experiments constitute further proof for the origin of osteoelasts from a

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circulatory monocyte-macrophage series. The importance of Walker's discovery was underscored recently by the successful bone marrow transplantation and cure of osteopetrosis in a 5-month old infant (Coccia et al., 1980). It is well known that beige mice (Chediak-Higashi syndrome) have giant lysosomes in their polymorphonuclear leukocytes and monocytes (Oliver and Essner, 1975). Recently this property was exploited in an incisive investigation of the origin of multinucleate osteoclasts from hematopoietic stern cells in radiated chimeras of osteopetrotic mice infused with bone marrow of beige mice (Ash et al., 1980). The osteopetrosis was cured and the osteoclasts in the osteopetrotic animals exhibited giant lysosomes by 6 weeks. It is noteworthy that osteoblasts were devoid of giant lysosomes. This example further underscores the role of blood-borne monocytes in osteoclast lineage and function. The foregoing evidence constitutes compelling arguments for the hematogeneous origin of osteoclasts. However, recently evidence to the contrary was presented based on the study of Fe and C3 receptors on macrophages and osteoclasts (Shapiro et al., 1979). The Fe receptor binds the Fe domain of the immunoglobulin moleeule, and the C3 receptor binds the third component of complement. In this investigation both receptors were present on macrophages but not on osteoclasts. While it is generally accepted that during endochondral bone formation cartilage is degraded and replaced by bone, the precise fate of hypertrophie chondrocytes is not known. Autoradiographie and morphological evidence has been presented to support the notion that hypertrophie chondrocytes may be transformed to osteoprogenitor cells (Crelin and Koch, 1967; Lutfi, 1971; Holtrop, 1972; Shimomura et al., 1973). Developmental Cascade The sequential proliferation and differentiation of cartilage, calcification of cartilage matrix, vascular invasion, resorption of cartilage, bone matrix formation and mineralization, bone resorption and remodeling, and finally hematopoiesis constitute a developmental cascade. It emphasizes the complicated nature of the entire endochondral bone development and challenges the student of bone biology to dissect and analyze in biochemical and molecular terms the various cellular phases. For example, are there local growth factors for cartilage? Wh at factor(s) initiates and terminates cartilage calcification? Are there chemotactic factors that signal vascular invasion? Are there similarities and/or differences between cartilage and bone resorption? What is the interrelationship between vascular invasion and bone formation? Is resorbing bone chemotactic for thc monocyte-macrophage series? Finally, what signals constitute the basis for the homing of hematopoietic stern cells to regions of osteogenesis? These are fundamental questions that need to be explored. A growth factor for chondrocytes was isolated and appears to be localized in the nuclear chromatin (Azizkhan and Klagsburn, 1980). Aspects of cartilage and bone mineralization are discussed in connection with the biochemistry of the inorganic phase. The importance of vascular invasion for bone formation is well known (Trueta, 1963). However, thc interaction and role of perivascular mesenchymal cells in bone formation is not clear. Bone resorption may involve more than once cell type (Heersehe, 1978). It has been proposed that multinucleate osteoclasts may demineralize bone and

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expose collagenous matrix. The collagen fibrils may then be phagocytosed by monocyte-macrophage cells. Mundy et al. (1978) have provided convincing evidence for chemotactic factors for monocytes in regions of resorbing bone. Cinematographic evidence for contact-mediated bone resorption by human monocytes in vitro is available (Kahn et al., 1978; Teitelbaum and Kahn, 1980). Further experiments in this area undoubtedly will expand our understanding of bone formation and resorption and its cellular regulation. Biochemistry of Endochondral Bone Development Another fruitful approach to a better knowledge of bone development is to explore the biochemistry of various distinct phases of endochondral bone development. In order to circumvent the technical difficulties of the epiphyseal growth plate for biochemical studies we have utilized the matrix-induced endochondral bone development system for our work.

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Organic Matrix

Collagen The occurrence and distribution of several genetically distinct collagens is now well established (Gay and Miller, 1978; Minor, 1980). The progressive transitions in the collagen types were first examined during the endochondral bone development of chick by biochemical (Linsenmeyer et al., 1973), and immunofluorescent techniques (von der Mark et al., 1976; von der Mark and Conrad, 1979). Mesenchymal tissue primarily consisted of type I collagen, but with chondrogenesis, type II appeared. Thereafter, with the advent of bone formation, type I collagen was present. We have examined by indirect immunofluorescence the localization of types I, II, III and IV collagen du ring matrix-induced endochondral bone formation (Reddi et al., 1977; Foidart and Reddi, 1980) and the results are summarized in Figure 5. On day 3, during mesenchymal cell proliferation, type III collagen was present. Subsenquently, type II collagen was detected in the cartilage matrix. Vascular invasion of the hypertrophie cartilage was marked by the appearance of type IV collagen (Foidart and Reddi, 1980). During bone formation type I collagen was demonstrated. More recently we have examined the biosynthesis of type land III collagen by pulse labeling with 3H-proline and gel electrophoresis (Steinmann and Reddi, 1980). Maximal biosynthesis of type III ---,-

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collagen was found on day 3 during mesenchym al cell proliferation. On days 9-20 during bone formation predominantly type I collagen was synthesized. In view of the extensive posttranslational modifications involved in collagen biosynthesis (Prockop et al., 1979), we examined the changes in prolyl 3 and 4 hydroxylases, lysyl hydroxyl ase, hydroxylysyl galactosyl transferase and galactosylhydroxylysyl glucosyl transferase (Myllyla et a1., 1981). The activities of all five enzymes reached a peak on day 9 during hypertrophy of chondrocytes and the early stages of bone formation (Figs.6, 7). Further, the tissue distribution and cellular localization of prolyl-4-hydroxylase was investigated by indirect immunofluorescence. Intense reactivity was found in hypertrophie chondrocytes on day 9and in osteoblasts on days 11-14. Proteoglycans Along with collagens, proteoglycans comprise a major dass of extracellularconnective tissue macromolecules. Proteoglycans have been extensively studied

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in cartilage (Hascall and Heinegard, 1979; Hascall, 1980) and Figure 8 summarizes the current model for proteoglycan structure in hyaline cartilage. The proteoglycans are extremely heterogeneous in their structure and consist of monomers (subunits) interacting noncovalently with hyaluronic acid to form large molecular weight aggregates that are stabilized by "link" glycoproteins. Each subunit consists of a protein core to which glycosaminoglycan (GAG) chains are covalently bound. In bovine cartilages the GAG consists of chondroitin sulfate CARTILAGE PROTEOGLYCAN AGGREGATE STRUCTURE

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and keratan sulfate chains. Recently certain N- and O-linked oligosaccharides were diseovered in the eore protein (Hascall, 1980). Despite these major advances in proteoglyean chemistry, virtually nothing is known about the bioehemical ehanges during endochondral bone development. We investigated the biosynthesis of proteoglycans labelIed in vivo at various stages of matrix-induced endochondral bone development (Reddi et al., 1978). During maximal chondrogenesis on day 7

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the elution profile of 35S04 -labelled proteoglycans were similar to other hyaline cartilage (Fig.9). On day 9 during calcification of cartilage matrix there was a decline in synthesis of cartilage type proteoglycans and supporting the electron microscopy observations (Matukas and Krikos, 1968). Then there was an increase in smaller size proteoglycans. The formation of these smaller bone-type proteoglycan increased on days 11 and 14. The constituent chondroitin sulfate chains of bone-type proteoglycan were larger (M. W. 50,000) than cartilage type proteoglycan (M. W. 25,000). Radioautography of 35S0 4 labelIed implants identified osteoblasts as the site of synthesis of bone-type proteoglycans. More recently, using indirect immunofluorescence, we have localized cartilage-specific proteoglycans and link pro tein (Reddi and Poole, unpublished observation) in cartilage matrix. The bone matrix was devoid of the fluorescence.

Fibronectin Fibronectin is a pericellular glycoprotein that is also present in a circulating form in the blood plasma (Yamada and Olden, 1978; Vaheri et al., 1978; Kleinman et al., 1979; Pearlstein et al., 1980). This glycoprotein is a disulfide bonded dimer of a molecular weight of 450,000 and has been shown to function in vitro as an adhesive protein for cell-substratum and cell - cell interaction, and has affinity for collagen, heparin and fibrin. It is conceivable that these interactions are crucial for early phases of wound healing, fracture repair and regeneration. During matrix-induced endochondral bone development one of the earliest responses is the interaction of the collagenous matrix with responding mesenchymai cells. In view of this we examined the biosynthesis and possible function of fibronectin during matrix-cell interactions in vivo. The subcutaneously implanted demineralized bone matrix bound circulating fibronectin, which may be an important initial requirement for cell attachment to the matrix (Weiss and Reddi, 1980a). Fibronectin appears to function as an a2-opsonic protein during phagocytosis and may be involved in wound debridement prior to the healing response (Saba et al., 1978). Very recently, fibronectin has been demonstrated to be a chemotactic macromolecule (V. Gauss-Muller, personal communication), and may therefore play a role in mesenchym al cell recruitment. The temporal appearance of fibronectin on day 3 correlates well with the increased biosynthesis of type III collagen (Steinmann and Reddi, 1980). This is noteworthy in view of the fact that f-ibrönectin binds avidly to native type III collagen in comparison to types I and II (Ruoslahti and Engvall, 1978; Hormann and Jileck, 1978).

Laminin and ather Glycoproteins Laminin is a recently described noncollagenous glycoprotein component of a basement membrane producing murine tumor (Timpl et al., 1979). It consists of at least two polypeptide chains (220,000 and 440,000) linked by disulfide bonds and was localized in the lamina rara of the basement membrane. During vascular invasion of hypertrophic cartilage laminin was localized by specific immunofluorescence in the invading endothelial cell basement membrane (Foidart and Reddi, 1980). The localization of laminin was paralleled by type IV collagen and blood coagulation factor VIII around the endothelial cells (Foidart and Reddi, 1980).

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a 2 HS-glycoprotein, a plasma glycoprotein was found to be present in human bone and dentin and is concentrated in the mineralizing tissues (Ashton et al., 1976). The plasma a2HS-glycoprotein concentration in patients with Paget's disease of bone was lower than controls. The possible role, if any, of this protein in bone is an open question.

y-Carboxyglutamic Acid Containing Pro teins Prompted by the discovery of y-carboxyglutamic acid (Gla) in vitamin K-dependent calcium-binding blood coagulation factors, calcified tissues were examined for Gla. Bone contained large amounts of a ~ 6,000 dalton Glacontaining protein (Hauschka et al., 1975; Priceet al., 1976). The Gla protein was named osteocalcin based on its affinity for calcium (Kd - 0.8 mM) (Hauschka, 1979). The appearance of y-carboxyglutamic acid during matrix-induced bone formation was investigated (Hauschka and Reddi, 1980). Gla levels were basal du ring chondrogenesis and began to increase gradually during mineralization. Recently, Price et al. (1979), employing a sensitive radioimmunoassay for the bone Gla protein (BGP), found that this protein may not be required for early mineralization. The human Gla pro tein was detected in human blood plasma by radioimmunoassay at a concentration of 4.5 ng/ml (Price and Nishimoto, 1980). In certain disease states involving bone, such as Paget's disease, primary hyperparathyroidism, and metastatic bone disease, the plasma Gla protein concentration is e1evated (Price et al., 1979). More recently we found that the BGP increased only at later stages of bone remodeling during matrix-induced bone development, raising the possibility that it may be a marker of bone turnover (Price and Reddi, 1980, unpublished).

Enzyme Transitions The role of alkaline phosphatase in mineralization is still far from clear. We have routinely employed this enzyme as an usdul marker for bone differentiation (Reddi and Huggins, 1972). The increase in enzyme activity precedes 45Ca incorporation. Lactic dehydrogenase (LDH) was a usdul enzyme to monitor cartilage formation as the levels are high; with bone formation the LDH levels declined (Reddi and Huggins, 1971). During endochondral bone formation, the cartilage is replaced by bone and in view of this considerable attention has been focused on lysosom al enzymes and their role therein (Vaes, 1969; Dingle, 1973; Doty and Schofield, 1976; Thyberg and Friberg, 1978). We have investigated the changes in acid phosphatase, ß-glucuronidase, and arylsulfatase by biochemical and histochemical techniques (Rath et al., 1981). We found that these enzymes, especially arylsulfatase is a good marker for bone resorption and remodeling and maximal levels were detected on day 13 (Fig.10).

Inorganic Phase and Mineralization The differentiation of osteoblasts and the biosynthesis, secretion and assembly of extracellular bone matrix is followed by mineralization. The major component of bone mineral is a crystalline calcium phosphate, with the structüre of hydroxyapatite (OHAp), Cal0(P04)6(OH)2 (Eanes and Posner, 1970; Glimcher, 1976;

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Eanes and Reddi, 1979). Mineralization of cartilage and bone was quantitated by 45Ca incorporation and 32 p incorporation and is depicted in Figure 5 (Reddi, 1976; Reddi, 1980). It is noteworthy that the matrix-induced endochondral bone forming system affords an opportunity to perform concurrent metabolic radiolabeling and enzyme assays to correlate the interrelationships. A detailed discussion of mineralization is outside the scope of this article and the reader is referred to the above articles.

Hormonal Regulation The availability of an experimental system with a predictable temporal sequence of endochondral bone development affords a useful model for the study of hormanal regulation of this process. Matrix-induced bone formation is pituitary growth hormone dependent (Reddi, 1974; Reddi and Sullivan, 1980). In hypophysectomized recipient rats, there is an additive influence of growth 6 Collagen 1/2

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hormone and thyroid stimulating hormone (Reddi and SuHivan, 1980). Although administration of growth hormone and thyroid stimulating hormone adequately corrected 45Ca incorporation in tibial metaphyses in hypophysectomized rats, it was unable to support initiation of mineralization in matrix-induced implants. It is likely that additional pituitary hormones regulate initiation of mineralization, as apposed to maintenance of mineralization that was already initiated. The incidence of osteoporosis in diabetics is higher than in age and sex matched controls (McNair et al., 1979). However, the underlying reasons are not clear. The influence of streptozotocin-induced diabetes on matrix-induced bone formation was studied (Weiss and Reddi, 1980b). Mesenchymal ceH proliferation was decreased and delayed in diabetes, but could be corrected by systemic insulin. Local injection of insulin at the site of implantation also corrected mesenchymal ceH proliferation and mineralization of bone (Weiss and Reddi, unpublished o bserva tions). It is weH known that massive doses of corticosteroids to control certain malignancies result in osteopenia. The local influence of dexamethasone was investigated using various biochemical parameters at discrete stages of development (Rath and Reddi, 1979b). Ornithine decarboxylase activity (ODC) is a useful indicator of mesenchym al ceH proliferation prior to chondrogenesis and osteogenesis (Rath and Reddi, 1978). Local dexamethasone inhibited ODC in day 3 implants and delayed chondrogenesis, thus indicating the site of action to be on mesenchymal ceH proliferation. Biomedical Implications Increasing the present understanding of the ceH biology and biochemistry of endochondral bone development has immense implications from the standpoint of metabolie bone disorders and pathological mineralization. The detailed disseetion of the complicated biological cascade of endochondral bone differentiation may result in a better knowledge of the role of extraceHular matrix in development. Determining the intricate ceH - ceH metabolie cooperation necessary for bone resorption and remodeling may provide new examples of regulation of matrix-mineral interaction, accretion, and dissolution. Elucidation of the intricate hormonal regulation of endochondral bone formation may reveal points of control hitherto unrecognized that may lead us to understand metabolie bone diseases, periodontal bone resorptive mechanisms and pathological skeletal metastases. FinaHy the phenomenon of matrix-induced new bone formation may be useful in the re alm of plastic and reconstructive surgery of acquired and congenital craniofacial and skeletal deformities.

References Ash, P., Loutit, J. F., and Townsend, K. M. 5.: Osteoclasts derived from haematopoeitic stern cells. Nature 283: 669-670, 1980. Ashton, B. A., Hohling, H. J., and Triffit, J. T.: Plasma proteins present in human cortical bone: Enrichment of the a 2 HS-Glycoprotein. Calcit. Tiss. Res. 22: 22-33, 1976. Azizkhan, J. C. and Klagsburn, M.: Chondrocytes contain growth factor that is locaIized

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