The role of TGF-β and BMP-7 in regenerating bone and soft tissues

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Materials Science and Engineering C2 (1994) 19-26

12

The role of TGF-/3 and BMP-7 in regenerating bone and soft tissues * Jaro

S o d e k a,c, I v a n W . S . L i a,c H o n g L i b C a r l t o n G . B e l l o w s a C h r i s t o p h e r A . G . M c C u l l o c h a,b, H o w a r d C. T e n e n b a u m a.b, R i c h a r d P. E l l e n b

a Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ont., M5S 1A8 Canada b Department of Periodontics, Faculty of Dentistry, University of Toronto, Toronto, Ont., Canada c Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ont., Canada

Abstract

The involvement of transforming growth factor-/3 (TGF-/3) in the formation and repair of connective tissues and the related bone morphogenic proteins (BMPs) in bone induction has been well established. However, the ability of TGF-/3 to promote rapid repair results in scarring whereas the effect of BMPs on soft connective tissues is largely unknown. In order to evaluate the potential of TGF-/3 and BMPs, alone and in combination, in regenerating hard and soft connective tissues, such as those found in the periodontium, we have been studying the influence of these cytokines on fibroblast and bone cell metabolism. In earlier studies we have shown that TGF-/31 stimulates matrix formation through increased transcription of matrix protein genes while suppressing genes coding for matrix degrading enzymes in both fibroblasts and bone cells. However, TGF-/31 alone suppresses the expression of alkaline phosphatase and the formation of mineralized tissue in vitro, whereas BMP-7 stimulates bone matrix protein expression and alkaline phosphatase in association with bone formation. Recent studies also indicate that BMP-7 stimulates the clonal expansion of pre-osteoblasts, thereby increasing the number of bone-forming cells. In contrast to osteogenic cells, however, fibroblasts appear refractory towards BMP-7. Thus, the effects of TGF-/3 and BMP are clearly discrete and are dependent upon the responding cell populations. If used in combination these cytokines could promote the formation of both hard and soft connective tissues. Keywords: TGF-/3; BMP-7; Regenerating bone; Soft tissue

1. Introduction

Transforming growth factor-/3 (TGF-/3), originally identified in tumour tissues, was characterized initially by its ability to induce anchorage-independent growth of cultured cells in the presence of EGF. The physiological importance of TGF-/3 was demonstrated when purified TGF-/3 was shown to promote wound healing in vivo [1]. Subsequently, TGF-/3 has been shown to affect proliferation and modulate the phenotype of a wide variety of cells, as well as being important in embryogenesis, development and tissue homeostasis [2,3]. Following the initial characterization of TGF-/3 as a dimeric protein comprising identical polypeptide subunits of 12.5 kDa, additional family members have been recognized (Fig. 1). The prominent species of TGF-/3, originally characterized as the major form in bone and platelets, is known as TGF-/31, while TGF* Paper presented at the Bionic Design Workshop '94, 22-23 February 1994, Tsukuba, Japan.

0928-4931/94/$07.00 © 1994 Elsevier Science S.A. All rights reserved SSDI 0928-4931 (94) 00038-T

/32 is present in the same tissues in generally lower amounts. Other TGF-/3 family members have been demonstrated using cloning techniques. Thus, TGF-/33 has been shown to be expressed primarily in embryonic tissue, while studies on TGF-/34 and TGF-/35 have been largely limited to their expression in chick embryos and in Xenopus. In each case the gene codes for a precursor protein of 390 amino acids from which the active protein is released by proteolytic cleavage (Fig. 2). TGF-/31 is secreted in a latent form in which the active protein dimer is complexed with the precursor protein (latent associated protein, LAP) which can be dissociated by chaotropes, acidic pH or proteolytic activity [3]. The importance of TGF-/3s in the fundamental regulation of biological processes is demonstrated in the highly conserved nature of the active proteins, the expression by a wide variety of cell types at various developmental stages, the ubiquitous distribution of receptors [4] and the multiple points in the expression of the protein that its activity can be regulated. The TGF-/3s are members of a superfamily of cytokines,

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J. Sodek et al. / Materials Science and Engineering C2 (1994) 19-26

BMPs to induce bone formation [8,9] has special significance for the repair of periodontal tissues. However, the ability of TGF-/3 to promote tissue repair can result in scarring and may abrogate regenerative processes [10]. Moreover, while BMPs may stimulate bone formation, little is known of their influence on the associated soft connective tissues. Thus, while there is a great potential for utilizing TGF-/3s and BMPs in connective tissue regeneration, it is important to determine their effects in the complex environment of mixed tissues and to understand the molecular basis of their activities. Here we summarize previous studies on TGF-/3 and describe more recent, preliminary studies on BMP-7 (human osteogenic protein-i; hOP-l), a recombinant form of one of the BMPs [11]. From these studies, we hope to dissect the molecular mechanisms involved in the regulation of fibroblast and bone cell phenotypes and to evaluate the utility of these cytokines in propagating cell populations with the appropriate characteristics for tissue regeneration. In particular, it is of interest to determine whether or not a combination of these cytokines can be used effectively in regenerating both hard and soft connective tissues.

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known as the TGF-/3 superfamily (Fig. 1), which includes other regulators of developmental processes and tissue repair, including the activins, Mullerian inhibitory substance (MIS) and the BMPs. Notably, the TGF-fls and BMPs are closely related to genes that code for developmental products such as decapentaplegic (dpp) gene in Drosophila and vegetal (Vg-1), and the activins in Xenopus (Fig. 1). Several receptors have been characterized for TGF-/3 that present (betaglycan, receptor 3, endoglin) and bind (receptors 1 and 2) TGF-/3. Following TGF-/3 binding, a complex of receptors 1 and 2 associate for signalling through a unique ser/thr kinase [5]. While some selectivity for the different isoforms is evident in the distribution of receptors 1 and 2 on different cells and in the binding affinity of receptor 3, essentially the same receptor molecules are utilized for the TGF-/3s. In contrast, distinct ser/thr kinase receptors appear to mediate BMP signalling [6], indicating that discrete signalling pathways influence the expression of different subsets of genes in different tissues. The involvement of TGF-/3 in the development and repair of connective tissues [3,7] and the ability of Protein cys

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Our interest in the potential of TGF-/3 to stimulate the repair of periodontal tissues was initiated ten years ago with the demonstration that purified TGF-/3 promoted wound healing [1]. Subsequently, we have analyzed the effects of TGF-/3 in stimulating tissue formation using human gingival fibroblasts as a model for soft connective tissue and fetal rat calvarial cells as an established model for bone formation. Studies on gingival fibroblasts revealed responses that were distinctly different from established cell lines which had been used in the initial characterization of TGF-/3. Thus, TGF-/3 was unable to stimulate anchorage-independent growth even in the presence of EGF but, in combination, these cytokines did stimulate anchorage-dependent growth of confluent cells [12]. Having shown that TGF/3 stimulates matrix protein synthesis, we subsequently studied the temporal aspects and the molecular basis

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of the stimulation, with particular emphasis on SPARC protein [13], which is elevated in periodontal tissues, and we have correlated its presence with rapidly remodelling tissues [14,15]. This study revealed a differential regulation of matrix proteins (PAl-l, COL I, FN) in a precise temporal pattern via transcriptional and post-transcriptional pathways that we believe are important aspects of the response of fibroblasts in a wound environment [13]. To determine whether TGF/3 stimulates a 'fibrotic' response in which ECM accumulates, or simply an increased remodelling of ECM, we studied the effects of TGF-fl on the expression of ECM-degrading enzymes, the matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs). We found that collagenase (MMP-1), gelatinase (MMP-2) and the tissue inhibitor of MMPs (TIMP-1) were regulated independently; the MMP-1 being suppressed, while the MMP-2 and TIMP were stimulated [16]. We proposed that the stimulation of MMP-2 might be important for degrading abnormal and denatured collagen in the wound site. However, the effects on collagenase and TIMP, together with the stimulation of PAI-1 indicate that TGF-/3 promotes matrix formation by blocking the activity of plasminogen activator and collagenase. These studies were extended to examine the transcriptional and post-transcriptional regulation of these proteins from which it was deduced that the expression of MMP2 was regulated in a similar manner to COL I and FN, involving altered transcription and mRNA stability, whereas MMP-1 and TIMP-1 were regulated (negatively and positively, respectively) through changes in transcription that required the synthesis of protein factors [17]. The ability of TGF-/3 to promote bone formation was indicated by the in vivo studies of Joyce et al. [18]. To analyze the effects of TGF-/3 on bone forming cells under controlled conditions we used fetal rat calvarial cells (FRCC) which form bone-like tissue nodules in vitro [19]. In this system TGF-/3 was found to stimulate bone matrix formation [20,21], but blocked the formation of mineralized tissue [22]. The apparent discrepancy could be explained by the inhibition of alkaline phosphatase expression by TGF-/3, since the synthesis of all the major matrix proteins that form the bone matrix, including COL I, and bone sialoproteins [20,21] was stimulated. In more detailed studies, we further showed that TGF-/3 stimulated the synthesis of different forms of the bone sialoprotein, osteopontin (OPN) [23] and that the form of OPN expressed and the degree of stimulation by TGF-/3 was characteristic of sub-types of bone cells [24], revealing the complexities of cytokine regulation in context with cellular hierarchy. We have also shown that TGF-fl modulates enzymes and inhibitors involved in bone ECM degradation in much the same way as observed in fibroblasts [25]. In related studies we have shown that the expression of

TGF-/3 can be correlated to cell shape and proliferation in endothelial cells [26]. To study the effects of BMP-7 on the growth of fibroblastic cells varying concentrations of the recombinant cytokine were added to gingival and ligament fibroblasts plated at low density. As shown in Fig. 3, the presence of BMP-7 did not have a mitogenic effect on either of the fibroblastic cell populations. Since periodontal ligament fibroblasts have some phenotypic characteristics of bone cells, that include the expression of high levels of alkaline phosphatase, and since BMPs are known to stimulate the expression of this enzyme in bone cell populations [27-32], we analyzed for alkaline phosphatase activity in both ligament and gingival fibroblast cells (Fig. 4). Notably, confluent periodontal ligament fibroblasts (PDLF) expressed constitutive alkaline phosphatase activities of approximately 33% of the bone cells whereas the level in gingival fibroblasts 2O0000

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J. Sodek et al. / Materials Science and Engineering C2 (1994) 19-26

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Days Fig. 4. Effects of BMP-7 on alkaline phosphatase activity in h u m a n periodontal fibroblasts. H u m a n periodontal ligament fibroblasts (PDLF) a n d h u m a n gingival fibroblasts (HGF) were grown in 35 m m tissue culture dishes and 25 ng ml-1 of BMP-7 added to both sub-confluent (SC) and confluent (C) cultures and the alkaline phosphatase activity m e a s u r e d 24 h later. Note the higher levels of alkaline p h o s p h a t a s e activity in the confluent cells with the ten-fold higher levels in the P D L F at day 1, However, these enzyme levels were not significantly changed by the presence of BMP-7 when analyzed after 1, 4 or 7 days.

(HGF) was only 3%. Even at concentrations of up to 50 ng m1-1 BMP-7, which strongly stimulate alkaline phosphatase in osteoblastic cells [11], there was no significant effect on the fibroblastic cells demonstrating distinct differences in the response of these cells compared with osteoblastic cells. To characterize the effects of BMP-7 on bone cells we studied the formation of mineralizing bone nodules in adult rat bone marrow cells [33] and FRCCs [19]. Concentrations of BMP-7 varying from 1 ng ml-I to 50 ng ml-~ administered as an initial pulse, as a pulse to confluent cells, or administered continuously, failed to induce the formation of mineralized bone nodules in rat bone marrow cell cultures in the absence of dexamethasone. In contrast, FRCCs responded by generating an increased number of tissue nodules, and an increased proportion of these nodules were mineralized (Fig. 5). Notably, the strongest response was obtained when the cytokine was administered after the cells had

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B M P - 7 C o n c . (ng/ml) Fig. 5. Stimulation of bone nodule formation by BMP-7. T h e effects of various concentrations of BMP-7 on the formation of mineralizing bone nodules were studied using fetal rat calvarial ceils (FRCCs). Both the n u m b e r of nodules produced and the percentage of nodules that were mineralized (Von Kossa positive staining) were significantly increased in the presence of at least 1.0 ng ml-1 BMP-7, with maximal responses observed between 10--30 ng ml -I BMP-7.

become confluent and optimal stimulation was attained with 25 ng ml- 1 BMP-7. Consistent with previous studies [11], BMP-7 also increased alkaline phosphatase activity and was especially effective when administered following the log phase of cell growth (Fig. 6). To determine the effects of BMP-7 on the expression of bone matrix protein genes, confluent cells treated with 25 ng ml-1 BMP-7 for time periods varying from 1 h to 8.5 days, were harvested and RNA extracted and utilized for Northern hybridization analysis of mRNA levels (Fig. 7). Clear differences were observed in the response of different matrix protein genes. Thus, OPN was stimulated early, reaching a maximal level after 6 h before declining to control levels. In comparison, bone sialoprotein, which we have shown previously to be a marker of fully differentiated osteoblasts, was induced after approximately 1 day, and was expressed at high levels after 8 days, similar to the effect of dexamethasone. In contrast to the effects on the

J. Sodek et al. / Materials Science and Engineering C2 (1994) 19-26 CONTROL 500

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sialoproteins, SPARC mRNA expression was not altered significantly in response to BMP-7.

3. Discussion TGF-/3s have been characterized as multifunctional cytokines that have a fundamental role in the spatio-temporal organization of developing tissues [34,35]. They also play a key role in the repair of both soft and hard connective tissues [3,7,18]. Thus, administration of TGF-/3 has been shown to induce granulation tissue in normal skin and to induce chondrogenesis with subsequent new bone formation in uninjured bone. Of the three TGF-/3 isoforms that have been found in mammalian tissues TGF-/31 accounts for most of the activities of the TGF-/3s in the repair of adult tissues. TGF-/31 is released initially from degranulating platelets and initiates a cascade of events including the recruitment of cells, formation of new blood vessels and the synthesis of ECM. Auto-inductive processes amplify

Fig. 7. Effects of BMP-7 on bone protein m R N A expression by fetal rat calvarial cells. Confluent FRCCs were treated with 25 ng ml -~ BMP-7 and triplicate cultures analyzed at various time points extending to 8.5 days. Following extraction and purification total R N A from the ceils was used for Northern hybridization analysis with radiolabelled cDNA probes for bone sialoprotein (BSP), osteopontin (OPN) and SPARC protein and comparisons made with 10 -8 M dexamethasone which is also known to stimulate bone nodule formation by FRCCs. Notably, the BSP m R N A s (1.6 and 2.0 kb) were induced after 1 day in the presence of BMP-7 or dexamethasone, and were markedly stimulated compared to controls at 8.5 days. However, there was no synergistic effect with BMP-7 and dex. In comparison, the expression of OPN m R N A (1.5 kb) was increased early, reaching maximal levels at 6 h and declining thereafter. Contrasting the effect on sialoproteins BMP-7 did not appear to alter the expression of SPARC m R N A (2.0 kb) significantly.

and further extend the activity of TGF-/31 at the wound site. However, the involvement of TGF-/3 in the rapid response of adult tissue repair has been implicated as a detriment to regeneration and scar-flee healing and may promote fibrosis [10]. This is supported by observations of (1) high levels of TGF-/3 expression, together with bFGF and PDGF, at wound healing sites as well as in fibrotic tissues in post-natal tissues; (2) low levels of TGF-/3 and FGF, but not PDGF, in fetal wounds [36]. In addition, antibodies that neutralize the activities of TGF-/31 and TGF-/32 improve the organization of the newly synthesized matrix in the wound bed and reduce scarring [37]. Moreover, the concentration and relative amounts of the TGF-/3 isoforms in the wound fluid of fetal wounds that heal without scarring are different from those of adult wounds which do form scar tissue. Thus, concentrations of TGF-/32 are dramatically reduced in adult compared with fetal wound fluid [38]. The TGF-/3s influence a number of processes in the tissue repair process. Initially, TGF-/3 released from the platelets at the site of injury initiates a sequence of events that includes chemoattraction of monocytes and leukocytes, induction of neo-vascularization and control of cytokine and inflammatory mediator production. The stimulation of matrix production by TGF/3 and the autoinduction of TGF-/3 production by cells at the wound site are especially relevant to scar formation and fibrosis. TGF-/3 stimulates expression of individual matrix components including collagen, fi-

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Z Sodek et al. / Materials Science and Engineering C2 (1994) 19-26

bronectin, SPARC and tenascin. At the same time it blocks matrix degradation by blocking proteases such as plasminogen activator [39] and collagenase [16,17] while stimulating the synthesis of PAI-1 and TIMPs [16,17,40] which are inhibitors of these enzymes. A fundamental problem of tissue repair in mature tissues is the disorganized matrix that is formed during the rapid formation of new 'scar' tissue. The rapidity of the repair process appears to compromise the regenerative processes. However, a unique feature of the periodontal tissues is the absence of scar formation following standard excisional wounds involving the dentino--gingival fibres. In this respect, the gingival tissues respond in a manner similar to fetal tissues. Notably, the soft periodontal tissues and alveolar bone also resemble fetal tissues in their rapid remodelling of ECM, high levels of SPARC expression and high cell turnover, certain aspects of which have also been equated with healing tissues. An interesting feature shared by periodontal tissues and wound healing tissue is the prominent phagocytic activity of fibroblasts, while there is little evidence of collagenolytic activity [41]. In comparison, collagenase expression is clearly associated with tissue development and regeneration of tissue in fetal wounds. It has been hypothesized that the breakdown of the ECM (histolysis) which occurs in regenerative processes does not occur in full thickness excision wounds [42]. The collagenolytic step is believed necessary to allow differentiated connective tissue cells to de-differentiate, migrate, reassociate, proliferate and redifferentiate, otherwise the cells retain their original phenotype. Thus in the wound site, populations of fibroblasts produce a fibrous matrix that is thought to lack the morphogenic instructions required to regenerate a normal tissue. In this regard it is notable that the expression of collagenase and other metalloproteinases can produce a rapid, extensive dissolution of ECM in developing tissues whereas the phagocytic pathway involved in wound repair and periodontal remodelling of collagenous matrix is a more selective process [41]. Moreover, that the expression of metalloproteinases is suppressed by TGF-/3 [16,17] is consistent with the presence of TGF-/3 in adult, but not in fetal wounds. The BMPs have a role in bone formation and fracture repair both of which are critical features of periodontal and reconstructive surgery. The existence of BMPs was indicated from the pioneering studies of Urist [43] and Reddi and Huggins [44] who showed that a factor(s) in demineralized bone and dentine particles was capable of inducing bone formation by undifferentiated mesenchymal cells in ectopic tissue sites. In such sites the formation of bone follows an endochondral pathway that has been well characterized [45]. The sequential process involves activation and migration of mesenchymal progenitors, fibronectin-mediated cell attach-

ment to a collagenous matrix, proliferation of mesenchymal stem cells, differentiation of cartilage, hypertrophy and mineralization of the cartilage, angiogenesis and vascular invasion, differentiation of bone cells, mineralization, remodelling and hematopoietic marrow formation. However, the molecular basis of bone induction and the precise roles of the BMPs are not known. Indeed, the nature of the protein capable of inducing the endochondral bone formation cascade has been demonstrated only recently following the purification and initial characterization of one form of BMP [46]. Subsequently, other forms of osteogenic proteins have been isolated [47,48] and the existence of others (BMP-2 to BMP-7) demonstrated using molecular cloning methods [8,47,49,50]. Notably BMP-7, which we are studying as a prototypic BMP, was identiffed initially as a novel human gene OP-1 (osteogenic protein 1) [11]. Studies of BMP activity on bone cells in vitro have shown that alkaline phosphatase activity is stimulated by BMP-2 and BMP-3 (osteogenin) in MC3T3-E1 cells [28,29], BMP-3 [30], BMP4 [31] and BMP-7 [11] in FRCCs, and BMP-3 in marrow cells [30]. Notably, this stimulation of alkaline phosphatase by BMPs contrasts with the activity of TGF-/31 on normal bone cell populations [30,32]. BMP-3 has also been shown to stimulate collagen and non-collagen protein synthesis and increase the cAMP response to PTH in bone cells [30]. However, little information is available on the specific effects of BMP on the different matrix proteins and how these are regulated. From in situ hybridization studies it is clear that BMPs have a more general role in developmental processes. Thus, BMP-2 and BMP-6 (Vgr-1) expression has been identified in tooth buds, developing heart and whisker follicles as well as in developing limb buds [51], while BMP-4 has also been shown in the nervous system as well as craniofacial tissues [52]. Moreover, BMP-7 is expressed at high levels in the kidney and has been suggested to exhibit an autocrine function [11]. Thus, BMPs like the TGF-/3s may have a broad ranging effect on epithelial-mesenchymal interactions that are important in periodontal tissue development. However, there remains a paucity of information on the effects of BMPs on different cell types in the repair and regeneration of tissues. The growth in culture of FRCCs [53], and adult rat bone marrow cells in the presence of glucocorticoid [54], follows a pattern of cellular differentiation that resembles bone formation in vivo. From the mixed population of bone cells, those with osteogenic potential differentiate and form bone nodules that will mineralize in the presence of organophosphates [19]. Early in culture pluripotent cells exist and the administration of BMP-7 to FRCCs that have been freshly plated promotes cartilage cell differentiation [55]. However, after 2-3 days in culture BMP-7 promotes osteogenesis

J. Sodek et al. / Materials Science and Engineering C2 (1994) 19-26

[11] as we have confirmed in our studies. Since the FRCCs respond to BMP-7 more strongly after they have attained confluence by increasing the number of nodules formed, it would appear that BMP-7 stimulates the growth of a pre-osteoblastic cell population. This is supported by the early stimulation of OPN and ALP, which is expressed by pre-osteoblasts. That the responding pre-osteoblastic cell is already well differentiated is indicated by the lack of response of the less-differentiated osteoblastic cells in the RBMC populations, which are induced to differentiate with glucocorticoids [33]. In contrast BSP, which is expressed by fully differentiated osteoblasts, is not expressed until several days after BMP-7 administration, consistent with the differentiation of the pre-osteoblasts into osteoblasts and the formation of mineralizing nodules. The increase in BSP expression and ALP activity is also consistent with the potential role of these proteins in bone matrix mineralization [56] and the increased number of mineralized nodules formed in the presence of BMP-7. In contrast to osteogenic cell populations fibroblasts from gingival and periodontal ligament appear to be refractory to BMP-7 with respect to cell proliferation and expression of alkaline phosphatase. Since PDLF have characteristics of osteogenic cells their lack of response to BMP-7 is surprising and indicates that these cells either lack receptors for BMP-7 or regulate genes such as alkaline phosphatase through alternate mechanisms. Clearly, a more extensive analysis of fibroblast populations needs to be completed before it can be concluded that these cells do not respond to BMPs. However, these preliminary results suggest that fibroblast populations may not be affected adversely when BMPs are used to promote bone formation, a consideration of particular importance in the periodontium. In summary, our preliminary results on the effects of BMP-7 on bone and fibroblast cell populations indicate that this cytokine has the potential to stimulate bone formation by expanding existing populations of pre-osteoblastic cells without adverse effects on fibroblastic cells. Consequently, it is possible that a combination of BMPs and TGF-/3 might be effective in promoting repair and regeneration of soft and hard connective tissues in the periodontium and other complex tissues as well as promoting tissue integration with implanted biomaterials.

References [1] M.B. Sporn, A.B. Roberts, J.H. Schull, J.M. Smith, J.M. Ward and J. Sodek, Science, 219 (1983) 1329. [2] J. Massagu6, Annu. Rev. Cell Biol., 6 (1990) 597. [3] A.B. Roberts and M.B. Sporn (eds.), in Handbook o f Experimental Pharmacology, Springer, Heidelberg, 1990, 419 pp. [4] J. Massagu6, Cell, 69 (1992) 1067.

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[5] J.L. Wrana, L. Attisano, J. C~ircamo, A. Zentella, J. Doody, M. Laigo, X.-F. Wang and J. Massagu6, Cell 71 (1992) 1003. [6] N. Yamaji, R.S. Theis, A.J. Celeste and J.M. Wozney, J. Bone Min. Res., 5 (1993) Abs 115. [7] T.A. Mustoe, G.F. Pierce, A. Thomason, P. Granates, A.B. Spore and T.F. Deuel, Science, 237 (1987) 1333. [8] J.M. Wozney, V. Rosen, A.J. Celeste, C.L.M. Mitsock, M.J. Whitters, R.W. Kriz, R.M. Hewick and E.A. Wang, Science, 242 (1988) 1528. [9] A.H. Reddi, Curr. Opin. Cell Biol., 4 (1992) 850. [10] W.A. Border and E.J. Ruoslahti, Clin. Invest., 90 (1992) 1. [11] K.T. Sampath, J.C. Maliakal, P.V. Hauschka, W.K. Jones, H. Sasak, R.F. Tucker, K.H. White, J.E. Coughlin, M.M. Tucker, R.H.L. Pang, C. Corbett, E. Ozkaynak, H. Oppermann and D.C. Rueger, J. Biol. Chem., 267 (1992) 20352. [12] J.L. Wrana, J. Sodek, R. Ber and C.G. Bellows, Eur. J. Biochem., 159 (1986) 69. [13] J.L. Wrana, C.M. Overall and J. Sodek, Eur. J. Biochem., 197 (1991) 519. [14] J. Salonen, C. Domenicucci, H.A. Goldberg and J. Sodek, J. Arch. Oral Biol., 35 (1990) 337. [15] S. Wasi, K. Otsuka, K.-L. Yao, P.S. Tung, J.E. Aubin, J. Sodek and J.D. Termine, Can. J. Biochem. Cell Biol., 62 (1984) 470. [16] C.M. Overall, J.L. Wrana and J. Sodek, J. Biol. Chem., 264 (1989) 1860. [17] C.M. Overall, J.L. Wrana and J. Sodek, Z Biol. Chem., 266 (1991) 14064. [18] M.E. Joyce, A.B. Roberts, M.B. Sporn and M.E. Bolander, J. Cell Biol., 119 (1990) 2195. [19] C.G. Bellows, J.E. Aubin, J.N.M. Heersche and M.E. Antosz, Calcif. Tiss. Int., 38 (1986) 143. [20] J.L. Wrana, M. Maeno, B. Hawrylyshn, K.-L. Yao, C. Domenicucci and J. Sodek, J. Cell Biol., 106 (1988) 915. [21] J.L. Wrana, T. Kubota and J. Sodek, in M.J. Glimcher and J.B. Lian (eds.), Proc. of the Third lnt. Conf. on the Chemistry and Biology o f Mineralized Tissues, Gordon and Breach, New York, 1988, p. 950. [22] M.A. Antosz, C.G. Bellows and J.E. Aubin, J. Cell Physiol., 140 (1989) 386. [23] J.L. Wrana, T. Kubota, Q. Zhang, C.M. Overall, J.E. Aubin, W.T. Butler and J. Sodek, Biochem. J., 273 (1991) 523. [24] R. Ber, T. Kubota, J. Sodek and J. Aubin, Biochem. Cell Biol., 69 (1991) 132. [25] C.M. Overall, J.L. Wrana and J. Sodek, in M.J. Glimcher and J.B. Lian (eds.), Proc. of the Third Int. Conf. on the Chemistry and Biology o f Mineralized Tissues, Gordon and Breach, New York, 1989, p. 289. [26] M.J. Merrilees and J. Sodek, J. Vasc. Res., 29 (1992) 376. [27] S. Vukicevic, F.P. Luyten and A.H. Reddi, Proc. Natl. Acad. Sci. USA, 86 (1989) 8793. [28] Y. Hiraki, H. Inoue, C. Shigeno, Y. Sanma, H. Bentz, D.M. Rosen, A. Asada and F. Suzuki, J. Bone Miner. Res., 12 (1991) 1373. [29] Y. Tokuwa, C. Ohse, E.A. Wand, J.M. Wozney and K. Yamashita, Biochem. Biophys. Res. Commun., 174 (1991) 96. [30] S. Vukicevic, F.P. Luyten and A.H. Reddi, Biochem. Biophys. Res. Commun., 166 (1990) 750. [31] H. Zhou, R.G. Hammonds, Jr., D.M. Findlay, J.T. Martin and K.W. Ng, J. Cell Physiol., 155 (1993) 112. [32] T.L. Chen, R.L. Bates, A. Dudley, R.G. Hammonds, Jr. and E.P. Amento, J. Bone Miner. Res., 6 (1991) 1387. [33] C. Maniatopoulos, J. Sodek and A.H. Melcher, Cell Tiss. Res., 254 (1988) 317. [34] A. Vaahtokari, S. Vainio and I. Thesleff, Development, 113 (1991) 985.

26

J. Sodek et al. / Materials Science and Engineering C2 (1994) 19-26

[35] U.I. Heine, E.F. Munoz, K.C. Flanders, L.R. Ellingsworth, H.Y.-P. Lam, N.L. Thompson, A.B. Roberts and M.B. Sporn, J. Cell Biol., 105 (1987) 2861. [36] D.J. Whitby and M.W.J. Ferguson, Development, 112 (1991) 651. [37] M. Shah, D.M. Foreman and M.W.J. Ferguson, Lancet, 339 (1992) 213. [38] A.B. Roberts and M.B. Sporn, Z Cell Biochem., 17E (1993) Abs R020. [39] M. Laibo, O. Saksela, P.A. Andreasen and J. Keski-Oja, J. Cell Biol., 103 (1986) 2403. [40] D.R. Edwards, G. Murphy, J.J. Reynolds, S.E. Wbitham, A.J.P Docherty, P. Anger and J.K. Heath, E M B O J., 6 (1987) 1899. [41] J. Sodek and C.M. Overall, in Z. Davidovitch (ed.), Biological Mechanisms o f Tooth Eruption and Root Resorption, EBSCO Media, Birmingham, AL, 1988, p. 303. [42] J. Gross, J. Cell Biochem., 17E (1993) Abs RZ002. [43] M.R. Urist, Science, 150 (1965) 893. [44] A.H. Reddi and C.B. Huggins, Proc. Natl. Acad. Sci. USA, 78 (1972) 7599. [45] A.H. Reddi, Collagen Relat. Res., 1 (1981) 209. [46] F.P. Luyten, N.S. Cunningham, N. Muthukumaran, R.G. Hammonds, W.B. Nevins, W.I. Wood and A.H. Reddi, J. Biol. Chem., 264 (1989) 13777.

[47] E. Ozkaynak, D.C. Rueger, E.A. Drier, C. Corbett, R.J. Ridge, T.K. Sarnpath and H. Oppermann, E M B O J., 9 (1990) 2085. [48] F.P. Luyten, Y.M. Yu, M. Yanagishita, S. Vukicevic, R.G. Hammonds and A.H. Reddi, J. Biol. Chem., 267 (1992) 3691. [49] A.J. Celeste, J.A. Iannazzi, R.C. Taylor, R.M. Hewick, V. Rosen, E.A. Wang and J.M. Wozney, Proc. Natl. Acad. Sci. USA, 87 (1990) 9843. [50] V. Rosen and R.S. Thies, Trends Genets., 8 (1992) 97. [51] K.M. Lyons, R.W. Peyton and B.L.M. Hogan, Genes Dev., 3 (1989) 1657. [52] C.M. Jones, K.M. Lyons and B.L.M. Hogan, Development, 111 (1991) 531. [53] T.A. Owen, M. Aronow, V. Shalhoub, L.M. Barone, L. Wilmin, M.S. Tassinari, M.B. Kennedy, S. Pockwinse, J.B. Lian and G. Stein, J. Cell Physiol., 143 (1990) 420. [54] K.-L. Yao, R. Todescan, Jr. and J. Sodek, J. Bone and Min. Res., 9 (1994) 231. [55] I. Asahina, T.K. Sampath, I. Nishimura and P.V. Hauschka, Z Cell Biol., 123 (1993) 921. [56] J. Sodek, J. Chen, S. Kasugai, T. Nagata, Q. Zhang, M.D. McKee and A. Nanci, in H. Slavkin and P. Price (eds.), The Chemistry and Biology o f Mineralized Tissues, Elsevier, Amsterdam, 1992, p. 297.