0013-7227/90/1271-0069$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 1 Printed in U.S.A.
Stimulation of Bone Matrix Apposition in Vitro by Local Growth Factors: A Comparison between Insulin-like Growth Factor I, Platelet-Derived Growth Factor, and Transforming Growth Factor /?* JOHANNES PFEILSCHIFTER, MARTIN OECHSNER, ANTJE NAUMANN, RAINER G. K. GRONWALD, HELMUT W. MINNE, AND REINHARD ZIEGLER Department of Internal Medicine I, University of Heidelberg, 69 Heidelberg, West Germany
in a dose-dependent manner up to 2-fold within 48 h. In addition, they partially or completely reversed the inhibition of bone matrix apposition observed with PTH. Exogenously added TGF0 was significantly more potent than equimolar concentrations of PDGF or IGF I in stimulating bone formation. Matrix apposition was greatest when IGF I, PDGF, and TGF/3 were added simultaneously to the culture medium, indicating that these factors can enhance each other in stimulating bone formation. In conclusion, our results provide direct evidence that IGF I, PDGF, and TGF/S are capable of stimulating bone formation in vitro. {.Endocrinology 127: 69-75, 1990)
ABSTRACT. Many recent in vitro studies have shown effects of insulin-like growth factor I (IGF I), platelet-derived growth factor (PDGF), and transforming growth factor-jS (TGF/3) on the proliferation and differential functions of bone-forming osteoblasts; however, the question whether these factors might ultimately lead to a net increase or decrease in bone formation has been difficult to assess. In this study, we have used an autoradiographic method based on the incorporation of [3H] proline into freshly synthesized bone matrix to determine the overall effects of these factors on bone matrix apposition in 21day-old fetal rat calvariae. IGF I, PDGF, and TGFjS increased bone matrix apposition
B
ONE is constantly remodeled by subsequent cycles of bone resorption and formation. There is increasing evidence that both phases of the remodeling process are controlled by locally released growth factors (1-3). Among the factors that are believed to play a major role during the remodeling process are insulin-like growth factor I (IGF I), platelet-derived growth factor (PDGF), and transforming growth factor-13 (TGF/3). All of the above factors are present in osteoblast-like cell cultures (4, 5) or bone matrix (6, 7) and have a variety of effects on the proliferation and differential functions of the osteoblast. IGF I has been shown to stimulate proliferation (812) and matrix synthesis (8, 10-12) in cultures of osteoblast-like cells or bone organ cultures. It also increases the expression of proteins, such as alkaline phosphatase and osteocalcin (9, 11), which are indicative of a differentiated osteoblastic phenotype. TGF/3 stimulates matrix synthesis in most culture systems (13-15) but has both
stimulatory (4, 13, 16, 17) and inhibitory (13, 14, 17-20) effects on the proliferation of osteoblast-like cells, depending on the cell type and the culture conditions. In addition, it can either stimulate (14, 19, 21) or inhibit (15, 18, 19, 22-26) the expression of various osteoblast cell products. PDGF stimulates proliferation of osteoblast-like cells (27, 28) and thereby indirectly increases collagen synthesis, but it also has a direct stimulatory effect on collagen degradation (28). Although these findings are consistent with a role for IGF I, PDGF, and TGF/3 as potential regulators of bone formation, they do not establish that these factors can induce a net change in bone formation. Therefore, in the present study, we have used an autoradiographic method to directly measure the effects of IGF I, PDGF, and TGF/3 or a combination of these factors on bone formation in fetal rat calvarial cultures. Materials and Methods Growth factors
Received February 5,1990. Address requests for reprints to: Dr. J. Pfeilschifter, Department of Internal Medicine I, Endocrinology and Metabolism, Bergheimer Strasse 58, D-6900 Heidelberg, West Germany. * This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
Human TGFft (TGF/3) was purchased from British Biotechnology (Oxford, U.K.); human recombinant IGF I and platelet-derived porcine PDGF BB (PDGF) were obtained from Boehringer Mannheim (Mannheim, West Germany). Human 69
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recombinant PDGF BB was a gift from Dr. Mark Murray (Zymogenetics, Seattle, WA) and was used in all experiments with PDGF, if not mentioned otherwise. Human PTH 1-34 was obtained from Bachem Biochemica (Heidelberg, West Germany). Organ culture The left and right parietal areas of 21-day-old fetal rat calvariae (Thomae, Biberach, West Germany) were cultured on steel grids at the interface between air and medium (serumfree Fitton-Jackson modified BGJ medium containing 1 mg/ ml BSA, both from Sigma, Deisenhofen, West Germany) in a 37 C incubator with 5% CO2 atmosphere. After a 24-h preculture period, the calvariae were transferred to fresh medium containing the growth factors and 5 ^Ci/ml [3,4-3H]proline (30 Ci/mmol; New England Nuclear, Boston, MA). Ascorbic acid (50 Mg/ml, Sigma) was added daily to the culture medium. A similar method has recently been used by Hock et al. (10) to assess bone matrix apposition in these cultures. Under conditions of continuous labeling, the area of labeled bone increased with time in culture up to an incubation period of 7 days, indicating continuous bone matrix apposition during that period, although most of the bone formation was observed during the first 48 h after preculture. In the present study labeling was therefore confined to this 48 h period. After incubation, the calvariae were rinsed with buffered saline and fixed in formaldehyde. In most experiments calvariae were then decalcified with 7.5% EDTA overnight and embedded in paraffin. In some experiments calvariae were fixed in 70% ethanol and embedded undecalcified in methylmethacrylate as described previously (29). A utoradiography Using a Jung Supercut microtome (Cambridge Instruments, Nu/?loch, West Germany), 3 /im sections were cut perpendicular to the midsagittal suture through the parietal areas of the calvariae. Sections were mounted on gelatin-coated slides and dried at 54 C overnight. They were then dipped into NTB 2 photographic emulsion (Eastman Kodak, Rochester, NY), dried, and stored for 3 days at 4 C. Autoradiographs were developed in Kodak Dektol developer and fixed in Kodak fixer. Afterward the sections were stained either with hematoxylineosin or according to the method of Goldner (30). Measurement of bone matrix apposition Preliminary experiments with insulin had shown that changes in bone matrix apposition can be most accurately measured 300 fira lateral to the midsagittal suture. Bone formation was quantitated by measuring the area containing silver grains resulting from the incorporation of [3H]proline into freshly synthesized bone matrix. Measurements were taken at 400-fold magnification in one field of view for the left and right parietal bone using a Zeiss III photomicroscope (Zeiss, Oberkochen, West Germany) attached to a semiautomatic image analysis system (Zeiss Morphomat 10). In addition, we measured the total area of bone in the same field of view. The area of unlabeled bone was calculated by subtraction of the labeled area from the total bone area. Although the thickness of the
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calvariae at the site of measurement varied slightly from specimen to specimen, we preferred to measure the absolute area of labeled bone instead of the ratio of labeled bone to total bone area. Thus, we were certain to exclude any interference due to bone resorption. Measurements in each field of view were repeated at least three times on each side of the calvaria, and all data from a single calvaria measurement were subsequently pooled. To avoid observer bias all specimens were assigned random numbers that were not decoded until data collection was complete. Statistical methods Data are expressed as the mean ± SEM. Differences between more than two groups were analyzed by analysis of variance and subsequent Scheffe test. Differences between two groups were analyzed by Student's t test.
Results At the site the measurement the 21-day-old calvariae consisted of a solid layer of bone matrix lined on both surfaces by osteoblasts and adjacent cells of the upper and lower periosteum (Fig. 1A). Autoradiographs from calvariae that had been cultured in the presence of [3H] proline revealed a distinct band of silver grains where labeled proline had been incorporated into the freshly synthesized bone matrix. This band was located between the upper osteoblast layer and the upper surface of the unlabeled bone (Fig. 1A). Labeling along the lower bone surface was sparse and usually confined to a few isolated areas. Most of the labeled bone, as well as up to 50% of the unlabeled bone, consisted of unmineralized osteoid, as judged by the red color evident when undecalcified sections were stained according to the method of Goldner (not shown). Human recombinant IGF I increased matrix apposition at concentrations of 1 and 10 nM. Higher concentrations were less stimulatory (Fig. 2A). Human recombinant PDGF stimulated bone formation in a dose-dependent manner from 0.1 nM to 10 nM (Fig. 2B). Similar results could also be obtained with purified PDGF from porcine platelets (not shown). Purified TGF/3 also stimulated matrix apposition in a dose-dependent manner from 0.1 nM to 10 nM (Fig. 2C). None of these factors changed the pattern of bone apposition observed in control calvariae (Fig. 1, A and B). In all calvariae where bone matrix apposition was strongly increased, some of the cells, originally located at the bone surface, became completely embedded in the new bone matrix, thus apparently becoming osteocytes (Fig. 1C). As with the control cultures, the newly formed, growth factor-stimulated bone matrix was mainly unmineralized. Although multinucleated osteoclasts were occasionally observed, no significant decreases in the area of unlabeled bone could be observed with IGF I, PDGF, or TGF/3 as compared to control cultures, indicating that
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FIG. 1. Autoradiographs of 3 nm sections of fetal rat calvariae cultured for 48 h in the presence of [3H]proline and the following factors: A, No factors added; UP, upper periosteum; LP, lower periosteum; OB, osteoblast layer. B, TGF/310"8 M. C, IGF I 10~9 M + PDGF 10"9 M + TGF/3 10"9 M. D, PTH 10~7 M. Hematoxylin-eosin staining. White bars = 25 /*m.
bone resorption was hardly affected (not shown). For a direct comparison of the capacity of IGF I, PDGF and TGF/3 to stimulate matrix apposition, the effects of equimolar concentrations (1 and 10 nM) of each of these growth factors on bone formation were compared using pooled data from four different experiments. We found that, at a concentration of 1 nM, TGF/3 stimulated matrix apposition significantly more than did PDGF or IGF I (Fig. 3). IGF I appeared to be the least effective, although the difference compared to PDGF was not significant. A similar tendency in the potency of these factors was also observed at a concentration of 10 nM, but the differences were not significant (not shown). Since IGF I, PDGF, and TGF/3 have all been extracted from bone matrix (6, 7), it is likely that they interact during bone formation. We observed that a combination of IGF I, PDGF, and TGF/3 was capable of inducing a significant increase in matrix apposition at concentrations which did not increase matrix apposition with each factor alone (Fig. 4A). At higher concentrations, the effects of IGF I, PDGF, and TGF/3 appeared to be addi-
tive (Fig. 4B). To examine whether TGF/3, IGF I, and PDGF are capable of reversing hormone-induced inhibitory effects on bone matrix apposition, we repeated some of the experiments in the presence of PTH, which is a potent inhibitor of collagen synthesis in vitro (31). As shown in Fig. 4A, PTH alone induced a dose-dependent inhibition of bone matrix apposition which was almost complete at 100 nM (see also Fig. ID). When IGF I, PDGF, or TGF/3 was added together with PTH, these factors were capable of partially or completely preventing the inhibitory effect of PTH (Fig. 5B). IGF I was the least effective, whereas TGF0 alone and combinations of TGF/3, IGF I, and PDGF were the most effective in reversing the PTHinduced inhibition of bone matrix synthesis (Fig. 5B).
Discussion Although the effects of IGF I, PDGF, and TGF0 on the metabolism of osteoblast-like cells have been extensively studied (1), few studies have examined the conse-
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STIMULATION OF BONE MATRIX APPOSITION IN VITRO
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0
0.1
1
10
100
IGF I (nM)
I
0
0.01
0.1
control
IGF I
PDGF
TGF/3
FIG. 3. Comparison between equimolar (1 nM) concentrations of IGF I, PDGF, and TGF/3 on bone matrix apposition in fetal rat calvariae. Data from 4 independent experiments were pooled and expressed as the mean ± SEM from a total of 15-20 calvariae per group. Statistical differences were determined by analysis of variance. TGF/? stimulated bone matrix apposition significantly more than PDGF and IGF I (P < 0.05).
1
PDGF (nM)
0
0.01
0.1
1
TGF/3 (nM) FIG. 2. Effects of IGF I (A), PDGF (B), and TGF0 (C) on bone matrix apposition in fetal rat calvariae within a 48 h incubation period. Bone matrix apposition was determined as the area of [3H]proline labeled bone as described in Materials and Methods. Data are shown as mean ± SEM for four calvariae per group. Statistical differences between treated groups and control group were determined by Student's t test. *, P < 0.05; **, P < 0.005.
quences of these effects on net bone matrix apposition (10, 32). Our study shows that PDGF and TGF0 have a dose-dependent stimulatory effect on bone matrix apposition in fetal rat calvarial cultures and confirms data of Hock et al. (10) on IGF I-mediated bone matrix apposition in these cultures. Significant increases in bone matrix apposition were observed with TGF/? at concentrations at low as 0.1 nM (2.5 ng/ml). The anabolic effect of these factors is stressed by the fact that they also reversed PTH-mediated decreases in bone matrix apposition. Incorporation of [3H]proline has been used in a number of recent studies to examine the effects of the above factors on collagen synthesis in fetal rat calvariae (8,11, 12, 16, 28, 31). In most of these studies extracellular
control
IGF I
TGF0
l+P+T
FlG. 4. Synergistic effects of IGF I, PDGF, and TGF/3 on bone matrix apposition in fetal rat calvariae. A, Effects of 0.1 nM IGF I, 0.01 nM PDGF, 0.01 nM TGF/3, or a combination of these factors at the indicated concentrations on bone matrix apposition in fetal rat calvariae. B, Effects of 1 nM IGF I, 1 nM PDGF, 1 nM TGF/3, or a combination of these factors on bone matrix apposition. I, IGF I; P, PDGF; T, TGF/3. Data are expressed as means ± SEM from four calvariae cultures per group. Statistical differences were determined by analysis of variance. In both panels A and B the combined effect of IGF I, PDGF, and TGF/3 on matrix apposition was significantly greater than the effects of each of these factors (P < 0.05).
matrix synthesis was analyzed biochemically after digestion with collagenase. This method is less time consuming than the autoradiographic method, but has a number of disadvantages compared to the latter: First, only a small part of the labeled proline is utilized by mature osteoblasts and is incorporated into the freshly synthesized bone matrix. A relatively greater part is incorporated into the extracellular matrix of the surrounding periosteal tissue. The problem of linking collagen syn-
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STIMULATION OF BONE MATRIX APPOSITION IN VITRO
1
10
100
PTH (nM)
control
IGF I
PDGF
TGF0
l+P+T
FIG. 5. Effects of PTH on bone matrix apposition in fetal rat calvariae. Data are expressed as the mean ± SEM from four calvariae per group. Statistical differences were determined by Student's t test. Significantly different from control: *, P < 0.05; **, P < 0.005. B, Reversal of PTHinduced decreases in matrix apposition by IGF I, PDGF, and TGF/3. IGF I, PDGF, TGF/3, or a combination of all three factors was added to calvariae cultures at a concentration of 1 nM in the presence of 100 nM PTH. Data are expressed as the mean ± SEM from four calvariae cultures per group. Statistical differences were determined by analysis of variance. At P < 0.05, PDGF, TGF/3, and the combination of IGF I, PDGF, and TGF/3 significantly increased bone matrix apposition in PTH-treated cultures.
thesis occurring during the incubation period to bone formation can only be incompletely solved by the timeconsuming removal of the periosteum from both sides of the calvariae before analysis of the labeled proteins. Second, collagen is a vital, but not the only, component of the bone matrix. Changes in the synthesis of collagen may not necessarily reflect overall changes in the synthesis of other matrix proteins, which may alter bone matrix apposition independent of the amount of collagen synthesized. Third, whether IGF I, PDGF, or TGF/3 stimulates orderly bone matrix apposition or merely generates fibrous tissue matrix cannot be judged without histological examination. It is therefore not surprising that there are distinctive differences between the effects of the above factors on bone formation as compared to their effects on collagen synthesis in the conventional collagen synthesis assay. Of all three factors tested in our study, TGF/3 proved to be the most effective in stimulating bone matrix apposition. In contrast, TGF/3 had little effect on collagen synthesis in fetal rat calvarial cultures in the conventional collagen synthesis assay (16). Our study confirms
73
data of Hock et al. (10) who showed that 100 nM IGF I stimulated bone matrix apposition less than 10 nM IGF I did, whereas in the conventional collagen synthesis assay, collagen synthesis was markedly higher at 100 nM IGF I than at 10 nM. These examples clearly show that the biochemical analysis of the rate of bone matrix production and the histological determination of bone matrix apposition do not necessarily correlate. Although some of the effects of IGF I, PDGF, and TGF/3 on the metabolism of osteoblasts are overlapping, each of these factors has a different spectrum of effects (1-3). Thus, PDGF may be more important for the proliferation of osteoblastic precursor cells, whereas TGF/3 may be more important for matrix synthesis in mature osteoblasts. These complementary effects on the osteoblast metabolism may explain why IGF I, PDGF, and TGF/3 enhanced the effects of each other on bone matrix apposition when added simultaneously to the cultures. Nevertheless, whether the combined effects of these factors are truly synergistic or merely additive, is difficult to show in our culture system. At low concentrations, the three factors appeared to be more than additive, since a combination of all three factors could induce an increase in bone matrix apposition at concentrations where each of these factors alone failed to stimulate matrix apposition. We could detect approximately 200 pM IGF I-like immunoreactive material in 48 h calvarial conditioned medium (data not shown). Since IGF I, PDGF, and TGF/3 seem to enhance the effect of each other on bone formation, we cannot exclude that some of the stimulatory effect of TGF/3 and PDGF on bone formation was due to the combined effect of the endogenously released IGF I and these factors. This may explain why these two growth factors were each more potent than IGF I alone. Similar interactions may have occurred between IGF I or PDGF and endogenously released TGF/3, but this is less likely since TGF/3 is predominantly released in an inactive form from calvarial cultures (33). In a preliminary report TGF/3 inhibited mineralization in osteoblast-like cell cultures (34). Most of the freshly synthesized bone matrix in our cultures consisted of unmineralized osteoid, but it is difficult to judge whether the tested factors might have impaired the mineralization process, since the freshly synthesized matrix in control cultures was also unmineralized. This lack of mineralization may have been due to the low phosphate concentration in the culture medium. While this study was under way, Gronowicz et al. (35) reported that 3 mM phosphate gives a much better mineralization rate in these cultures than the 1 mM phosphate present in ordinary BGJ medium. To determine whether a factor can stimulate bone formation under physiological conditions, in vivo exper-
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STIMULATION OF BONE MATRIX APPOSITION IN VITRO
iments are certainly preferable to in vitro assays; however, local growth factors would have to be applied in a way that closely mimics their autocrine or paracrine effects in a defined microenvironment. This makes it extremely difficult to perform such experiments at the present time. In a recent study Noda and Camilliere (32) injected TGF/J and PDGF into the periosteum of newborn rat calvariae in vivo (32). Although in our study TGF/3 and PDGF increased bone matrix apposition up to 100% within 48 h, Noda and Camilliere did not observe any increases in bone formation unless TGF/3 was injected daily for at least 5 days, and PDGF did not increase bone formation at all when injected in vivo. It is possible that this might have been due to the more complex in vivo environment or to the fact that the calvariae in their study were older and had a reduced potential for growth. Furthermore, it is possible that the mechanism of bone formation differed in the two experiments. Preliminary data from Joyce et al. (36) show that part of the bone tissue formed after injection of TGF/3 into the periosteum of newborn rat tibia is preceded by cartilage formation. Thus, part of the TGF /3-induced increase in bone formation in vivo may have been due to osteoinduction rather than to an increase in matrix apposition at the preexisting bone surface. This would also explain the delayed response of bone formation in the in vivo experiments.
Acknowledgments We wish to thank Dr. Mark Murray (Zymogenetics, Seattle, WA) for the generous gift of human recombinant PDGF BB.
References 1. Canalis E, McCarthy T, Centrella M 1988 Growth factors and the regulation of bone remodeling. J Clin Invest 81:277 2. Raisz LG 1988 Local and systemic factors in the pathogenesis of osteoporosis. N Engl J Med 318:8182 3. Marks SC, Popoff SN 1988 Bone cell biology: the regulation of development, structure, and function in the skeleton. Am J Anat 183:1 4. Gehron Robey P, Young MF, Flanders KC, Roche NS, Kondaiah P, Reddi AH, Termine JD, Sporn MB, Roberts AB 1987 Osteoblasts synthesize and respond to transforming growth factor-type 0 (TGF-0) in vitro. J. Cell Biol 105:457 5. Canalis E, McCarthy T, Centrella M 1988 Isolation and characterization of insulin-like growth factor I (somatomedin-C) from cultures of fetal rat calvariae. Endocrinology 122:22 6. Hauschka PV, Mavrakos AE, Iafrati MD, Doleman SE, Klagsbrun M 1986 Growth factors in bone matrix: isolation of multiple types by affinity chromatography on heparin-sepharose. J Biol Chem 261:12665 7. Seyedin SM, Thompson AY, Bentz H, Rosen DM, McPherson JM, Conti A, Siegel NR, Gallup GR, Piez KA 1986 Cartilage inducing factor A: apparent identity to TGF-/3. J Biol Chem 261:5693 8. Canalis E 1980 Effect of insulin-like growth factor I on DNA and protein synthesis in cultured rat calvaria. J Clin Invest 66:709 9. Schmid C, Steiner T, Froesch ER 1984 Insulin-like growth factor 1 supports differentiation of cultured osteoblast-like cells. FEBS Lett 173:48 10. Hock JM, Centrella M, Canalis E 1988 Insulin-like growth factor
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has independent effects on bone matrix formation and cell replication. Endocrinology 122:254 11. Canalis E, Lian JB 1988 Effects of bone associated growth factors on DNA, collagen and osteocalcin synthesis in cultured fetal rat calvariae. Bone 9:243 12. McCarthy TL, Centrella M, Canalis E 1989 Regulatory effects of insulin-like growth factors I and II on bone collagen synthesis in rat calvarial cultures. Endocrinology 124:301 13. Centrella M, McCarthy TL, Canalis E 1987 Transforming growth factor beta (TGF/3) is a bifunctional regulator of replication and collagen synthesis in osteoblast-enriched cell cultures from fetal rat bone. J Biol Chem 262:2869 14. Pfeilschifter J, D'Souza SM, Mundy GR 1987 Effects of transforming growth factor-/3 on osteoblastic osteosarcoma cells. Endocrinology 121:212 15. Wrana JL, Maeno M, Hawrylyshyn B, Yao K-L, Domenicucci C, Sodek J 1988 Differential effects of transforming growth factor-/? on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations. J Cell Biol 106:915 16. Centrella M, Massague J, Canalis E 1986 Human platelet-derived transforming growth factor-/3 stimulates parameters of bone growth in fetal rat calvariae. Endocrinology 119:2306 17. Ibbotson KJ, Orcutt CM, Anglin A-M, D'Souza SM 1989 Effects of transforming growth factors /?i and /?2 on a mouse clonal, osteoblastlike cell line MC3T3-E1. J Bone Mineral Res 4:37 18. Noda M, Rodan GA 1986 Type-/? transforming growth factor inhibits proliferation and expression of alkaline phosphatase in murine osteoblast-like cells. Biochem Biophys Res Commun 140:56 19. Noda M, Rodan GA 1987 Type /? transforming growth factor (TGF/3) regulation of alkaline phosphatase expression and other phenotype-related mRNAs in osteoblastic rat osteosarcoma cells. J Cell Physiol 133:426 20. Antosz ME, Bellows CG, Aubin JE 1989 Effects of transforming growth factor /? and epidermal growth factor on cell proliferation and the formation of bone nodules in isolated fetal rat calvaria cells. J Cell Physiol 140:386 21. Noda M, Yoon K, Prince CW, Butler WT, Rodan GA 1988 Transcriptional regulation of osteopontin production in rat osteosarcoma cells by type /? transforming growth factor. J Biol Chem 263:13916 22. Elford PR, Guenther HL, Felix R, Ceccini MG, Fleisch H 1987 Transforming growth factor-/? reduces the phenotypic expression of osteoblastic MC3T3-E1 cells in monolayer culture. Bone 8:259 23. Rosen DM, Stempien SU, Thompson AY, Seyedin SM 1988 Transforming growth factor-beta modulates the expression of osteoblast and chondroblast phenotypes in vitro. J Cell Physiol 134:337 24. Globus RK, Patterson-Buckendahl P, Gospodarowicz D 1988 Regulation of bovine bone cell proliferation by fibroblast growth factor and transforming growth factor /?. Endocrinology 123:98 25. Noda M 1989 Transcriptional regulation of osteocalcin production by transforming growth factor-/? in rat osteoblast-like cells. Endocrinology 124:612 26. Bertolini DR, Buono L 1989 The effects of transforming growth factor beta and basic fibroblast growth factor on adult human bone cells. J Bone Mineral Res 4 [Suppl 1]:25 (abstract) 27. Centrella M, McCarthy TL, Canalis E 1989 Platelet-derived growth factor enhances deoxyribonucleic acid and collagen synthesis in osteoblast-enriched cultures from fetal rat parietal bone. Endocrinology 125:13 28. Canalis E, McCarthy TL, Centrella M 1989 Effects of plateletderived growth factor on bone formation in vitro. J Cell Physiol 140:530 29. Minne HW, Pfeilchifter J, Scharla S, Mutschelknauss S, Schwarz A, Krempien B, Ziegler R 1984 Inflammation-mediated osteopenia in the rat: a new animal model for pathological loss of bone mass. Endocrinology 115:50 30. Goldner J 1938 A modification of the trichrom-technique for routine laboratory purpose. Am J Pathol 14:237 31. Kream BE, Rowe DW, Gworek S, Raisz LG 1980 Parathyroid hormone alters collagen synthesis and procollagen mRNA levels in fetal rat calvaria. Proc Natl Acad Sci USA 77:5654 32. Noda M, Camilliere JJ 1989 In vivo stimulation of bone formation
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STIMULATION OF BONE MATRIX APPOSITION IN VITRO by transforming growth factor-^. Endocrinology 124:2991 33. Pfeilschifter J, Bonewald L, Mundy GR 1990 Characterization of the latent transforming growth factor 0 complex in bone. J Bone Mineral Res, in press 34. Talley DJ, Lajiness EJ 1988 Transforming growth factor type beta (TGF0) inhibition of mineralization by normal bone cells in culture is independent of its effects on alkaline phosphatase. J Bone
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Mineral Res 3 [Suppl 1]:438 (abstract) 35. Gronowicz G, Woodiel FN, McCarthy M-B, Raisz LG 1989 In vitro mineralization of fetal rat parietal bones in defined serum-free medium: effect of /S-glycerol phosphate. J Bone Mineral Res 4:313 36. Joyce ME, Jingushi S, Roberts AB, Sporn MB, Bolander ME 1989 Transforming growth factor-/3 initiates cartilage and bone formation in vivo. J Bone Mineral Res 4 [Suppl 1]:566 (abstract)
Symposium on the Regulation and Actions of Follicle Stimulating Hormone October 25-28, 1990 Northwestern University Evanston, Illinois Chairmen: Mary Hunzicker-Dunn, Ph.D., and Neena Schwartz, Ph.D. Sponsored by Serono Symposia, USA This conference will address recent advances in the physiology, biochemistry, and molecular biology of FSH, as well as the clinical implications of FSH and gonadal peptide secretion. Major sessions are (I) Neuroendocrinology of FSH Secretion; (II) Synthesis and Secretion of FSH: Molecular Regulation; (III) Molecular Mechanisms of FSH Action in the Ovary; (IV) Molecular Mechanisms of FSH Action in the Testis; (V) Clinical Implications of FSH and Gonadal Peptide Secretion. The program will include plenary sessions with invited expert speakers and poster sessions for presentation of original abstracts. Deadline for submission of abstracts will be August 31, 1990. Category I CME credits will be available. For further information and abstract forms, please contact: L. Lisa Kern, Ph.D., Serono Symposia, USA, 100 Longwater Circle, Norwell, MA 02061. Telephone: 800-283-8088; 617-982-9000. Telefax: 617-982-9481.
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Vol. 127, No. 1 Printed in U.S.A.
Stimulation of Bone Matrix Apposition in Vitro by Local Growth Factors: A Comparison between Insulin-like Growth Factor I, Platelet-Derived Growth Factor, and Transforming Growth Factor /?* JOHANNES PFEILSCHIFTER, MARTIN OECHSNER, ANTJE NAUMANN, RAINER G. K. GRONWALD, HELMUT W. MINNE, AND REINHARD ZIEGLER Department of Internal Medicine I, University of Heidelberg, 69 Heidelberg, West Germany
in a dose-dependent manner up to 2-fold within 48 h. In addition, they partially or completely reversed the inhibition of bone matrix apposition observed with PTH. Exogenously added TGF0 was significantly more potent than equimolar concentrations of PDGF or IGF I in stimulating bone formation. Matrix apposition was greatest when IGF I, PDGF, and TGF/3 were added simultaneously to the culture medium, indicating that these factors can enhance each other in stimulating bone formation. In conclusion, our results provide direct evidence that IGF I, PDGF, and TGF/S are capable of stimulating bone formation in vitro. {.Endocrinology 127: 69-75, 1990)
ABSTRACT. Many recent in vitro studies have shown effects of insulin-like growth factor I (IGF I), platelet-derived growth factor (PDGF), and transforming growth factor-jS (TGF/3) on the proliferation and differential functions of bone-forming osteoblasts; however, the question whether these factors might ultimately lead to a net increase or decrease in bone formation has been difficult to assess. In this study, we have used an autoradiographic method based on the incorporation of [3H] proline into freshly synthesized bone matrix to determine the overall effects of these factors on bone matrix apposition in 21day-old fetal rat calvariae. IGF I, PDGF, and TGFjS increased bone matrix apposition
B
ONE is constantly remodeled by subsequent cycles of bone resorption and formation. There is increasing evidence that both phases of the remodeling process are controlled by locally released growth factors (1-3). Among the factors that are believed to play a major role during the remodeling process are insulin-like growth factor I (IGF I), platelet-derived growth factor (PDGF), and transforming growth factor-13 (TGF/3). All of the above factors are present in osteoblast-like cell cultures (4, 5) or bone matrix (6, 7) and have a variety of effects on the proliferation and differential functions of the osteoblast. IGF I has been shown to stimulate proliferation (812) and matrix synthesis (8, 10-12) in cultures of osteoblast-like cells or bone organ cultures. It also increases the expression of proteins, such as alkaline phosphatase and osteocalcin (9, 11), which are indicative of a differentiated osteoblastic phenotype. TGF/3 stimulates matrix synthesis in most culture systems (13-15) but has both
stimulatory (4, 13, 16, 17) and inhibitory (13, 14, 17-20) effects on the proliferation of osteoblast-like cells, depending on the cell type and the culture conditions. In addition, it can either stimulate (14, 19, 21) or inhibit (15, 18, 19, 22-26) the expression of various osteoblast cell products. PDGF stimulates proliferation of osteoblast-like cells (27, 28) and thereby indirectly increases collagen synthesis, but it also has a direct stimulatory effect on collagen degradation (28). Although these findings are consistent with a role for IGF I, PDGF, and TGF/3 as potential regulators of bone formation, they do not establish that these factors can induce a net change in bone formation. Therefore, in the present study, we have used an autoradiographic method to directly measure the effects of IGF I, PDGF, and TGF/3 or a combination of these factors on bone formation in fetal rat calvarial cultures. Materials and Methods Growth factors
Received February 5,1990. Address requests for reprints to: Dr. J. Pfeilschifter, Department of Internal Medicine I, Endocrinology and Metabolism, Bergheimer Strasse 58, D-6900 Heidelberg, West Germany. * This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
Human TGFft (TGF/3) was purchased from British Biotechnology (Oxford, U.K.); human recombinant IGF I and platelet-derived porcine PDGF BB (PDGF) were obtained from Boehringer Mannheim (Mannheim, West Germany). Human 69
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recombinant PDGF BB was a gift from Dr. Mark Murray (Zymogenetics, Seattle, WA) and was used in all experiments with PDGF, if not mentioned otherwise. Human PTH 1-34 was obtained from Bachem Biochemica (Heidelberg, West Germany). Organ culture The left and right parietal areas of 21-day-old fetal rat calvariae (Thomae, Biberach, West Germany) were cultured on steel grids at the interface between air and medium (serumfree Fitton-Jackson modified BGJ medium containing 1 mg/ ml BSA, both from Sigma, Deisenhofen, West Germany) in a 37 C incubator with 5% CO2 atmosphere. After a 24-h preculture period, the calvariae were transferred to fresh medium containing the growth factors and 5 ^Ci/ml [3,4-3H]proline (30 Ci/mmol; New England Nuclear, Boston, MA). Ascorbic acid (50 Mg/ml, Sigma) was added daily to the culture medium. A similar method has recently been used by Hock et al. (10) to assess bone matrix apposition in these cultures. Under conditions of continuous labeling, the area of labeled bone increased with time in culture up to an incubation period of 7 days, indicating continuous bone matrix apposition during that period, although most of the bone formation was observed during the first 48 h after preculture. In the present study labeling was therefore confined to this 48 h period. After incubation, the calvariae were rinsed with buffered saline and fixed in formaldehyde. In most experiments calvariae were then decalcified with 7.5% EDTA overnight and embedded in paraffin. In some experiments calvariae were fixed in 70% ethanol and embedded undecalcified in methylmethacrylate as described previously (29). A utoradiography Using a Jung Supercut microtome (Cambridge Instruments, Nu/?loch, West Germany), 3 /im sections were cut perpendicular to the midsagittal suture through the parietal areas of the calvariae. Sections were mounted on gelatin-coated slides and dried at 54 C overnight. They were then dipped into NTB 2 photographic emulsion (Eastman Kodak, Rochester, NY), dried, and stored for 3 days at 4 C. Autoradiographs were developed in Kodak Dektol developer and fixed in Kodak fixer. Afterward the sections were stained either with hematoxylineosin or according to the method of Goldner (30). Measurement of bone matrix apposition Preliminary experiments with insulin had shown that changes in bone matrix apposition can be most accurately measured 300 fira lateral to the midsagittal suture. Bone formation was quantitated by measuring the area containing silver grains resulting from the incorporation of [3H]proline into freshly synthesized bone matrix. Measurements were taken at 400-fold magnification in one field of view for the left and right parietal bone using a Zeiss III photomicroscope (Zeiss, Oberkochen, West Germany) attached to a semiautomatic image analysis system (Zeiss Morphomat 10). In addition, we measured the total area of bone in the same field of view. The area of unlabeled bone was calculated by subtraction of the labeled area from the total bone area. Although the thickness of the
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calvariae at the site of measurement varied slightly from specimen to specimen, we preferred to measure the absolute area of labeled bone instead of the ratio of labeled bone to total bone area. Thus, we were certain to exclude any interference due to bone resorption. Measurements in each field of view were repeated at least three times on each side of the calvaria, and all data from a single calvaria measurement were subsequently pooled. To avoid observer bias all specimens were assigned random numbers that were not decoded until data collection was complete. Statistical methods Data are expressed as the mean ± SEM. Differences between more than two groups were analyzed by analysis of variance and subsequent Scheffe test. Differences between two groups were analyzed by Student's t test.
Results At the site the measurement the 21-day-old calvariae consisted of a solid layer of bone matrix lined on both surfaces by osteoblasts and adjacent cells of the upper and lower periosteum (Fig. 1A). Autoradiographs from calvariae that had been cultured in the presence of [3H] proline revealed a distinct band of silver grains where labeled proline had been incorporated into the freshly synthesized bone matrix. This band was located between the upper osteoblast layer and the upper surface of the unlabeled bone (Fig. 1A). Labeling along the lower bone surface was sparse and usually confined to a few isolated areas. Most of the labeled bone, as well as up to 50% of the unlabeled bone, consisted of unmineralized osteoid, as judged by the red color evident when undecalcified sections were stained according to the method of Goldner (not shown). Human recombinant IGF I increased matrix apposition at concentrations of 1 and 10 nM. Higher concentrations were less stimulatory (Fig. 2A). Human recombinant PDGF stimulated bone formation in a dose-dependent manner from 0.1 nM to 10 nM (Fig. 2B). Similar results could also be obtained with purified PDGF from porcine platelets (not shown). Purified TGF/3 also stimulated matrix apposition in a dose-dependent manner from 0.1 nM to 10 nM (Fig. 2C). None of these factors changed the pattern of bone apposition observed in control calvariae (Fig. 1, A and B). In all calvariae where bone matrix apposition was strongly increased, some of the cells, originally located at the bone surface, became completely embedded in the new bone matrix, thus apparently becoming osteocytes (Fig. 1C). As with the control cultures, the newly formed, growth factor-stimulated bone matrix was mainly unmineralized. Although multinucleated osteoclasts were occasionally observed, no significant decreases in the area of unlabeled bone could be observed with IGF I, PDGF, or TGF/3 as compared to control cultures, indicating that
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FIG. 1. Autoradiographs of 3 nm sections of fetal rat calvariae cultured for 48 h in the presence of [3H]proline and the following factors: A, No factors added; UP, upper periosteum; LP, lower periosteum; OB, osteoblast layer. B, TGF/310"8 M. C, IGF I 10~9 M + PDGF 10"9 M + TGF/3 10"9 M. D, PTH 10~7 M. Hematoxylin-eosin staining. White bars = 25 /*m.
bone resorption was hardly affected (not shown). For a direct comparison of the capacity of IGF I, PDGF and TGF/3 to stimulate matrix apposition, the effects of equimolar concentrations (1 and 10 nM) of each of these growth factors on bone formation were compared using pooled data from four different experiments. We found that, at a concentration of 1 nM, TGF/3 stimulated matrix apposition significantly more than did PDGF or IGF I (Fig. 3). IGF I appeared to be the least effective, although the difference compared to PDGF was not significant. A similar tendency in the potency of these factors was also observed at a concentration of 10 nM, but the differences were not significant (not shown). Since IGF I, PDGF, and TGF/3 have all been extracted from bone matrix (6, 7), it is likely that they interact during bone formation. We observed that a combination of IGF I, PDGF, and TGF/3 was capable of inducing a significant increase in matrix apposition at concentrations which did not increase matrix apposition with each factor alone (Fig. 4A). At higher concentrations, the effects of IGF I, PDGF, and TGF/3 appeared to be addi-
tive (Fig. 4B). To examine whether TGF/3, IGF I, and PDGF are capable of reversing hormone-induced inhibitory effects on bone matrix apposition, we repeated some of the experiments in the presence of PTH, which is a potent inhibitor of collagen synthesis in vitro (31). As shown in Fig. 4A, PTH alone induced a dose-dependent inhibition of bone matrix apposition which was almost complete at 100 nM (see also Fig. ID). When IGF I, PDGF, or TGF/3 was added together with PTH, these factors were capable of partially or completely preventing the inhibitory effect of PTH (Fig. 5B). IGF I was the least effective, whereas TGF0 alone and combinations of TGF/3, IGF I, and PDGF were the most effective in reversing the PTHinduced inhibition of bone matrix synthesis (Fig. 5B).
Discussion Although the effects of IGF I, PDGF, and TGF0 on the metabolism of osteoblast-like cells have been extensively studied (1), few studies have examined the conse-
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0
0.1
1
10
100
IGF I (nM)
I
0
0.01
0.1
control
IGF I
PDGF
TGF/3
FIG. 3. Comparison between equimolar (1 nM) concentrations of IGF I, PDGF, and TGF/3 on bone matrix apposition in fetal rat calvariae. Data from 4 independent experiments were pooled and expressed as the mean ± SEM from a total of 15-20 calvariae per group. Statistical differences were determined by analysis of variance. TGF/? stimulated bone matrix apposition significantly more than PDGF and IGF I (P < 0.05).
1
PDGF (nM)
0
0.01
0.1
1
TGF/3 (nM) FIG. 2. Effects of IGF I (A), PDGF (B), and TGF0 (C) on bone matrix apposition in fetal rat calvariae within a 48 h incubation period. Bone matrix apposition was determined as the area of [3H]proline labeled bone as described in Materials and Methods. Data are shown as mean ± SEM for four calvariae per group. Statistical differences between treated groups and control group were determined by Student's t test. *, P < 0.05; **, P < 0.005.
quences of these effects on net bone matrix apposition (10, 32). Our study shows that PDGF and TGF0 have a dose-dependent stimulatory effect on bone matrix apposition in fetal rat calvarial cultures and confirms data of Hock et al. (10) on IGF I-mediated bone matrix apposition in these cultures. Significant increases in bone matrix apposition were observed with TGF/? at concentrations at low as 0.1 nM (2.5 ng/ml). The anabolic effect of these factors is stressed by the fact that they also reversed PTH-mediated decreases in bone matrix apposition. Incorporation of [3H]proline has been used in a number of recent studies to examine the effects of the above factors on collagen synthesis in fetal rat calvariae (8,11, 12, 16, 28, 31). In most of these studies extracellular
control
IGF I
TGF0
l+P+T
FlG. 4. Synergistic effects of IGF I, PDGF, and TGF/3 on bone matrix apposition in fetal rat calvariae. A, Effects of 0.1 nM IGF I, 0.01 nM PDGF, 0.01 nM TGF/3, or a combination of these factors at the indicated concentrations on bone matrix apposition in fetal rat calvariae. B, Effects of 1 nM IGF I, 1 nM PDGF, 1 nM TGF/3, or a combination of these factors on bone matrix apposition. I, IGF I; P, PDGF; T, TGF/3. Data are expressed as means ± SEM from four calvariae cultures per group. Statistical differences were determined by analysis of variance. In both panels A and B the combined effect of IGF I, PDGF, and TGF/3 on matrix apposition was significantly greater than the effects of each of these factors (P < 0.05).
matrix synthesis was analyzed biochemically after digestion with collagenase. This method is less time consuming than the autoradiographic method, but has a number of disadvantages compared to the latter: First, only a small part of the labeled proline is utilized by mature osteoblasts and is incorporated into the freshly synthesized bone matrix. A relatively greater part is incorporated into the extracellular matrix of the surrounding periosteal tissue. The problem of linking collagen syn-
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STIMULATION OF BONE MATRIX APPOSITION IN VITRO
1
10
100
PTH (nM)
control
IGF I
PDGF
TGF0
l+P+T
FIG. 5. Effects of PTH on bone matrix apposition in fetal rat calvariae. Data are expressed as the mean ± SEM from four calvariae per group. Statistical differences were determined by Student's t test. Significantly different from control: *, P < 0.05; **, P < 0.005. B, Reversal of PTHinduced decreases in matrix apposition by IGF I, PDGF, and TGF/3. IGF I, PDGF, TGF/3, or a combination of all three factors was added to calvariae cultures at a concentration of 1 nM in the presence of 100 nM PTH. Data are expressed as the mean ± SEM from four calvariae cultures per group. Statistical differences were determined by analysis of variance. At P < 0.05, PDGF, TGF/3, and the combination of IGF I, PDGF, and TGF/3 significantly increased bone matrix apposition in PTH-treated cultures.
thesis occurring during the incubation period to bone formation can only be incompletely solved by the timeconsuming removal of the periosteum from both sides of the calvariae before analysis of the labeled proteins. Second, collagen is a vital, but not the only, component of the bone matrix. Changes in the synthesis of collagen may not necessarily reflect overall changes in the synthesis of other matrix proteins, which may alter bone matrix apposition independent of the amount of collagen synthesized. Third, whether IGF I, PDGF, or TGF/3 stimulates orderly bone matrix apposition or merely generates fibrous tissue matrix cannot be judged without histological examination. It is therefore not surprising that there are distinctive differences between the effects of the above factors on bone formation as compared to their effects on collagen synthesis in the conventional collagen synthesis assay. Of all three factors tested in our study, TGF/3 proved to be the most effective in stimulating bone matrix apposition. In contrast, TGF/3 had little effect on collagen synthesis in fetal rat calvarial cultures in the conventional collagen synthesis assay (16). Our study confirms
73
data of Hock et al. (10) who showed that 100 nM IGF I stimulated bone matrix apposition less than 10 nM IGF I did, whereas in the conventional collagen synthesis assay, collagen synthesis was markedly higher at 100 nM IGF I than at 10 nM. These examples clearly show that the biochemical analysis of the rate of bone matrix production and the histological determination of bone matrix apposition do not necessarily correlate. Although some of the effects of IGF I, PDGF, and TGF/3 on the metabolism of osteoblasts are overlapping, each of these factors has a different spectrum of effects (1-3). Thus, PDGF may be more important for the proliferation of osteoblastic precursor cells, whereas TGF/3 may be more important for matrix synthesis in mature osteoblasts. These complementary effects on the osteoblast metabolism may explain why IGF I, PDGF, and TGF/3 enhanced the effects of each other on bone matrix apposition when added simultaneously to the cultures. Nevertheless, whether the combined effects of these factors are truly synergistic or merely additive, is difficult to show in our culture system. At low concentrations, the three factors appeared to be more than additive, since a combination of all three factors could induce an increase in bone matrix apposition at concentrations where each of these factors alone failed to stimulate matrix apposition. We could detect approximately 200 pM IGF I-like immunoreactive material in 48 h calvarial conditioned medium (data not shown). Since IGF I, PDGF, and TGF/3 seem to enhance the effect of each other on bone formation, we cannot exclude that some of the stimulatory effect of TGF/3 and PDGF on bone formation was due to the combined effect of the endogenously released IGF I and these factors. This may explain why these two growth factors were each more potent than IGF I alone. Similar interactions may have occurred between IGF I or PDGF and endogenously released TGF/3, but this is less likely since TGF/3 is predominantly released in an inactive form from calvarial cultures (33). In a preliminary report TGF/3 inhibited mineralization in osteoblast-like cell cultures (34). Most of the freshly synthesized bone matrix in our cultures consisted of unmineralized osteoid, but it is difficult to judge whether the tested factors might have impaired the mineralization process, since the freshly synthesized matrix in control cultures was also unmineralized. This lack of mineralization may have been due to the low phosphate concentration in the culture medium. While this study was under way, Gronowicz et al. (35) reported that 3 mM phosphate gives a much better mineralization rate in these cultures than the 1 mM phosphate present in ordinary BGJ medium. To determine whether a factor can stimulate bone formation under physiological conditions, in vivo exper-
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STIMULATION OF BONE MATRIX APPOSITION IN VITRO
iments are certainly preferable to in vitro assays; however, local growth factors would have to be applied in a way that closely mimics their autocrine or paracrine effects in a defined microenvironment. This makes it extremely difficult to perform such experiments at the present time. In a recent study Noda and Camilliere (32) injected TGF/J and PDGF into the periosteum of newborn rat calvariae in vivo (32). Although in our study TGF/3 and PDGF increased bone matrix apposition up to 100% within 48 h, Noda and Camilliere did not observe any increases in bone formation unless TGF/3 was injected daily for at least 5 days, and PDGF did not increase bone formation at all when injected in vivo. It is possible that this might have been due to the more complex in vivo environment or to the fact that the calvariae in their study were older and had a reduced potential for growth. Furthermore, it is possible that the mechanism of bone formation differed in the two experiments. Preliminary data from Joyce et al. (36) show that part of the bone tissue formed after injection of TGF/3 into the periosteum of newborn rat tibia is preceded by cartilage formation. Thus, part of the TGF /3-induced increase in bone formation in vivo may have been due to osteoinduction rather than to an increase in matrix apposition at the preexisting bone surface. This would also explain the delayed response of bone formation in the in vivo experiments.
Acknowledgments We wish to thank Dr. Mark Murray (Zymogenetics, Seattle, WA) for the generous gift of human recombinant PDGF BB.
References 1. Canalis E, McCarthy T, Centrella M 1988 Growth factors and the regulation of bone remodeling. J Clin Invest 81:277 2. Raisz LG 1988 Local and systemic factors in the pathogenesis of osteoporosis. N Engl J Med 318:8182 3. Marks SC, Popoff SN 1988 Bone cell biology: the regulation of development, structure, and function in the skeleton. Am J Anat 183:1 4. Gehron Robey P, Young MF, Flanders KC, Roche NS, Kondaiah P, Reddi AH, Termine JD, Sporn MB, Roberts AB 1987 Osteoblasts synthesize and respond to transforming growth factor-type 0 (TGF-0) in vitro. J. Cell Biol 105:457 5. Canalis E, McCarthy T, Centrella M 1988 Isolation and characterization of insulin-like growth factor I (somatomedin-C) from cultures of fetal rat calvariae. Endocrinology 122:22 6. Hauschka PV, Mavrakos AE, Iafrati MD, Doleman SE, Klagsbrun M 1986 Growth factors in bone matrix: isolation of multiple types by affinity chromatography on heparin-sepharose. J Biol Chem 261:12665 7. Seyedin SM, Thompson AY, Bentz H, Rosen DM, McPherson JM, Conti A, Siegel NR, Gallup GR, Piez KA 1986 Cartilage inducing factor A: apparent identity to TGF-/3. J Biol Chem 261:5693 8. Canalis E 1980 Effect of insulin-like growth factor I on DNA and protein synthesis in cultured rat calvaria. J Clin Invest 66:709 9. Schmid C, Steiner T, Froesch ER 1984 Insulin-like growth factor 1 supports differentiation of cultured osteoblast-like cells. FEBS Lett 173:48 10. Hock JM, Centrella M, Canalis E 1988 Insulin-like growth factor
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has independent effects on bone matrix formation and cell replication. Endocrinology 122:254 11. Canalis E, Lian JB 1988 Effects of bone associated growth factors on DNA, collagen and osteocalcin synthesis in cultured fetal rat calvariae. Bone 9:243 12. McCarthy TL, Centrella M, Canalis E 1989 Regulatory effects of insulin-like growth factors I and II on bone collagen synthesis in rat calvarial cultures. Endocrinology 124:301 13. Centrella M, McCarthy TL, Canalis E 1987 Transforming growth factor beta (TGF/3) is a bifunctional regulator of replication and collagen synthesis in osteoblast-enriched cell cultures from fetal rat bone. J Biol Chem 262:2869 14. Pfeilschifter J, D'Souza SM, Mundy GR 1987 Effects of transforming growth factor-/3 on osteoblastic osteosarcoma cells. Endocrinology 121:212 15. Wrana JL, Maeno M, Hawrylyshyn B, Yao K-L, Domenicucci C, Sodek J 1988 Differential effects of transforming growth factor-/? on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations. J Cell Biol 106:915 16. Centrella M, Massague J, Canalis E 1986 Human platelet-derived transforming growth factor-/3 stimulates parameters of bone growth in fetal rat calvariae. Endocrinology 119:2306 17. Ibbotson KJ, Orcutt CM, Anglin A-M, D'Souza SM 1989 Effects of transforming growth factors /?i and /?2 on a mouse clonal, osteoblastlike cell line MC3T3-E1. J Bone Mineral Res 4:37 18. Noda M, Rodan GA 1986 Type-/? transforming growth factor inhibits proliferation and expression of alkaline phosphatase in murine osteoblast-like cells. Biochem Biophys Res Commun 140:56 19. Noda M, Rodan GA 1987 Type /? transforming growth factor (TGF/3) regulation of alkaline phosphatase expression and other phenotype-related mRNAs in osteoblastic rat osteosarcoma cells. J Cell Physiol 133:426 20. Antosz ME, Bellows CG, Aubin JE 1989 Effects of transforming growth factor /? and epidermal growth factor on cell proliferation and the formation of bone nodules in isolated fetal rat calvaria cells. J Cell Physiol 140:386 21. Noda M, Yoon K, Prince CW, Butler WT, Rodan GA 1988 Transcriptional regulation of osteopontin production in rat osteosarcoma cells by type /? transforming growth factor. J Biol Chem 263:13916 22. Elford PR, Guenther HL, Felix R, Ceccini MG, Fleisch H 1987 Transforming growth factor-/? reduces the phenotypic expression of osteoblastic MC3T3-E1 cells in monolayer culture. Bone 8:259 23. Rosen DM, Stempien SU, Thompson AY, Seyedin SM 1988 Transforming growth factor-beta modulates the expression of osteoblast and chondroblast phenotypes in vitro. J Cell Physiol 134:337 24. Globus RK, Patterson-Buckendahl P, Gospodarowicz D 1988 Regulation of bovine bone cell proliferation by fibroblast growth factor and transforming growth factor /?. Endocrinology 123:98 25. Noda M 1989 Transcriptional regulation of osteocalcin production by transforming growth factor-/? in rat osteoblast-like cells. Endocrinology 124:612 26. Bertolini DR, Buono L 1989 The effects of transforming growth factor beta and basic fibroblast growth factor on adult human bone cells. J Bone Mineral Res 4 [Suppl 1]:25 (abstract) 27. Centrella M, McCarthy TL, Canalis E 1989 Platelet-derived growth factor enhances deoxyribonucleic acid and collagen synthesis in osteoblast-enriched cultures from fetal rat parietal bone. Endocrinology 125:13 28. Canalis E, McCarthy TL, Centrella M 1989 Effects of plateletderived growth factor on bone formation in vitro. J Cell Physiol 140:530 29. Minne HW, Pfeilchifter J, Scharla S, Mutschelknauss S, Schwarz A, Krempien B, Ziegler R 1984 Inflammation-mediated osteopenia in the rat: a new animal model for pathological loss of bone mass. Endocrinology 115:50 30. Goldner J 1938 A modification of the trichrom-technique for routine laboratory purpose. Am J Pathol 14:237 31. Kream BE, Rowe DW, Gworek S, Raisz LG 1980 Parathyroid hormone alters collagen synthesis and procollagen mRNA levels in fetal rat calvaria. Proc Natl Acad Sci USA 77:5654 32. Noda M, Camilliere JJ 1989 In vivo stimulation of bone formation
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STIMULATION OF BONE MATRIX APPOSITION IN VITRO by transforming growth factor-^. Endocrinology 124:2991 33. Pfeilschifter J, Bonewald L, Mundy GR 1990 Characterization of the latent transforming growth factor 0 complex in bone. J Bone Mineral Res, in press 34. Talley DJ, Lajiness EJ 1988 Transforming growth factor type beta (TGF0) inhibition of mineralization by normal bone cells in culture is independent of its effects on alkaline phosphatase. J Bone
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Mineral Res 3 [Suppl 1]:438 (abstract) 35. Gronowicz G, Woodiel FN, McCarthy M-B, Raisz LG 1989 In vitro mineralization of fetal rat parietal bones in defined serum-free medium: effect of /S-glycerol phosphate. J Bone Mineral Res 4:313 36. Joyce ME, Jingushi S, Roberts AB, Sporn MB, Bolander ME 1989 Transforming growth factor-/3 initiates cartilage and bone formation in vivo. J Bone Mineral Res 4 [Suppl 1]:566 (abstract)
Symposium on the Regulation and Actions of Follicle Stimulating Hormone October 25-28, 1990 Northwestern University Evanston, Illinois Chairmen: Mary Hunzicker-Dunn, Ph.D., and Neena Schwartz, Ph.D. Sponsored by Serono Symposia, USA This conference will address recent advances in the physiology, biochemistry, and molecular biology of FSH, as well as the clinical implications of FSH and gonadal peptide secretion. Major sessions are (I) Neuroendocrinology of FSH Secretion; (II) Synthesis and Secretion of FSH: Molecular Regulation; (III) Molecular Mechanisms of FSH Action in the Ovary; (IV) Molecular Mechanisms of FSH Action in the Testis; (V) Clinical Implications of FSH and Gonadal Peptide Secretion. The program will include plenary sessions with invited expert speakers and poster sessions for presentation of original abstracts. Deadline for submission of abstracts will be August 31, 1990. Category I CME credits will be available. For further information and abstract forms, please contact: L. Lisa Kern, Ph.D., Serono Symposia, USA, 100 Longwater Circle, Norwell, MA 02061. Telephone: 800-283-8088; 617-982-9000. Telefax: 617-982-9481.
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