PGE2 stimulates both resorption and formation of bone in vitroDifferential responses of the periosteum and the endosteum in fetal rat long bone cultures.код для вставкиСкачать
THE ANATOMICAL RECORD 211:9-16 (1985) PGE2 Stimulates Both Resorption and Formation of Bone In Vitro: Differential Responses of the Periosteum and the Endosteum in Fetal Rat Long Bone Cultures JEAN-RAPHAEL NEFUSSI AND ROLAND BARON Yale University School of Medicine, Departments of Cell Biology and Internal Medicine, New Haven, CT 06510 ABSTRACT The ability of PGE2 to stimulate bone resorption in vitro and in vivo is well established but the effects of this compound on bone formation are still controversial. Recent clinical reports have suggested that long-term infusion of PGE in infants with cyanotic heart diseases led to a stimulation of periosteal bone formation and to hyperostosis. M) in bone organ In the present report, we describe the effects of PGE2 cultures on bone resorption, measured by the release of 45Calcium and the number of osteoclasts in sections of cultured bones, and bone volume, by measuring separately medullary and cortical areas. PGE2 induced a marked increase in 45Ca release and in cortical and medullary osteoclast numbers over 4 days in vitro; despite this increase in bone resorption, cortical bone volume remained constant, indicating a parallel increase in bone resorption and formation a t this site. Morphological and quantitative data demonstrated a higher extent of osteoblastic surface along the periosteum of PGE2-treated bones when compared with control cultures. Medullary bone volume, on the other hand, decreased sharply during the culture period, demonstrating a lack of parallel increase in bone formation at this site. It is concluded that, under these experimental conditions, prostaglandin E2 stimulated both resorption and formation along the periosteum and only bone resorption along the endosteum of the cultured bones. The overall effect of PGE2 on bone a s a whole, however, was net bone loss. During bone remodeling, bone resorption and bone formation normally occur as two successive steps of the same sequence of events (Frost, 1964; Baron, 1977). Nevertheless, studies on the effects of prostaglandins (PG) on bone have usually considered separately the changes in bone resorption and in bone formation both in vitro (Klein and Raisz, 1970; Dietrich et al., 1975; Holtrop and Raisz, 1979) and in vivo (Santoro et al., 1977; Goodson et al., 1974; Tashjian et al., 1972). The effects of PG of the E series on bone resorption are rather well characterized: in vitro a s well as in vivo after administration of PGE or in various pathological conditions (Powles et al., 1973; Seyberth et a1.,1975; Eilon and Mundy, 1978; Tashjian et al., 1972; Franklin and Tashjian, 1975; Voelkel et al., 19751, PGE have been shown to increase bone resorption through a n increase in the number of osteoclasts (Rifkin et al., 1980)and/or in their activity (Holtrop and Raisz, 1979). More controversial are the effects of PGE on bone formation. It was suggested (Raisz and Koolemans-Beynen, 1974) that in vitro they inhibited bone formation as measured by the incorporation of labeled proline into bone matrix. Similarly, both a n increase in resorption and a decrease in formation were reported in rabbits bearing the prostaglandin-producing VX2 carcinoma (Wolfe et al., 1978) and in rats injected daily for up to 9 0 1985 ALAN R. LISS, INC. days with PGE (Yonaga and Morimoto, 1979). On the other hand, a n opposite effect on bone formation has been suggested by some experimental and clinical studies (Blumenkrantz and Sondergard, 1972; Ueda et al., 1980; Ringel et al., 1982). Similarly, the results of Vanderwiel and Talmage (1979) have suggested a n increase in calcium flow to the bone under PGE2 infusion. The present study was therefore undertaken to evaluate in a single culture system (Raisz and Niemann, 1969; Nefussi et al., 1982) the effects of PGE2 on both bone volume and bone resorption using a concentration (lop5M ) l that was shown in previous studies to induce maximum stimulation of bone resorption and inhibition of collagen synthesis (Klein and Raisz, 1970; Raisz and Koolemans-Beynen, 1974). Owing to the fact that histo- Received October 17, 1983; accepted August 6,1984. Jean-Raphael Nefussi's present address is University of Paris 7, Paris, France. Address reprint request to Dr. Roland Baron, Endocrine Section, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510. 'A typographical error was present in a previously published abstract of part of this work (Calcif. Tissue Int. 33:298, 1981)mentioning erroneously a concentration of M PGE2. It should have been M PGE2. 10 J:R. NEFUSSI AND R. BARON morphometric methods allow a distinction between different areas of the explants which might react differently to the same stimulus, the results demonstrate that 1) bone resorption is stimulated by PGE2 through a n increase in the number of osteoclasts; 2) the amount of the bone present in the cortical bone, however, remains constant despite the increase in resorption and in 45Ca release, indirectly implying that, a t this concentration, bone formation is also stimulated along the periosteum, as confirmed by a measured increase in osteoblastic surface; and 3) medullary bone resorption is rapid and results in a n overall net bone loss. MATERIALS AND METHODS Bone Culture System Following the technique of Raisz and Niemann (1969) for bone cultures, three Sprague-Dawley rats were injected on the 18th day of pregnancy with 500 pCi of 45CaC1z (specific activity - 30 Ci/gm; New England Nuclear, Boston, MA). One day later, the rats were sacrificed and the fetal radii and ulnae were dissected out. The cartilage ends were removed and the remaining midshafts were individually cultured in a 24-well culture dish with modified BGJb medium (Gibco, Grand Island, NY) supplemented with ascorbic acid (0.1 mg/ml; Fischer Sci. Co.), streptomycin (50 pg/ml; Gibco), penicillin (50 units/ml; Gibco) bovine serum albumin (1mgiml, Fraction V; Sigma, St. Louis, MO) and L-glutamine (2 x lop3 M, Gibco). The dishes were placed in a humidified incubator a t 37°C in a n atmosphere of 5% COz and air. The bones were precultured from the first day either in supplemented BGJb medium or in the same medium but containing M PGE2 for the experimental bones. After a change in medium, the bones were kept in culture for 4 additional days and the medium was changed after 48 hours of culture. PGEz was present in the culture medium of the experimental bones throughout the culture period. Preparation of the PGE2 Medium Four milligrams of PGE2 (MW = 352; Upjohn, Kalamazoo, MI) were dissolved in 1 ml of absolute alcohol, and 20 p1 of this solution was added to 19.98 ml of supplemented BGJb medium. The final concentration of M1 in PGE2 in the medium therefore was 1.14 x 0.01% alcohol. A similar amount of alcohol was added to the control medium. Bone Histomorphometric Studies and 45Ca Release Measurement At the end of each day, five control and experimental paired bones were removed for histomorphometric study and aliquots of 50 pI of cultured medium of each well was diluted in 10 ml of scintillation fluid and counted for the presence of radioactive calcium. The data for 45Ca release are expressed a t the ratio of treated over control bones. Control and PGE2-treated bones were fixed in 40% ethanol, dehydrated in graded alcohol, and embedded without decalcification in methyl-methacrylate. Three micron thick longitudinal sections were cut with an Autocut Microtome (Jung, Germany) and stained with toluidine blue (pH 3.0). A morphological study was performed to evaluate the cellularity of the explant. Measurements of the internal diameter of the midshaft with a n ocular micrometer was used to select the widest (most central) section for histomorphometry. The measurements were made at x 500 magnification with a Manual Optical Planimeter (MOP 3, Carl Zeiss, Germany). The following parameters were recorded separately for cortical and medullary bone: bone volume (% of whole tissue and cubic microns [p3] number of osteoclasts per field (0.05 mm2) and extent of osteoblastic surface (osteoblasts lining osteoid tissue) along the periosteal surface (%I. Statistical comparison between control and PGE2treated cultures was performed using the Student's t test and the difference was considered significant when P < 0.05. RESULTS Control Bone Cultures A slight but unsignificant increase in bone volume (% and cubic microns, p3) was recorded during the 4 days of culture, both in the cortical and medullary bone. Medullary and cortical osteoclast numbers were low and remained unchanged during the experiment. Similarly, no changes were observed in the extent of periosteal osteoblastic surface (Table 1). PG&Treated Bone Cultures In contrast, significant changes were recorded in the bones cultured with PGE2 (Figs. 1, 2). Over the total culture period (cumulative data of the 4 days in culture), the number of cortical osteoclasts was significantly higher than in the control bones (0.46 5 0.28 vs. 0.05 f 0.07, OCifield, P < 0.01) (all numbers are given as mean standard deviation). However, despite this increase in the number of osteoclasts, no significant changes occurred in the cortical bone volume (%) and amount (Fig. 1).Similarly, a significant increase in osteoclast numbers was recorded in the medullary bone (0.3 k 0.3 vs. 0.8 % 0.4,OC/field, P < 0.05). In this case, however, this was associated with a significant decrease in medullary bone (185 k 52 vs. 114 & 20, p3, P < 0.01) and bone volume (17.7 f 3.3 vs. 9.8 f 2.0%, P < 0.01) (Fig. 2). The extent of periosteal osteoblastic surface was significantly higher in the PGEz-treated bones (Table 1)and numerous active osteoblasts, characterized by large Golgi areas in a very basophilic cytoplasm, and new bone formation, attested by increased osteoid, was prominent along the periosteal surface of the bones cultured with PGE2 (Figs. 3,4). Although osteoclast numbers increased both in the medullary and cortical bone, the kinetics of that increase was different. A significant increase in medullary osteoclasts was first observed after 24 hours in culture (1.1 0.2 vs. 0.3 5 0.2, OC/field, P < 0.001); this number remained unchanged and returned toward the control level within the last 2 days (Fig. 2). In contrast, the increase in cortical osteoclasts was slower and delayed in time, reaching a peak only after the third day in culture (0.7 -+_ 0.4 vs. 0.5 f 0.05, OC/field, P < 0.01) and then decreased (Fig. 1). When the 45Ca release was compared with changes in osteoclast numbers, a positive linear and significant correlation (r = 0.72, P < 0.01) was found with the cortical osteoclasts but not with the medullary osteoclasts (Fig. 5). Finally, when the total bone volume was calculated without separating cortical and medullary bone, a sig- * * 11 PGEz STIMULATES BONE FORMATION AND RESORPTION CORTICAL BONE y MEDULLARY BONE *** p<ooo1/c ** p<oo1 /c - Control -- PGE, - Control -- PGE, * p<O.OI / c 1.0 tt ku) a J 0 0.5 0 W !u) 0 b 0 t T ' I 1 I I I L I TI I 0' I T -s W I W 3 J 0 i , 50 0 > > W 45 I : i m I I I t I 0 I 2 3 4 DAYS lo/ 1 I L 0 1 2 1 3 4 DAYS Fig. 2. Effects of PGE2 on medullary bone in culture over a 4-day Fig. 1. Effects of PGE2 on cortical bone in culture over a 4-day period. Bars represent standard deviations; P < O.Ol/C = significantly differ- period. Bars represent standard deviations; /C = significantly different ent from controls. from controls. medullary bone, this led to a significant bone loss, therefore indicating that bone formation was decreased relative to bone resorption. On the other hand, despite the increase in the number of osteoclasts, no decrease in cortical bone was measured and a n increase in periosteal osteoblastic surface was present, therefore demonstrating a parallel increase in bone formation a t this site. The overall effect on the amount of bone present in the cultured bones, however, was a net bone loss. Our observations regarding the stimulation of bone resorption by PGE2 are in agreement with those in much of the literature (Klein and Raisz, 1970; Dietrich et al., 1975; Rifkin et al., 1980; Holtrop and Raisz, 1979). Few reports, however, have mentioned an increase in bone formation. The maintenance of a normal bone mass in the cortical bone could be accounted for in two different ways. The first would be to assume that the newly differentiated osteoclasts are unable to resorb bone at a normal rate: this is, however, unlikely, given the parallel increase in DISCUSSION 45Ca release in the culture medium, strongly correlated PGE2, added to the bone culture medium a t a concen- with osteoclast numbers in the cortex. tration of M, increased the number of osteoclasts The only alternative hypothesis to explain the mainboth in the medullary and the cortical bone. In the tenance of cortical bone would be to assume that PGEz nificant decrease in the bone volume of the bones cul3.4%,P < 0.01) tured with PGE2 (35.2 4.4 vs. 27.5 was observed together with a significant increase in the total number of osteoclasts (0.13 0.09 vs. 0.58 + 0.24, OCIfield, P < 0.01) which was correlated with the release of 45Ca (r = 0.60 P < 0.001). In summary, PGE2 added to the culture medium stimulated the differentiation of new osteoclasts both in the cortical and medullary bone. Despite the increase in cortical osteoclasts, a slight increase in the amount of cortical bone was recorded and active osteoblasts forming new bone matrix along the periosteal surface were prominent (Figs. 3, 4).On the other hand, the increase in medullary osteoclasts was associated with a decrease in medullary bone. An increase in 45Ca release in the culture medium was also recorded and correlated to the total and cortical number of osteoclasts but not with the medullary osteoclasts. 12 J.-R. NEFUSSI AND R. BARON 13 PGEz STIMULATES BONE FORMATION AND RESORPTION TABLE 1. Effects of PGE2 on 45Carelease and bone histomorphometry of fetal rat long bones in culture Day 0 45CaT/C ratio Day 1 1.04 (0.13) - Bone volume (%) Cortical bone C 49.6 (3.2) T Medullary bone C 15.3 (1.0) T Combined C 31.5 (6.3) T - Amount of bone (.urn3) Cortical bone C 206 (76) T Medullary bone C 105 (26) T Combined C 312(92) T - 50.5 (2.6) 49.8 (2.2) 16.2 (1.6) 12.1 (2.5) 33.0 (3.3) 27.8 (6.2) 241 (42) 205 (68) 174 (35)',* 114 (34) 415(9) 319 (97) No. of osteoclasts/field Cortical bone C 0 T Medullary bone C 0.6 (0.4) T Combined C 0.23 (0.16) T Periostal C 25.5 (10.4) osteoblastic T - Day 2 1.45 (0.23)' 50.6 (1.6) 49.8 (1.2) 14.6 (2.6) 9.2 (1.0)'."* 32.4 (5.4) 26.3 (2.6) 199 (17) 251 (68) 133 (45)'.* 110 (17)'.* 332 (53) 361 (84) 0.05 (0.05) 0.08 (0.1) 0.5 (0.1)'z2,** 0.2 (0.2) 0.3 (0.2) 0.3 (0.3) 1.1(0.2)'.*** 1.0 (0.4)'.* 0.17 (0.09) 0.16 (0.14) 0.46 (0.10)'-** 0.64 (0.09)'.2.*** 20.7 (7.4) 23.1 (8.7) 27.5 (7.2) 36.7 (12.5)'.* Day 3 1.91 (0.18)',2 51.7 (2.5) 51.2 (0.4) 16.8 (2.4) 7.8 (0.6)'**** 35.7 (2.9) 27.2 (0.5)"** 281 (86) 288 (65) 194 (27) 105 (16)'.*** 475 (72) 394 (72) 0.05 (0.05) 0.7 (0.4)'.** 0.2 (0.2) 0.7 (0.5) 0.12 (0.06) 0.74 (0.35)',* 22.2 (11.4) 35.1 (14.5)',* Day 4 1.79 (0.43)' 51.8 (3.3) 50.7 (2.7) 20.2 (4.4) 10.5 (0.6)'.2**" 38.2 (4.1) 28.9 (4.5)a* 273 (95) 304 (98) 226 (49) 131 (9)'.** 499 (88) 435 (95) Cumulative data 1.6 (0.4)' 51.2 (2.5) 50.5 (1.7) 17.7 (3.3) 9.8 (2.0)'3** 35.2 (4.4) 27.5 (3.4F"* 250 (73) 264 176) 185 (52) 114 (20)'%** 435 (91) 379 (86) 0.05 (0.07) 0.05 (0.07) 0.4 (0.2)',** 0.5 (0.3)',** 0.2 (0.3) 0.3 (0.3) 0.5 (0.2)',* 0.8 (0.4)'.* 0.13 (0.09) 0.09 (0.05) 0.44 (0.12)',*** 0.58 (0.24)'*4'* 21.2 (10.4) 19.1 (9.1)',* 34.3 (13.2)',"" 38.0 (19.5) surface (%) M)-treated bones, C , control bones. Numbers are means with standard deviations in parentheses. 'Significantly different from control. :Significantly different from previous day. P < 0.05. *I T,PGE, P < 0.01. < 0.001 "'P a t the concentration used in this study stimulates not only bone resorption but also bone formation along the periosteum. We have indeed observed a significantly higher periosteal osteoblastic surface in PGEz-treated bones. This quantitative result is strongly supported by the morphological observation of numerous active osteoblasts and new bone formation along the periosteum of the bones cultured in the presence of prostaglandins. This envelope-specific effect could be related to the concentration of PGE2 used in our study since Raisz and Koolemans-Beynen (1974) and Goldhaber et al. (1973) did not observe a stimulation of bone formation in vitro at lower concentrations. However, it is important to point out here that our results are actually not in contradiction with these previous reports since we observe an overall bone loss that could correspond to a n overall decrease in collagen synthesis when measurements are made on the whole explant. This does not exclude a local stimulatory effect a t the periosteum which these methods would not be able to detect. On the other hand, Blumenkrantz and Sondergard (1972) reported a n in vitro stimulation of bone collagen formation by PGEl and similar observations were made in vivo in various circumstances: Kafrawy and Mitchell (1977)observed a n increase in both bone resorption and bone formation after five injections of PGE2 per week for 2 weeks in alveolar bone; Goodson et al. (19741, who reported some extensive, although not osteoclastic, resorption areas after seven daily injections over the calvaria of rats, also mentioned intense bone formation along the periosFig. 3.Morphology of fetal rat long bones at the end of the culture teum, an observation that we have later confirmed usperiod. A,B) General views of the bones ( x 100). A) PGEz-treated; B) ing fluorescent labels (Baron et al., 1978). Finally, and control medium. The medullary bone (M) is markedly decreased in t,he cultures containing PGEz (A) as compared to control @3); ostewlasts more recently, long-term infusions of PGEl in children (straight arrows) are large and numerous in PGEz-treated bones but have led to increased periosteal bone formation as asare also frequent in control bones. Along the periosteum (P), intense sessed by X rays (Ueda et al., 1980; Ringel et al., 1982). proliferation of osteoblasts and formation of bone can be observed Therefore, it seems that PGE are able to stimulate bone (curved arrows) in PGEz-treated bones. C ) Higher magnification formation, both in vivo and in vitro, at given concentra( X 1,000)of large and numerous ostewlasts in the medullary bone of PGEz-treated bones. tions and maybe preferentially along the periosteum. 14 J.-R. NEFUSSI AND R. BARON Fig. 4. Higher magnifications ( ~ 2 5 0of) the periosteal osteoblastic reaction in PGEz-treated bones. A) Highly proliferative area along the periosteum of PGEz-treated bones with large numbers of osteoblasts (open arrows) and newly produced matrix (black arrows). B) Similar bone-forming area in a control bone: presence of matrix (black arrows) lined with osteoblasts but lack of intense cell proliferation. C) Osteoblastic proliferation (open arrow) along the periosteum (p) of PGE2treated bones with production of matrix (small black arrows); the endosteum (e) shows little formation and large ostewlasts (black arrow). PGEz STIMULATES BONE FORMATION AND RESORPTION “ ’ ~ a RELEASE AND OSTEOCLAST NUMBER -*.-. 3.O - Cortical Bone -- Medullary Bone - 45C0 Release 3M p<o.o01/c * p<0.02 / c ** p c o . 0 1 /c 2.5 0 \ N g -n W v) U W -I W E 2.0 1.5 1.0 0 0 n 0 0.5 0 Fig. 5. Comparison of 45Ca release and osteoclast numbers in the cortical and medullary areas of PGEz-treated bones in culture. Bars represent standard deviations. /C = significantly different from controls. However, there was a clearly different response a t the level of the endosteum: the total amount of bone present in the medullary space decreased markedly during the experiment and this was associated with a marked increase in the number of osteoclasts. Morphologically, no signs of bone formation could be detected at this level. A morphometric study performed on trabecular bone in VX2 carcinoma-bearing rabbits also showed decreased bone formation at this site (Wolfe et al., 1978). These results would therefore indicate a differential response of the periosteum and the endosteum to prostaglandins under our experimental conditions: both resorption and formation (bone turnover) would be increased in the cortex, leaving the balance between these two activities in equilibrium whereas only bone resorption would be increased along the endosteum, leading to marked medullary bone loss as well as an overall bone loss when considering cortical and medullary bone together. A similar, but reversed, envelopespecific action has also been suggested for parathyroid hormone and for thyroid hormones (Mosekilde and Melsen, 1978a,b). Since similar studies have not been performed with other bone-resorbing factors, it is not possible to make conclusions on the specificity of P G E 2 in inducing this differential response but our results nevertheless demonstrate that, under these experimental conditions, prostaglandins can stimulate both formation and resorption in organ cultures. 15 ACKNOWLEDGMENTS This work was supported by grant #DE 04724 from the NIH. The authors are grateful to Mrs. Lynn Neff for her expert technical help and to Mrs. Barbara Devlin for typing the manuscript. LITERATURE CITED Baron, R. (1977) Importance of the intermediate phases between resorption and formation in the measurement and understanding of the bone remodeling sequence. In: Bone Histomorphometry, P.J. Meunier, ed. Lab Armour Montagu, Paris, pp. 179-183. Baron, R., J.R. Nefussi, A. Duflot-Vignery, J.J. Lasfargues, F. Cohen, and E. Puzas (1978) Failure of prostaglandins E to induce local bone resorption in vivo (Abstract). 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