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Effects of Leukemia Inhibitory Factor and Oncostatin M on Bone Mineral Formed inin VitroRat Bone-Marrow Stromal Cell Culture: Physicochemical Aspects
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
253, 506 –513 (1998)
RC989781
Effects of Leukemia Inhibitory Factor and Oncostatin M on Bone Mineral Formed in in Vitro Rat Bone-Marrow Stromal Cell Culture: Physicochemical Aspects S. Bohic,* P. Pilet,* and D. Heymann*,†,1 *UPRES EA 2159, Faculte´ de Chirurgie Dentaire, 1 place A. Ricordeau, 44042 Nantes cedex 1, France; and †Laboratoire d’Histologie-Embryologie, Faculte´ de Me´decine, 1 rue Gaston Veil, 44093 Nantes, France
Received October 29, 1998
Leukemia inhibitory factor (LIF) and oncostatin M (OSM), two pleiotropic cytokines involved in bone remodeling, have both anabolic and catabolic activities. This study analyzed the effects of LIF and OSM on the physicochemical characteristics of mineral phases formed in a rat bone-marrow stromal cell culture model. Stromal cells were cultured for three weeks in the presence of 1028 M dexamethasone, 50 mg/mL ascorbic acid and 10 mM Na b-glycerophosphate with or without 10 ng/ml LIF or OSM. Subsequently, the physicochemical characteristics of the mineralization nodules formed were analyzed by energy dispersive X ray microanalysis (EDX) and Fourier transforminfrared (FT-IR) and FT-Raman spectroscopy. EDX and FT-IR spectroscopy revealed the influence of LIF and OSM on the physicochemical characteristics of mineral phases. FT-Raman spectroscopy showed modifications of the main vibrational modes of the organic matrix. These alterations induced by growth factors could help define new strategies for the prevention and treatment of skeletal disorders. © 1998 Academic Press Key Words: leukemia inhibitory factor; oncostatin M; Fourier transform-infrared microspectroscopy; Raman spectroscopy; mineralization.
Bone volume is maintained by the intense and continuous activity of many cell lineages (osteoblasts, osteoclasts, monocytes, lymphocytes) which communicate by various soluble (i.e. cytokines) or membrane mediators (i.e. adhesion molecules) (1, 2). These molecules initiate and promote the recruitment and proliferation of appropriate cells at the right site and time. The main substances implicated in these mechanisms belong to the cytokine and growth factor families which are (glyco)proteins produced most often after cellular activation (i.e. cytokines) and that act via their specific 1 To whom correspondence should be addressed. Fax: 1 33 (0) 2 40 08 37 12. E-mail: [email protected].
0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
receptors, whether soluble or located at the cell membrane (3). Moreover, the same cytokines which can influence physiological bone remodeling also play a key role in various bone pathologies [Paget’s disease (4), osteopetrosis (5), osteoporosis (6), etc.]. LIF and OSM belong to a large family of multifunctional cytokines which includes interleukin-6 (7), ciliary neurotrophic factor (8) and interleukin-11 (9). LIF and OSM show structural similarities, and both have induced similar biological activities on a variety of target cells, including hematopoietic cells, hepatocytes, neurons, embryonic stem cells and bone cells (10, 11). The redundancy of their activities has been attributed to the existence of common transducing receptor subunits (gp130 and gp190) (12, 13). LIF and OSM have been identified as resorption factors in a mouse bonemarrow-osteoblast co-culture (14) or, on the contrary, as inhibitors of resorption (15, 16). OSM induced proliferation and collagen synthesis and inhibited alkaline phosphatase activity in primary neonatal murine or fetal rat calvaria osteoblastic cultures (16), and LIF showed similar activities on osteoblastic cells (15, 17). However, it is not clear whether both factors have a direct effect on bone formation or whether LIF and OSM exert their influence through the mineral phases formed, as suggested by a recent study in an in vivo model (18). In vitro models provide useful tools for analyzing mineralization processes. Mineral deposition has been observed in fetal calvaria cell cultures (19 –22) or in bone cell cultures isolated from adult skeletal tissues (23–25). Similarly, it has been determined that bone-marrow tissue contains cells which can differentiate to the osteogenic direction (26 –28). An optimal and reproducible mineralization process has been achieved by inclusion of inorganic phosphates such as b-glycerophosphate, ascorbic acid and the glucocorticoid dexamethasone in the culture medium (27–31).
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Although these cytokines are clearly involved in the differentiation/proliferation mechanisms of osteogenic progenitors, their impact on the quality of the mineral phases generated in in vitro models has not been extensively investigated. Most of the data collected relate to cellular events, mainly to matrix formation during the mineralization process. Thus, little is known about the influence of cytokines on the mineral phases formed in an in vitro model. The purpose of the present work was to use a rat bone-marrow stromal cell culture model to study the influence of LIF and OSM on the physicochemical characteristics of mineral phases formed during an in vitro mineralization process. MATERIALS AND METHODS Materials. Human recombinant LIF (specific activity 4 3 107 units/mg) was purified from serum-free conditioned medium of CHO cells transfected with a full-length cDNA, as described in Godard et al. (32). Human recombinant OSM (specific activity 0.33– 0.66 3 107 units/mg) was purchased from R&D System (Oxon, UK). Alpha minimum essential medium (a-MEM) and fetal bovine serum were purchased from Gibco (Eragny, France) and DAP (Vogelgrun, France). Petri dishes were obtained from Falcon (AES Laboratories, Combourg, France). Antibiotics, dexamethasone, ascorbic acid and Na b-glycerophosphate were purchased from Sigma (Saint-Quentin Fallavier, France), and alizarin red-S from Merck (Nogent sur Marne, France). Cell isolation and culture. Bone-marrow cells were isolated from adult male Wistar rats (Centre d’Elevage Janvier, Le Genest SaintIsle, France) (approximately 150 g). Briefly, for each rat, both femora and tibias were dissected aseptically and cleaned of soft tissues. The epiphyses were removed and the bone-marrow cells flushed out with a syringe fitted with a 21-gauge needle containing a-MEM supplemented with an antibiotic mixture (100 U/mL penicillin; 100 mg/mL streptomycin). A single cell suspension was prepared by repeated pipetting. Unfractioned bone-marrow cells (107) were isolated in a 35-mm Petri dish with 4 mL of a-MEM supplemented with antibiotics, 15% fetal bovine serum, 1028 M dexamethasone and 50 mg/mL of freshly-prepared ascorbic acid. After one week of culture, 10 mM Na b-glycerophosphate was added to the medium together with recombinant LIF (10 ng/mL) and OSM (10 ng/mL). The cell cultures were then placed at 37°C in a 5% CO2 humidified atmosphere and maintained for three weeks, with total renewal of the culture medium every two days. The specific concentrations of the culture medium used were those reported by Maniatopoulos et al. (27), and the concentrations of LIF and OSM were based on results described in a recent study (18). Alizarin red-S staining. Alizarin red-S staining was used to detect the mineralization nodules formed in vitro (33). After three weeks of culture, adherent cells were washed with phosphatebuffered saline (PBS) followed by fixation in ice-cold 70% ethanol for 1 h. The ethanol was then removed, and the fixed cells were washed with distilled water and incubated with alizarin red-S (40 mM, pH 7.4) for 10 min at room temperature. The alizarin red-S solution was eliminated by washing with distilled water and the cells were observed under light microscopy. Scanning electron microscopy (SEM) and electron dispersive spectroscopic (EDS) microanalysis. After three weeks of culture, cell cultures were washed three times with PBS and fixed for 24 h at 4°C in ice-cold ethanol 70%. The samples were then dehydrated through a graded alcohol series and conserved in ethanol 100% until analysis. After fixation, dehydrated cell samples were carbon-coated (JEOL JEE 4B, Japan) for SEM observations at 15 kV (JEOL 6300, Japan)
by secondary electron imaging (SEI) and/or backscattered electrons (BSE). Semi-quantitative EDX microanalysis was done using the EXL Oxford Link System. Forty random fields were analyzed for each condition. Fourier transform-infrared and Raman spectroscopy. FT-Raman spectra were recorded in the 4000-200 cm21 range on a Bruker RFS 100 FT-Raman spectrometer equipped with an InGaAs detector cooled with liquid nitrogen. A few milligrams of culture sample powder were loaded into a cup, and spectra were obtained using the focused beam of an Nd-YAG laser operating at 1064 nm with 180° backscattering geometry. Spectral parameters were 4 cm21 resolution, 100 scans and 370 mW laser power. One milligram of dry powder of the 21-day cell culture containing bone nodules was mixed with 300 mg of infrared grade KBr (Sigma) in an agate mortar and pelletized under pressure. The same sample concentration in KBr was used to perform all spectra in order to obtain valid relative data. FT-IR spectra were recorded from the KBr pellets with a Magna 550 FTIR spectrometer (Nicolet, Trappes, France) continuously purged with dry air. One hundred and twenty-eight scans were collected, co-added and apodized with a Happ-Genzel function and Fourier transform to yield spectra with 4 cm21 resolution. Data analysis. The methodology used was modified essentially from that described by Paschalis et al. (34, 35). Individual peaks between 1800 and 800 cm21 were baseline-corrected prior to area calculations. Accordingly, ratios were calculated for the integrated area of n1, n3 PO4 (1200-910 cm21), characteristic of the mineral phases, to the amide I absorption band (1750-1590 cm21, relative to the organic component, and for the integrated area of the n2 CO3 absorption band (892-850 cm21) to the n1, n3 PO4 absorption band. As previously reported (34, 35), these ratios indicated the relative amounts of mineral and carbonate content in the samples analyzed. Fourier self-deconvolution (FSD) was also used and allow the separation of underlying peaks in the n1, n3 PO4, n2 CO3 and n4 PO4 (650-500 cm21) domains. The bandwidth parameters (s) and enhancement (K) were optimized, so that no sidelobes or negative-going bands occurred. Infrared indexes were calculated from these deconvoluted spectra in the n2 CO3 domain according to methods described by Rey et al. (36). In this domain, spectra were resolved into 3 components, with peaks at 879 cm21 and 872 cm21 assigned to the apatitic location of carbonate at OH2 (CO3 type-A) and PO32 (CO3 4 type-B) sites respectively. The shoulder at 866 cm-1 was assigned to non-apatitic carbonate ions (“labile” carbonate). Changes in these carbonate ion environments in the mineral phases formed in rat bone-marrow cell culture were monitored by evaluating the ratio of the integrated intensity of type-A to type-B carbonate and the ratio of labile carbonate to type-B carbonate. Deconvoluted spectra and second-derivative spectroscopy also provided information on the existence and the position of overlapping peaks. A curve-fitting technique was applied to resolve these components and calculate their relative percentage area by means of a commercially non-linear curve-fitting program (Peaksolve, Galactic Inc., Salem, USA). The number of Lorentzian-shaped and linear-baselined bands were used as inputs for the curve-fitting program. Positions were allowed to vary within 6 4 cm21 for the n1, n3 PO4 domain and 6 2 cm21 for the n2 CO3 and n4 PO4 domains. Finally, the program iterated the curvefitting process, varying the width, height and position of the curves until the best solution was reached. The quality of the fit was controlled by means of a x2-test. The same procedure for the study of each infrared domain was carried out on all spectra. Statistical analysis. All analyses were performed at least in triplicate. Data are reported as the mean 6 SD. Statistical comparisons performed with Statview software (Abacus Concept Inc., Berkeley, CA, USA) were based on analysis of variance (ANOVA) following multiple comparisons by the Fisher test. A probability (p) value of less than 0.05 was considered significant.
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FIG. 1. Morphological observations of mineralized-nodules formed in rat bone-marrow primary cell culture. Rat bone-marrow primary cells were cultured for 3 weeks in a-MEM supplemented with dexamethasone and ascorbic acid in the presence or absence of OSM (10 ng/ml). (A) Culture dish containing alizarin red-S-positive nodules. Arrow: typical stained area demonstrating the presence of calcium. (B) SEM observation with secondary electron imaging showing nodules formed in culture dishes. (C) SEM dual representation with secondary electron imaging (right) of the inner layer of the nodule and the corresponding backscattered electron image (left). Note the presence of globular calcified accretions intimately associated with the organic matrix. (D) Energy dispersive X-ray microanalysis spectrum of mineralization nodules showing the presence of prominent peaks of calcium (Ca) and phosphorus (P) and smaller peaks of magnesium (Mg), sodium (Na), chloride (Cl) and sulfur (S). Similar results were obtained in the presence of 10 ng/ml LIF.
RESULTS SEM and EDX Analysis After three weeks of culture of rat bone-marrow stromal cells, numerous cell clusters formed threedimensional nodular structures positve by alizarin red-S staining were observed (Fig. 1A). Intense staining was indicative of calcium mineral deposition in the nodular structure. SEM observations of the nodules clearly revealed their mineral nature (Fig. 1B, C). SEM analysis performed with a secondary electron imaging and the corresponding backscattered electron image showed that the inner layer of the nodule was composed of globular calcified accretions intimately associated with the organic matrix. EDX analysis of the mineral phases with or without cytokine revealed the presence of prominent calcium and phosphorus peaks
and to a lesser extent of magnesium, sodium, sulfur and chloride (Fig. 1D). The Ca/P, Mg/Ca and Na/Ca molar ratios of these mineralization nodules were determined for the different conditions tested (Fig. 2). The Ca/P ratio was 12% lower (p , 0.05) in the presence of 10 ng/ml OSM or 10 ng/ml LIF as compared to the control without cytokine. The Mg/Ca molar ratio increased by about 60% (p , 0.001) and 100% (p , 0.001) respectively in the presence of 10 ng/ml OSM and 10 ng/ml LIF as compared to the control. Similarly, 10 ng/ml OSM and LIF induced an increase in the Na/Ca molar ratio of about 100 % and 200 % respectively as compared to the control (p , 0.001). FT-Raman Spectroscopy FT-Raman spectra of the mineral phases formed in in vitro rat bone-marrow cell cultures showed the main
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FIG. 2. Elemental composition of mineral phases formed after 21 days of rat bone-marrow primary cell cultures. Rat bone-marrow primary cells were cultured for 3 weeks in a-MEM supplemented with dexamethasone and ascorbic acid in the presence or absence of LIF (10 ng/ml) or OSM (10 ng/ml). Semi-quantitative EDS microanalysis was performed using the EXL Oxford Link System. Results are expressed as the mean 6 SD of 40 random fields analyzed for each condition. Ca/P, Mg/Ca and Na/Ca are expressed as molar ratios deduced from elemental microanalysis. *: P , 0.05 compared to the control without cytokine.
vibrational band characteristic of the apatitic phosphate phase in a protein aqueous matrix (data not shown). The dominant 960 cm21 peak was characteristic of the presence of the phosphate group (symmetrical stretching mode, n1 mode). No other phases than apatite were detected since the position of this band was found to vary among different calcium phosphates (947 cm21 for amorphous calcium phosphate, 960 cm21 for carbonated hydroxyapatite, 970 cm21 for magnesium-substituted b-tricalcium phosphate, 980 cm21 for calcium hydrogen phosphate, 985 cm21 for dicalcium phosphate dihydrate). Moreover, the full width at half maximum (FWHM) of this band, which depends on the degree of carbonate substitution (37, 38), was used as a determinant of crystallinity. In all conditions, FWHM
was found to vary slightly but not significantly (around 19 cm21), which is close to the value of 20 cm21 found in mature rat bone mineral. This width was found to be 14 cm21 for mature human enamel or 35 cm21 for amorphous calcium phosphate (39). The main vibrational modes of the organic matrix corresponded to those of protein bands: for the control or mature rat bone n (CO) amide I band (1670 cm21), d (CH2) scissors modes (1463, 1488 cm21) and amide III d (NH) band (1270, 1244 cm21) were found. The shift in the position of the amide I (1663 cm21) and amide III (1250 cm21) bands with 10 ng/ml LIF or 10 ng/ml OSM may indicate a change in the conformational state of the proteins. Bands at 504 and 897 cm21 (currently unassigned) were found in all conditions but not in Raman spectra of mature rat bone mineral or mature enamel. In the presence of 10 ng/ml LIF and 10 ng/ml OSM, bands at around 525 and 1003 cm21 (assigned to the n4 HPO4 and n1 HPO4 respectively according Fowler et al. (40)) were enhanced by 30% or more (p , 0.05) as compared to the control. The 1003 cm21 has also been assigned to the n1 mode of organic phosphate associated with phosphoprotein occurring mainly in the region of low mineralization (41). Small but noticeable differences were also noted in the structure of the observed band, particularly in the n3 phosphate region (1030-1080 cm21). In control conditions, bands of comparable intensity at 1032 and 1046 cm21 and a band at 1072 cm21 of higher intensity were well-resolved. The n3 phosphate and n1 carbonate peaks both contributed to the band at 1072 cm21. In the presence of 10 ng/ml LIF or 10 ng/ml OSM, broad featureless peaks of comparable intensity were found around 1038 and 1080 cm21. Fourier Transform-Infrared Analysis The infrared parameters are reported in Table 1 for each condition tested in the n4 PO4 (650-500 cm21), n2 CO3 (892-850 cm21) and n1, n3 PO4 (1200-900 cm21) domains. Qualitatively, the relative amount of mineral deposited on the matrix, as determined from the ratio of the phosphate band to the amide I band, was increased by about 40% (p , 0.001) in the presence of 10 ng/ml LIF or 10 ng/ml OSM as compared to the control, whereas the carbonate-to-phosphate ratio, indicative of the relative amount of carbonate in the mineral, remained stable. Infrared parameters obtained from deconvoluted spectra in the n2 CO3 domain indicated that the type A-to-type B carbonate ratio was similar in all conditions tested, whereas the labile-to-type B ratio increased by about 25% (p , 0.05) with 10 ng/ml of LIF or OSM as compared to the control. Moreover, deconvoluted spectra showed more prominent shoulders of labile phosphate or carbonate groups as well as acid phosphate groups in the presence of 10 ng/ml of LIF or OSM (Fig. 3).
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*: Calculated from deconvoluted spectra obtained by Fourier self-deconvolution. : percentage of underlying peaks in the n4 PO4, n2 CO3 and n1, n3 PO4 domains obtained from curve-fitting analysis. Peaks reported are those found to have a significant variation (p , 0.05) in LIF or OSM conditions as compared to the control. †
2.7 6 1 4.8 6 0.7 5.1 6 1.5 27.6 6 1.5 24.7 6 1.4 20 6 0.5 27.1 6 3 34.9 6 0.7 35.3 6 3 7.4 6 2 3.7 6 0.7 2.5 6 0.4 0.0079 6 0.0007 0.0084 6 0.0018 0.008 6 0.0009 Control LIF (10 ng/ml) OSM (10 ng/ml)
2.8 6 0.05 3.9 6 0.07 3.8 6 0.03
0.92 6 0.003 0.91 6 0.018 0.92 6 0.08
0.45 6 0.05 0.56 6 0.06 0.55 6 0.04
10.8 6 2.4 14.5 6 1.5 14.3 6 1.4
17.6 6 0.9 21 6 1.7 22.5 6 1.5
879 cm21 866 cm21 520 cm21 540 cm21 623 cm21 † CO3* type A/ CO3* labile/ type B type B CO3/PO4 PO4/Amide I
Infrared Characteristics of Carbonate and Phosphate Environments of Mineral Phases Deposited in Rat Bone Marrow Primary Cell-Culture
TABLE 1
2.2 6 0.7 5.2 6 0.6 6.6 6 2.2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1150 cm21 1018/1035 cm21
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On a semi-quantitative basis, variations of phosphate and carbonate environments in mineralized nodules formed in the presence or absence of cytokines were investigated by curve-fitting (Fig. 3). Six bands were used in the n4 PO4 domain. Those at 600, 576 and 559 cm21 corresponded to the apatitic environment of phosphate, and those at 623 cm21, 520 cm21 and 545 cm21 were assigned respectively to labile non-apatitic phosphate, labile non-apatitic HPO22 groups and the 4 apatitic environment of HPO4. The relative percent area of labile phosphate and labile HPO4 increased by about 30% (p , 0.05) in the presence of 10 ng/ml LIF or 10 ng/ml OSM as compared to the control, whereas the relative apatitic HPO4 component decreased by about 60% (p , 0.01). Among the three carbonate environments detected in n2 CO3, labile carbonate from the mineral formed after 10 ng/ml LIF or OSM treatment increased by about 30% (p , 0.05) as compared to the control value. This is in agreement with results for the labile-to-type B carbonate ratio for deconvoluted spectra. In n1, n3 PO4, nine underlying bands were used to fit the contour. The most significant data were an increase in the HPO4 band at 1150 cm21, which corroborated the observation for deconvoluted spectra, and a large increase (about 3-fold) in the 1018-to-1035 cm21 ratio when LIF or OSM was added to cultures. DISCUSSION The main purpose of this study was to analyze the effects of LIF and OSM on mineral phases formed in in vitro primary rat bone-marrow stromal cell cultures. Raman data on these mineral phases, reported here for the first time, showed spectral modifications of the amide I and III bands of the organic matrix synthesized in the presence of LIF and OSM. Both infrared and Raman spectroscopy assessed the apatitic nature of the mineral deposited after 21 days of culture, and its immature state was revealed by the presence of environment rich in labile non-apatitic phosphate, carbonate and HPO4. These features, related to the surface reactivity of the bone mineral and probably to the homeostasis of calcium-phosphate metabolism, were present to a greater extent in cell cultures treated with LIF or OSM. These results indicate that LIF and OSM influenced the physicochemical characteristics of mineral phases formed in this in vitro mineralization model. Numerous studies have already demonstrated the role of soluble molecules during the mineralization process. The effect of dexamethasone on the stimulation of osteogenesis by osteoprogenitors has been reported by Tenenbaum and Heersche (42). This glucocorticoid increased collagen synthesis and alkaline phosphatase activity. Ascorbic acid is another important protagonist in the mineralization required for the collagen synthe510
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FIG. 3. Deconvoluted infrared spectra and curve-fitting analysis of phosphate and carbonate infrared absorption bands from mineral phases formed in rat bone-marrow primary cell cultures. The bottom set of spectra shows typical Fourier self-deconvoluted spectra in the n4 PO4, n2 CO3, n1, n3 PO4 domains consecutively. Bandwidth (s) and enhancement (K) were respectively s 5 18 cm21, K 5 2.2; s 5 9 cm21, K 5 2; and s 5 30 cm21, K 5 2.2. This allowed us to resolve the intrinsically overlapping components, with their position being supported in second-derivative spectra. (1) Deconvoluted spectra of mineral phases formed under control conditions; (2) 10 ng/ml LIF; (3) 10 ng/ml OSM. Among the apatitic sites of phosphate or carbonate ions, several non-apatitic environments were labeled, such as labile phosphate (620 cm21 and 1125 cm21), labile carbonate (866 cm21) and acid phosphate (520 cm21). HPO4 was also present at 1150 cm21 in the n1, n3 PO4 domain. Thus, the curve-fitting technique was performed on a semi-quantitative basis, and the relative percent areas (upper set of spectra) of the individual components were calculated in each spectrum. Spectral decomposition of the corresponding original spectrum was displayed in the three infrared domains investigated.
sis (43), and b-glycerophosphate has often been used in vitro as a source of inorganic phosphate ions (44), although the mineralization process has also been described in its absence (45). A considerable number of growth factors participate in mineral formation, such as fibroblast growth factor, insulin-like growth factor, transforming growth factor, bone morphogenetic proteins (46 – 48), etc. However, the influence of these factors on the physicochemical characteristics of the mineral formed in vitro had not been studied previously. LIF and OSM are among the factors which exert an intense activity on osteoblastic cells. However, the activities of such factors are largely influenced by the differentiation state of the osteoblasts, as noted by Malaval et al. (49) for LIF. A recent study demonstrated the influence of both cytokines on the mineral phases formed after bone-marrow implantation under a mouse kidney capsule (18). The present results confirm the probable involvement of cytokines in the mineralization process. Raman spectroscopy is complementary to infrared spectroscopy since inactive infrared modes are often
active in Raman. Moreover, as water is a weak Raman scatter, adsorbed water has minimal effects on the spectrum. The high background fluorescence of the organic matrix in biological samples alters the quality of the spectrum. However, when the near-infrared range was used in our study instead of the visible range, fluorescence was virtually eliminated since electronic absorption bands are much less likely to occur in this region. The small shifts in the positions of the amide I and III bands of the organic matrix in Raman spectra observed after LIF or OSM treatment may be indicative of changes in the conformational state of the proteins of the organic matrix as compared to that of the control. The shape and position of the bands in vitro relative to the organic matrix were also different from those of rat bone mineral. LIF and OSM probably influenced the synthesis of the matrix, which may have some effect on the formation and nature of the mineral deposited at later stage. formed For mineral phases, it has been shown that the full width at half maximum (FWHM) of the Raman band located at 961 cm21 (characteristic of apatite from mineralized tissues) depends
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on the degree of carbonate substitution (37, 38). Although this feature has also been used as a measurement of changes in crystallinity, there was no apparent changes in our study. The value (19 cm21) found was close to that of mature rat bone mineral and between that of well-crystallized human enamel (14 cm21) and amorphous calcium phosphate (35 cm21), as reported by Leung et al. (39). The changes observed for bands at around 525 and 1003 cm21 could be explain by an increase in HPO4 mineral content formed by bone cells in presence of cytokines. In addition, the band at 1003 cm21 has also been assigned to the n1 mode of organic phosphate associated with phosphoprotein occurring mainly in the region of low mineralization (41). Raman bands at 505 and 897 cm21, as well as a shoulder at around 400 cm21 and an infrared band at 780 cm21 (H2O rocking mode), have been found in newberyite (MgHPO4 z 3 H2O) (50, 51). However the very strong Raman peaks of this phase at 987 cm21 were not found. Infrared spectroscopy in conjunction with curvefitting can improve our understanding of variations in different phosphate and carbonate mineral environments, particularly the labile non-apatitic environment at the surface of the crystals which is very sensitive to bone mineral alteration and interacts with specific proteins associated with crystals (52). Although the lack of a standard similar to that for bone apatite has made it difficult to evaluate carbonate and the HPO4 fraction quantitatively, this technique has proved useful (34, 35, 53) and provides further support for the notion that LIF and OSM influence mineral environments formed in vitro as well as in vivo (18). It is also possible that this difference is due to a completely distinct mechanism of organization of mineralized deposits in cell cultures in the presence of cytokines as compared to the development of mineral in control conditions (i.e. variations of the nature and of the level of extracellular matrix proteins). In all conditions, mineral phases formed with the present model showed an apatitic nature, which appeared to be very immature, with a low carbonate content and the presence of acid phosphate groups. These observations are in agreement with data previously reported by Rey et al. for cell cultures of chicken osteoblasts (54). It is interesting to note that relative mineral amount of mineral deposited was higher in presence of LIF or OSM compared to the control, and that that mineral phases are more immature. The uptake of Mg or Na which appear to be increase in presence of LIF and OSM, would also play a role in these differences of crystal maturity. These observations are in agreement with data reported in vivo. Mg and CO3 are known to hinder the formation of crystalline apatite while they stabilize the amorphous phases. Furthermore the increase of the 1018 –1032 cm21 ratio after LIF and OSM treatment may indicate changes in cristallinity/ maturity of the crystals deposited in vitro. This last
parameter has been shown to provide a measure of mineral crystallinity and maturity (35). Data reported clearly showed changes of the mineral environment (mainly the labile component). These environment have been found closely related to bone mineral reactivity and consequently have been shown to be sensitive indicator of the maturity of biological apatite crystals or some skeletal alterations (52). However, the relations between variations of these labile non apatitic environments and the extracellular matrix are not well understood. The slow rate of maturation of the mineral formed in vitro contrast with in vivo conditions even for a very long-term culture, according to the observations of Rey et al. (54). The chemical environment (composition of the cell culture medium compared to the biological fluids) may influence the way by which cytokines act on cellular behavior and on bone mineral phases. Our results indicate that this new approach (particularly infrared spectroscopy and microspectroscopy) could lead to a better knowledge of the pathophysiology of numerous bone diseases involving a potentially hormonal and/or growth factor pathway. However, other growth factors have been implicated in bone remodeling, and their role in the formation of the mineral phase as well as of the organic matrix also needs to be elucidated. REFERENCES 1. Heymann, D., Guicheux, J., Gouin, F., Passuti, N., Daculsi, G., and Heymann, D. (1998) Cytokine 10, 155–158. 2. Rodan, G. A., and Rodan, S. B. (1995) in Osteoporosis: Etiology, Diagnosis, and Management (Riggs, B. L., and Melton L. J., III, Eds.), 2nd ed., pp. 1–39. Mayo Foundation, Lippincott–Raven, Philadelphia. 3. Mu¨ller-Newen, G., Ko¨hne, C., and Heinrich, P. C. (1996) Int. Arch. Allergy Immunol. 111, 99 –109. 4. Roodman, G. D. (1995) Bone 17, 57S– 61S. 5. Kodama, H., Yamasaki, A., Nose, M., Niida, S., Ohgame, Y., Abe, M., Kumegawa, M., and Suda, T. (1991) J. Exp. Med. 173, 269 – 272. 6. Pacifi, R., and Avioli, L. V. (1993) Osteoporosis Int. 1, 106 –107. 7. Rose, T. M., and Bruce, A. G. (1991) Proc. Natl. Acad. Sci. USA 88, 8641– 8645. 8. Lin, L. F. H., Mismer, D., Lile, J. D., Armes, L. G., Butler, E. T., Vannice, J. L., and Collins, F. (1989) Science 246, 1023–1025. 9. Hilton, D. J., Hilton, A. A., Raicevic, A., Harrison-Smith, L., Gough, N. M., Begley, C. G., Metcalf, D., Nicola, N. A., and Willson, T. A. (1994) EMBO J. 13, 4765– 4775. 10. Piquet-Pellorce, C, Grey, L., Mereau, A., and Heath, J. K. (1994) Exp. Cell Res. 213, 340 –347. 11. Thoma, B., Bird, T. A., Friend, D. J., Gearing, D. P., and Dower, S. K. (1994) J. Biol. Chem. 269, 6215– 6222. 12. Gearing, D. P., Comeau, M. R., Friend, D. J., Gimpel, S. D., Thut, C. J., McGourty, J., Brasher, K. K., King, J. A., Mosley, B., Ziegler, S. F., and Cosman, D. (1992) Science 255, 1434 –1437. 13. Heymann, D., Godard, A., Raher, S., Bentouimou, N., Blanchard, F., Che´rel, M., Hallet, M. M., and Jacques, Y. (1996) Cytokine 8, 197–205.
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14. Tamura, T., Udagawa, N., Takahashi, N., Miyauri, C., Tanka, S., Yamada, Y., Koishihara, Y., Ohsugi, Y., Kumaki, K., Taga, T., Kishimoto, T,, and Suda, T. (1993) Proc. Natl. Acad. Sci. USA 90, 11924 –11928. 15. Van Beek, E., Van Der Wee-Pals, L., Van de Ruit, M., Nijweide, P., Papopoulos, S., and Lo¨wik, C. (1993) J. Bone Miner. Res. 8, 191–199. 16. Jay, P. R., Schindler, C., Raz, R., Levy, D. E., Baron, R., and Horowitz, M. C. (1996) Endocrinology 137, 1159 –1165. 17. Reid, I. R., Lowe, C., Cornish, J., Skinner, S. J. M., Hilton, D. J., Willson, T. A., Gearing, D. P., and Martin, T. J. (1990) Endocrinology 126, 1416 –1420. 18. Bohic, S., Rohanizadeh, R., Touchais, S., Godard, A., Daculsi, G., and Heymann, D. (1998) J. Bone Miner. Res., in press. 19. Binderman, I., Duksin, D., Harrell, A., Ketzir, E., and Sahs, L. (1974) J. Cell Biol. 61, 427– 439. 20. Burger, E. H., Boonekamp, P. M., and Nijweide, P. J. (1986) Anat. Rec. 214, 32– 40. 21. Escarot-Charrier, B., Glorieux, F. H., van der Rest, M., and Pereira, G. (1983) J. Cell Biol. 96, 639 – 643. 22. Bellow, C. G., Aubin, J. E., Heersche, J. N. M., and Antosz, M. E. (1986) Calcif. Tissue Int. 38, 143–154. 23. Anderson, R. E., Kemp, J. W., Jee, W. S. S., and Woodbury, D. M. (1984) In Vitro 20, 837– 846. 24. Gehron Robey, P., and Termine, J. D. (1985) Calcif. Tissue Int. 37, 453– 460. 25. Koshihara, Y., Kawamura, M., Oda, H., and Higaki, S. (1987) Biochem. Biophys. Res. Commun. 145, 651– 657. 26. Ashton, B. A., Allen, T. D., Howlett, C. R., Eaglesom, C. C., Hattori, A., and Owen, M. E. (1980) Clin. Orthop. 151, 294 –307. 27. Maniatopoulos, C., Sodek, J., and Melcher, A. H. (1988) Cell Tissue Res. 254, 317–330. 28. Satomura, K., and Nagayama, M. (1991) Anat. Rec. 142, 97–104. 29. Kasugai, S., Todescan, R., Nagata, T., Yao, K., Butler, W. T., and Sodek, J. (1991) J. Cell Physiol. 147, 111–120. 30. Malaval, L., Modrowski, D., Gupta, A. K., and Aubin, J. E. (1994) J. Cell Physiol. 138, 555–572. 31. Tenenbaum, H. C., and Heersche, J. N. M. (1982) Calcif. Tissue Int. 34, 76 –79. 32. Godard, A., Heymann, D., Raher, S., Anegon, I., Peyrat, M. A., Le Mauff, B., Mouray, E., Gre´goire, M., Virdee, K., Soulillou, J. P., Moreau, J. F., and Jacques, Y. (1992) J. Biol. Chem. 267, 3214 –3222.
33. Stanford, C. M., Jacobson, P. A., Eanes, E. D., Lambke, L. A., and Midura, R. J. (1995) J. Biol. Chem. 270, 9420 –9428. 34. Paschalis, E. P., Jacenko, O., Olsen, B., Mendelsohn, R., and Boskey, A. L. (1996) Bone 19, 151–156. 35. Paschalis, E. P., Betts, F., DiCarlo, E., Mendelsohn, R., and Boskey, A. L. (1997) Calcif. Tissue Int. 61, 480 – 487. 36. Rey, C., Shimizu, M., Collins, B., and Glimcher, M. J. (1991) Calcif. Tissue Int. 49, 383–388. 37. Nelson, D. G., and Bryce, E. W. (1982) Aust. J. Chem. 35, 715–727. 38. Clarke, R. H., Hanlon, E. B., Brody, H., and Isner, J. M. (1988) J. Raman Spectrosc. 19, 183–188. 39. Leung, Y., Walters, M. A., Blumenthal, N. C., Ricci, J. L., and Spivak, J. M. (1995) J. Biomed. Mat. Res. 29, 591–594. 40. Fowler, B. O., Markovic, M., and Brown, W. E. (1993) Chem. Mater. 5, 1417–1423. 41. Sauer, G. R., Zunic, W. B., Durig, J. R., and Wuthier, R. E. (1994) Calcif. Tissue Int. 54, 414 – 420. 42. Tenenbaum, H. C., and Heersche, J. N. M. (1985) Endocrinology 177, 2211–2217. 43. Bellows, C. G., Aubin, J. E., Heersche, J. N. M., and Antosz, N. M. (1986) Calicif. Tissue Int. 38, 143–154. 44. Chung, C. H., Golub, E. E., Forbes, E., Tokuoka, T. and Shapiro, I. M. (1992) Calcif. Tissue Int. 51, 305–311. 45. Satomura, K., Hiraiwa, K., and Nagayama, M. (1991) Bone Miner. 14, 41–54. 46. Canalis E., McCarthy, T., and Centrella, M. (1988) J. Clin. Invest. 81, 277–281. 47. Whitson, S. W., Whitson, M. A., Bowers, D. E., and Falk, M. C. (1992) J. Bone Miner. Res. 7, 727–741. 48. Urist M. R. (1997) J. Bone Miner. Res. 12, 343–346. 49. Malaval, L., Gupta, A. K., and Aubin, J. E. (1995) Endocrinology 136, 1411–1418. 50. Mitchell, P. C. H., Parker, S. F., Simkiss, K., Simmons, J., and Taylor, M. G. (1996) J. Inorg. Biochem. 62, 183–197. 51. Sudlow, K., and Woolf, A. (1991) Clin. Chim. Acta 203, 387–394. 52. Rey, C., Strawich, E., and Glimcher, M. J. (1994) Bull. l’Inst. Oce´anogr Monaco, n° spe´cial 14(1), 55– 65. 53. Boskey, A. L., Guidon, P., Doty, S. B., Stiner, D., Leboy, P., and Binderman, I. (1996) J. Bone Miner. Res. 11, 1694 –1702. 54. Rey, C., Kim, Gerstenfeld, L., and Glimcher, M. J. (1995) J. Bone Miner. Res. 10, 1577–1588.
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RC989781
Effects of Leukemia Inhibitory Factor and Oncostatin M on Bone Mineral Formed in in Vitro Rat Bone-Marrow Stromal Cell Culture: Physicochemical Aspects S. Bohic,* P. Pilet,* and D. Heymann*,†,1 *UPRES EA 2159, Faculte´ de Chirurgie Dentaire, 1 place A. Ricordeau, 44042 Nantes cedex 1, France; and †Laboratoire d’Histologie-Embryologie, Faculte´ de Me´decine, 1 rue Gaston Veil, 44093 Nantes, France
Received October 29, 1998
Leukemia inhibitory factor (LIF) and oncostatin M (OSM), two pleiotropic cytokines involved in bone remodeling, have both anabolic and catabolic activities. This study analyzed the effects of LIF and OSM on the physicochemical characteristics of mineral phases formed in a rat bone-marrow stromal cell culture model. Stromal cells were cultured for three weeks in the presence of 1028 M dexamethasone, 50 mg/mL ascorbic acid and 10 mM Na b-glycerophosphate with or without 10 ng/ml LIF or OSM. Subsequently, the physicochemical characteristics of the mineralization nodules formed were analyzed by energy dispersive X ray microanalysis (EDX) and Fourier transforminfrared (FT-IR) and FT-Raman spectroscopy. EDX and FT-IR spectroscopy revealed the influence of LIF and OSM on the physicochemical characteristics of mineral phases. FT-Raman spectroscopy showed modifications of the main vibrational modes of the organic matrix. These alterations induced by growth factors could help define new strategies for the prevention and treatment of skeletal disorders. © 1998 Academic Press Key Words: leukemia inhibitory factor; oncostatin M; Fourier transform-infrared microspectroscopy; Raman spectroscopy; mineralization.
Bone volume is maintained by the intense and continuous activity of many cell lineages (osteoblasts, osteoclasts, monocytes, lymphocytes) which communicate by various soluble (i.e. cytokines) or membrane mediators (i.e. adhesion molecules) (1, 2). These molecules initiate and promote the recruitment and proliferation of appropriate cells at the right site and time. The main substances implicated in these mechanisms belong to the cytokine and growth factor families which are (glyco)proteins produced most often after cellular activation (i.e. cytokines) and that act via their specific 1 To whom correspondence should be addressed. Fax: 1 33 (0) 2 40 08 37 12. E-mail: [email protected].
0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
receptors, whether soluble or located at the cell membrane (3). Moreover, the same cytokines which can influence physiological bone remodeling also play a key role in various bone pathologies [Paget’s disease (4), osteopetrosis (5), osteoporosis (6), etc.]. LIF and OSM belong to a large family of multifunctional cytokines which includes interleukin-6 (7), ciliary neurotrophic factor (8) and interleukin-11 (9). LIF and OSM show structural similarities, and both have induced similar biological activities on a variety of target cells, including hematopoietic cells, hepatocytes, neurons, embryonic stem cells and bone cells (10, 11). The redundancy of their activities has been attributed to the existence of common transducing receptor subunits (gp130 and gp190) (12, 13). LIF and OSM have been identified as resorption factors in a mouse bonemarrow-osteoblast co-culture (14) or, on the contrary, as inhibitors of resorption (15, 16). OSM induced proliferation and collagen synthesis and inhibited alkaline phosphatase activity in primary neonatal murine or fetal rat calvaria osteoblastic cultures (16), and LIF showed similar activities on osteoblastic cells (15, 17). However, it is not clear whether both factors have a direct effect on bone formation or whether LIF and OSM exert their influence through the mineral phases formed, as suggested by a recent study in an in vivo model (18). In vitro models provide useful tools for analyzing mineralization processes. Mineral deposition has been observed in fetal calvaria cell cultures (19 –22) or in bone cell cultures isolated from adult skeletal tissues (23–25). Similarly, it has been determined that bone-marrow tissue contains cells which can differentiate to the osteogenic direction (26 –28). An optimal and reproducible mineralization process has been achieved by inclusion of inorganic phosphates such as b-glycerophosphate, ascorbic acid and the glucocorticoid dexamethasone in the culture medium (27–31).
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Although these cytokines are clearly involved in the differentiation/proliferation mechanisms of osteogenic progenitors, their impact on the quality of the mineral phases generated in in vitro models has not been extensively investigated. Most of the data collected relate to cellular events, mainly to matrix formation during the mineralization process. Thus, little is known about the influence of cytokines on the mineral phases formed in an in vitro model. The purpose of the present work was to use a rat bone-marrow stromal cell culture model to study the influence of LIF and OSM on the physicochemical characteristics of mineral phases formed during an in vitro mineralization process. MATERIALS AND METHODS Materials. Human recombinant LIF (specific activity 4 3 107 units/mg) was purified from serum-free conditioned medium of CHO cells transfected with a full-length cDNA, as described in Godard et al. (32). Human recombinant OSM (specific activity 0.33– 0.66 3 107 units/mg) was purchased from R&D System (Oxon, UK). Alpha minimum essential medium (a-MEM) and fetal bovine serum were purchased from Gibco (Eragny, France) and DAP (Vogelgrun, France). Petri dishes were obtained from Falcon (AES Laboratories, Combourg, France). Antibiotics, dexamethasone, ascorbic acid and Na b-glycerophosphate were purchased from Sigma (Saint-Quentin Fallavier, France), and alizarin red-S from Merck (Nogent sur Marne, France). Cell isolation and culture. Bone-marrow cells were isolated from adult male Wistar rats (Centre d’Elevage Janvier, Le Genest SaintIsle, France) (approximately 150 g). Briefly, for each rat, both femora and tibias were dissected aseptically and cleaned of soft tissues. The epiphyses were removed and the bone-marrow cells flushed out with a syringe fitted with a 21-gauge needle containing a-MEM supplemented with an antibiotic mixture (100 U/mL penicillin; 100 mg/mL streptomycin). A single cell suspension was prepared by repeated pipetting. Unfractioned bone-marrow cells (107) were isolated in a 35-mm Petri dish with 4 mL of a-MEM supplemented with antibiotics, 15% fetal bovine serum, 1028 M dexamethasone and 50 mg/mL of freshly-prepared ascorbic acid. After one week of culture, 10 mM Na b-glycerophosphate was added to the medium together with recombinant LIF (10 ng/mL) and OSM (10 ng/mL). The cell cultures were then placed at 37°C in a 5% CO2 humidified atmosphere and maintained for three weeks, with total renewal of the culture medium every two days. The specific concentrations of the culture medium used were those reported by Maniatopoulos et al. (27), and the concentrations of LIF and OSM were based on results described in a recent study (18). Alizarin red-S staining. Alizarin red-S staining was used to detect the mineralization nodules formed in vitro (33). After three weeks of culture, adherent cells were washed with phosphatebuffered saline (PBS) followed by fixation in ice-cold 70% ethanol for 1 h. The ethanol was then removed, and the fixed cells were washed with distilled water and incubated with alizarin red-S (40 mM, pH 7.4) for 10 min at room temperature. The alizarin red-S solution was eliminated by washing with distilled water and the cells were observed under light microscopy. Scanning electron microscopy (SEM) and electron dispersive spectroscopic (EDS) microanalysis. After three weeks of culture, cell cultures were washed three times with PBS and fixed for 24 h at 4°C in ice-cold ethanol 70%. The samples were then dehydrated through a graded alcohol series and conserved in ethanol 100% until analysis. After fixation, dehydrated cell samples were carbon-coated (JEOL JEE 4B, Japan) for SEM observations at 15 kV (JEOL 6300, Japan)
by secondary electron imaging (SEI) and/or backscattered electrons (BSE). Semi-quantitative EDX microanalysis was done using the EXL Oxford Link System. Forty random fields were analyzed for each condition. Fourier transform-infrared and Raman spectroscopy. FT-Raman spectra were recorded in the 4000-200 cm21 range on a Bruker RFS 100 FT-Raman spectrometer equipped with an InGaAs detector cooled with liquid nitrogen. A few milligrams of culture sample powder were loaded into a cup, and spectra were obtained using the focused beam of an Nd-YAG laser operating at 1064 nm with 180° backscattering geometry. Spectral parameters were 4 cm21 resolution, 100 scans and 370 mW laser power. One milligram of dry powder of the 21-day cell culture containing bone nodules was mixed with 300 mg of infrared grade KBr (Sigma) in an agate mortar and pelletized under pressure. The same sample concentration in KBr was used to perform all spectra in order to obtain valid relative data. FT-IR spectra were recorded from the KBr pellets with a Magna 550 FTIR spectrometer (Nicolet, Trappes, France) continuously purged with dry air. One hundred and twenty-eight scans were collected, co-added and apodized with a Happ-Genzel function and Fourier transform to yield spectra with 4 cm21 resolution. Data analysis. The methodology used was modified essentially from that described by Paschalis et al. (34, 35). Individual peaks between 1800 and 800 cm21 were baseline-corrected prior to area calculations. Accordingly, ratios were calculated for the integrated area of n1, n3 PO4 (1200-910 cm21), characteristic of the mineral phases, to the amide I absorption band (1750-1590 cm21, relative to the organic component, and for the integrated area of the n2 CO3 absorption band (892-850 cm21) to the n1, n3 PO4 absorption band. As previously reported (34, 35), these ratios indicated the relative amounts of mineral and carbonate content in the samples analyzed. Fourier self-deconvolution (FSD) was also used and allow the separation of underlying peaks in the n1, n3 PO4, n2 CO3 and n4 PO4 (650-500 cm21) domains. The bandwidth parameters (s) and enhancement (K) were optimized, so that no sidelobes or negative-going bands occurred. Infrared indexes were calculated from these deconvoluted spectra in the n2 CO3 domain according to methods described by Rey et al. (36). In this domain, spectra were resolved into 3 components, with peaks at 879 cm21 and 872 cm21 assigned to the apatitic location of carbonate at OH2 (CO3 type-A) and PO32 (CO3 4 type-B) sites respectively. The shoulder at 866 cm-1 was assigned to non-apatitic carbonate ions (“labile” carbonate). Changes in these carbonate ion environments in the mineral phases formed in rat bone-marrow cell culture were monitored by evaluating the ratio of the integrated intensity of type-A to type-B carbonate and the ratio of labile carbonate to type-B carbonate. Deconvoluted spectra and second-derivative spectroscopy also provided information on the existence and the position of overlapping peaks. A curve-fitting technique was applied to resolve these components and calculate their relative percentage area by means of a commercially non-linear curve-fitting program (Peaksolve, Galactic Inc., Salem, USA). The number of Lorentzian-shaped and linear-baselined bands were used as inputs for the curve-fitting program. Positions were allowed to vary within 6 4 cm21 for the n1, n3 PO4 domain and 6 2 cm21 for the n2 CO3 and n4 PO4 domains. Finally, the program iterated the curvefitting process, varying the width, height and position of the curves until the best solution was reached. The quality of the fit was controlled by means of a x2-test. The same procedure for the study of each infrared domain was carried out on all spectra. Statistical analysis. All analyses were performed at least in triplicate. Data are reported as the mean 6 SD. Statistical comparisons performed with Statview software (Abacus Concept Inc., Berkeley, CA, USA) were based on analysis of variance (ANOVA) following multiple comparisons by the Fisher test. A probability (p) value of less than 0.05 was considered significant.
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FIG. 1. Morphological observations of mineralized-nodules formed in rat bone-marrow primary cell culture. Rat bone-marrow primary cells were cultured for 3 weeks in a-MEM supplemented with dexamethasone and ascorbic acid in the presence or absence of OSM (10 ng/ml). (A) Culture dish containing alizarin red-S-positive nodules. Arrow: typical stained area demonstrating the presence of calcium. (B) SEM observation with secondary electron imaging showing nodules formed in culture dishes. (C) SEM dual representation with secondary electron imaging (right) of the inner layer of the nodule and the corresponding backscattered electron image (left). Note the presence of globular calcified accretions intimately associated with the organic matrix. (D) Energy dispersive X-ray microanalysis spectrum of mineralization nodules showing the presence of prominent peaks of calcium (Ca) and phosphorus (P) and smaller peaks of magnesium (Mg), sodium (Na), chloride (Cl) and sulfur (S). Similar results were obtained in the presence of 10 ng/ml LIF.
RESULTS SEM and EDX Analysis After three weeks of culture of rat bone-marrow stromal cells, numerous cell clusters formed threedimensional nodular structures positve by alizarin red-S staining were observed (Fig. 1A). Intense staining was indicative of calcium mineral deposition in the nodular structure. SEM observations of the nodules clearly revealed their mineral nature (Fig. 1B, C). SEM analysis performed with a secondary electron imaging and the corresponding backscattered electron image showed that the inner layer of the nodule was composed of globular calcified accretions intimately associated with the organic matrix. EDX analysis of the mineral phases with or without cytokine revealed the presence of prominent calcium and phosphorus peaks
and to a lesser extent of magnesium, sodium, sulfur and chloride (Fig. 1D). The Ca/P, Mg/Ca and Na/Ca molar ratios of these mineralization nodules were determined for the different conditions tested (Fig. 2). The Ca/P ratio was 12% lower (p , 0.05) in the presence of 10 ng/ml OSM or 10 ng/ml LIF as compared to the control without cytokine. The Mg/Ca molar ratio increased by about 60% (p , 0.001) and 100% (p , 0.001) respectively in the presence of 10 ng/ml OSM and 10 ng/ml LIF as compared to the control. Similarly, 10 ng/ml OSM and LIF induced an increase in the Na/Ca molar ratio of about 100 % and 200 % respectively as compared to the control (p , 0.001). FT-Raman Spectroscopy FT-Raman spectra of the mineral phases formed in in vitro rat bone-marrow cell cultures showed the main
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FIG. 2. Elemental composition of mineral phases formed after 21 days of rat bone-marrow primary cell cultures. Rat bone-marrow primary cells were cultured for 3 weeks in a-MEM supplemented with dexamethasone and ascorbic acid in the presence or absence of LIF (10 ng/ml) or OSM (10 ng/ml). Semi-quantitative EDS microanalysis was performed using the EXL Oxford Link System. Results are expressed as the mean 6 SD of 40 random fields analyzed for each condition. Ca/P, Mg/Ca and Na/Ca are expressed as molar ratios deduced from elemental microanalysis. *: P , 0.05 compared to the control without cytokine.
vibrational band characteristic of the apatitic phosphate phase in a protein aqueous matrix (data not shown). The dominant 960 cm21 peak was characteristic of the presence of the phosphate group (symmetrical stretching mode, n1 mode). No other phases than apatite were detected since the position of this band was found to vary among different calcium phosphates (947 cm21 for amorphous calcium phosphate, 960 cm21 for carbonated hydroxyapatite, 970 cm21 for magnesium-substituted b-tricalcium phosphate, 980 cm21 for calcium hydrogen phosphate, 985 cm21 for dicalcium phosphate dihydrate). Moreover, the full width at half maximum (FWHM) of this band, which depends on the degree of carbonate substitution (37, 38), was used as a determinant of crystallinity. In all conditions, FWHM
was found to vary slightly but not significantly (around 19 cm21), which is close to the value of 20 cm21 found in mature rat bone mineral. This width was found to be 14 cm21 for mature human enamel or 35 cm21 for amorphous calcium phosphate (39). The main vibrational modes of the organic matrix corresponded to those of protein bands: for the control or mature rat bone n (CO) amide I band (1670 cm21), d (CH2) scissors modes (1463, 1488 cm21) and amide III d (NH) band (1270, 1244 cm21) were found. The shift in the position of the amide I (1663 cm21) and amide III (1250 cm21) bands with 10 ng/ml LIF or 10 ng/ml OSM may indicate a change in the conformational state of the proteins. Bands at 504 and 897 cm21 (currently unassigned) were found in all conditions but not in Raman spectra of mature rat bone mineral or mature enamel. In the presence of 10 ng/ml LIF and 10 ng/ml OSM, bands at around 525 and 1003 cm21 (assigned to the n4 HPO4 and n1 HPO4 respectively according Fowler et al. (40)) were enhanced by 30% or more (p , 0.05) as compared to the control. The 1003 cm21 has also been assigned to the n1 mode of organic phosphate associated with phosphoprotein occurring mainly in the region of low mineralization (41). Small but noticeable differences were also noted in the structure of the observed band, particularly in the n3 phosphate region (1030-1080 cm21). In control conditions, bands of comparable intensity at 1032 and 1046 cm21 and a band at 1072 cm21 of higher intensity were well-resolved. The n3 phosphate and n1 carbonate peaks both contributed to the band at 1072 cm21. In the presence of 10 ng/ml LIF or 10 ng/ml OSM, broad featureless peaks of comparable intensity were found around 1038 and 1080 cm21. Fourier Transform-Infrared Analysis The infrared parameters are reported in Table 1 for each condition tested in the n4 PO4 (650-500 cm21), n2 CO3 (892-850 cm21) and n1, n3 PO4 (1200-900 cm21) domains. Qualitatively, the relative amount of mineral deposited on the matrix, as determined from the ratio of the phosphate band to the amide I band, was increased by about 40% (p , 0.001) in the presence of 10 ng/ml LIF or 10 ng/ml OSM as compared to the control, whereas the carbonate-to-phosphate ratio, indicative of the relative amount of carbonate in the mineral, remained stable. Infrared parameters obtained from deconvoluted spectra in the n2 CO3 domain indicated that the type A-to-type B carbonate ratio was similar in all conditions tested, whereas the labile-to-type B ratio increased by about 25% (p , 0.05) with 10 ng/ml of LIF or OSM as compared to the control. Moreover, deconvoluted spectra showed more prominent shoulders of labile phosphate or carbonate groups as well as acid phosphate groups in the presence of 10 ng/ml of LIF or OSM (Fig. 3).
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*: Calculated from deconvoluted spectra obtained by Fourier self-deconvolution. : percentage of underlying peaks in the n4 PO4, n2 CO3 and n1, n3 PO4 domains obtained from curve-fitting analysis. Peaks reported are those found to have a significant variation (p , 0.05) in LIF or OSM conditions as compared to the control. †
2.7 6 1 4.8 6 0.7 5.1 6 1.5 27.6 6 1.5 24.7 6 1.4 20 6 0.5 27.1 6 3 34.9 6 0.7 35.3 6 3 7.4 6 2 3.7 6 0.7 2.5 6 0.4 0.0079 6 0.0007 0.0084 6 0.0018 0.008 6 0.0009 Control LIF (10 ng/ml) OSM (10 ng/ml)
2.8 6 0.05 3.9 6 0.07 3.8 6 0.03
0.92 6 0.003 0.91 6 0.018 0.92 6 0.08
0.45 6 0.05 0.56 6 0.06 0.55 6 0.04
10.8 6 2.4 14.5 6 1.5 14.3 6 1.4
17.6 6 0.9 21 6 1.7 22.5 6 1.5
879 cm21 866 cm21 520 cm21 540 cm21 623 cm21 † CO3* type A/ CO3* labile/ type B type B CO3/PO4 PO4/Amide I
Infrared Characteristics of Carbonate and Phosphate Environments of Mineral Phases Deposited in Rat Bone Marrow Primary Cell-Culture
TABLE 1
2.2 6 0.7 5.2 6 0.6 6.6 6 2.2
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On a semi-quantitative basis, variations of phosphate and carbonate environments in mineralized nodules formed in the presence or absence of cytokines were investigated by curve-fitting (Fig. 3). Six bands were used in the n4 PO4 domain. Those at 600, 576 and 559 cm21 corresponded to the apatitic environment of phosphate, and those at 623 cm21, 520 cm21 and 545 cm21 were assigned respectively to labile non-apatitic phosphate, labile non-apatitic HPO22 groups and the 4 apatitic environment of HPO4. The relative percent area of labile phosphate and labile HPO4 increased by about 30% (p , 0.05) in the presence of 10 ng/ml LIF or 10 ng/ml OSM as compared to the control, whereas the relative apatitic HPO4 component decreased by about 60% (p , 0.01). Among the three carbonate environments detected in n2 CO3, labile carbonate from the mineral formed after 10 ng/ml LIF or OSM treatment increased by about 30% (p , 0.05) as compared to the control value. This is in agreement with results for the labile-to-type B carbonate ratio for deconvoluted spectra. In n1, n3 PO4, nine underlying bands were used to fit the contour. The most significant data were an increase in the HPO4 band at 1150 cm21, which corroborated the observation for deconvoluted spectra, and a large increase (about 3-fold) in the 1018-to-1035 cm21 ratio when LIF or OSM was added to cultures. DISCUSSION The main purpose of this study was to analyze the effects of LIF and OSM on mineral phases formed in in vitro primary rat bone-marrow stromal cell cultures. Raman data on these mineral phases, reported here for the first time, showed spectral modifications of the amide I and III bands of the organic matrix synthesized in the presence of LIF and OSM. Both infrared and Raman spectroscopy assessed the apatitic nature of the mineral deposited after 21 days of culture, and its immature state was revealed by the presence of environment rich in labile non-apatitic phosphate, carbonate and HPO4. These features, related to the surface reactivity of the bone mineral and probably to the homeostasis of calcium-phosphate metabolism, were present to a greater extent in cell cultures treated with LIF or OSM. These results indicate that LIF and OSM influenced the physicochemical characteristics of mineral phases formed in this in vitro mineralization model. Numerous studies have already demonstrated the role of soluble molecules during the mineralization process. The effect of dexamethasone on the stimulation of osteogenesis by osteoprogenitors has been reported by Tenenbaum and Heersche (42). This glucocorticoid increased collagen synthesis and alkaline phosphatase activity. Ascorbic acid is another important protagonist in the mineralization required for the collagen synthe510
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FIG. 3. Deconvoluted infrared spectra and curve-fitting analysis of phosphate and carbonate infrared absorption bands from mineral phases formed in rat bone-marrow primary cell cultures. The bottom set of spectra shows typical Fourier self-deconvoluted spectra in the n4 PO4, n2 CO3, n1, n3 PO4 domains consecutively. Bandwidth (s) and enhancement (K) were respectively s 5 18 cm21, K 5 2.2; s 5 9 cm21, K 5 2; and s 5 30 cm21, K 5 2.2. This allowed us to resolve the intrinsically overlapping components, with their position being supported in second-derivative spectra. (1) Deconvoluted spectra of mineral phases formed under control conditions; (2) 10 ng/ml LIF; (3) 10 ng/ml OSM. Among the apatitic sites of phosphate or carbonate ions, several non-apatitic environments were labeled, such as labile phosphate (620 cm21 and 1125 cm21), labile carbonate (866 cm21) and acid phosphate (520 cm21). HPO4 was also present at 1150 cm21 in the n1, n3 PO4 domain. Thus, the curve-fitting technique was performed on a semi-quantitative basis, and the relative percent areas (upper set of spectra) of the individual components were calculated in each spectrum. Spectral decomposition of the corresponding original spectrum was displayed in the three infrared domains investigated.
sis (43), and b-glycerophosphate has often been used in vitro as a source of inorganic phosphate ions (44), although the mineralization process has also been described in its absence (45). A considerable number of growth factors participate in mineral formation, such as fibroblast growth factor, insulin-like growth factor, transforming growth factor, bone morphogenetic proteins (46 – 48), etc. However, the influence of these factors on the physicochemical characteristics of the mineral formed in vitro had not been studied previously. LIF and OSM are among the factors which exert an intense activity on osteoblastic cells. However, the activities of such factors are largely influenced by the differentiation state of the osteoblasts, as noted by Malaval et al. (49) for LIF. A recent study demonstrated the influence of both cytokines on the mineral phases formed after bone-marrow implantation under a mouse kidney capsule (18). The present results confirm the probable involvement of cytokines in the mineralization process. Raman spectroscopy is complementary to infrared spectroscopy since inactive infrared modes are often
active in Raman. Moreover, as water is a weak Raman scatter, adsorbed water has minimal effects on the spectrum. The high background fluorescence of the organic matrix in biological samples alters the quality of the spectrum. However, when the near-infrared range was used in our study instead of the visible range, fluorescence was virtually eliminated since electronic absorption bands are much less likely to occur in this region. The small shifts in the positions of the amide I and III bands of the organic matrix in Raman spectra observed after LIF or OSM treatment may be indicative of changes in the conformational state of the proteins of the organic matrix as compared to that of the control. The shape and position of the bands in vitro relative to the organic matrix were also different from those of rat bone mineral. LIF and OSM probably influenced the synthesis of the matrix, which may have some effect on the formation and nature of the mineral deposited at later stage. formed For mineral phases, it has been shown that the full width at half maximum (FWHM) of the Raman band located at 961 cm21 (characteristic of apatite from mineralized tissues) depends
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on the degree of carbonate substitution (37, 38). Although this feature has also been used as a measurement of changes in crystallinity, there was no apparent changes in our study. The value (19 cm21) found was close to that of mature rat bone mineral and between that of well-crystallized human enamel (14 cm21) and amorphous calcium phosphate (35 cm21), as reported by Leung et al. (39). The changes observed for bands at around 525 and 1003 cm21 could be explain by an increase in HPO4 mineral content formed by bone cells in presence of cytokines. In addition, the band at 1003 cm21 has also been assigned to the n1 mode of organic phosphate associated with phosphoprotein occurring mainly in the region of low mineralization (41). Raman bands at 505 and 897 cm21, as well as a shoulder at around 400 cm21 and an infrared band at 780 cm21 (H2O rocking mode), have been found in newberyite (MgHPO4 z 3 H2O) (50, 51). However the very strong Raman peaks of this phase at 987 cm21 were not found. Infrared spectroscopy in conjunction with curvefitting can improve our understanding of variations in different phosphate and carbonate mineral environments, particularly the labile non-apatitic environment at the surface of the crystals which is very sensitive to bone mineral alteration and interacts with specific proteins associated with crystals (52). Although the lack of a standard similar to that for bone apatite has made it difficult to evaluate carbonate and the HPO4 fraction quantitatively, this technique has proved useful (34, 35, 53) and provides further support for the notion that LIF and OSM influence mineral environments formed in vitro as well as in vivo (18). It is also possible that this difference is due to a completely distinct mechanism of organization of mineralized deposits in cell cultures in the presence of cytokines as compared to the development of mineral in control conditions (i.e. variations of the nature and of the level of extracellular matrix proteins). In all conditions, mineral phases formed with the present model showed an apatitic nature, which appeared to be very immature, with a low carbonate content and the presence of acid phosphate groups. These observations are in agreement with data previously reported by Rey et al. for cell cultures of chicken osteoblasts (54). It is interesting to note that relative mineral amount of mineral deposited was higher in presence of LIF or OSM compared to the control, and that that mineral phases are more immature. The uptake of Mg or Na which appear to be increase in presence of LIF and OSM, would also play a role in these differences of crystal maturity. These observations are in agreement with data reported in vivo. Mg and CO3 are known to hinder the formation of crystalline apatite while they stabilize the amorphous phases. Furthermore the increase of the 1018 –1032 cm21 ratio after LIF and OSM treatment may indicate changes in cristallinity/ maturity of the crystals deposited in vitro. This last
parameter has been shown to provide a measure of mineral crystallinity and maturity (35). Data reported clearly showed changes of the mineral environment (mainly the labile component). These environment have been found closely related to bone mineral reactivity and consequently have been shown to be sensitive indicator of the maturity of biological apatite crystals or some skeletal alterations (52). However, the relations between variations of these labile non apatitic environments and the extracellular matrix are not well understood. The slow rate of maturation of the mineral formed in vitro contrast with in vivo conditions even for a very long-term culture, according to the observations of Rey et al. (54). The chemical environment (composition of the cell culture medium compared to the biological fluids) may influence the way by which cytokines act on cellular behavior and on bone mineral phases. Our results indicate that this new approach (particularly infrared spectroscopy and microspectroscopy) could lead to a better knowledge of the pathophysiology of numerous bone diseases involving a potentially hormonal and/or growth factor pathway. However, other growth factors have been implicated in bone remodeling, and their role in the formation of the mineral phase as well as of the organic matrix also needs to be elucidated. REFERENCES 1. Heymann, D., Guicheux, J., Gouin, F., Passuti, N., Daculsi, G., and Heymann, D. (1998) Cytokine 10, 155–158. 2. Rodan, G. A., and Rodan, S. B. (1995) in Osteoporosis: Etiology, Diagnosis, and Management (Riggs, B. L., and Melton L. J., III, Eds.), 2nd ed., pp. 1–39. Mayo Foundation, Lippincott–Raven, Philadelphia. 3. Mu¨ller-Newen, G., Ko¨hne, C., and Heinrich, P. C. (1996) Int. Arch. Allergy Immunol. 111, 99 –109. 4. Roodman, G. D. (1995) Bone 17, 57S– 61S. 5. Kodama, H., Yamasaki, A., Nose, M., Niida, S., Ohgame, Y., Abe, M., Kumegawa, M., and Suda, T. (1991) J. Exp. Med. 173, 269 – 272. 6. Pacifi, R., and Avioli, L. V. (1993) Osteoporosis Int. 1, 106 –107. 7. Rose, T. M., and Bruce, A. G. (1991) Proc. Natl. Acad. Sci. USA 88, 8641– 8645. 8. Lin, L. F. H., Mismer, D., Lile, J. D., Armes, L. G., Butler, E. T., Vannice, J. L., and Collins, F. (1989) Science 246, 1023–1025. 9. Hilton, D. J., Hilton, A. A., Raicevic, A., Harrison-Smith, L., Gough, N. M., Begley, C. G., Metcalf, D., Nicola, N. A., and Willson, T. A. (1994) EMBO J. 13, 4765– 4775. 10. Piquet-Pellorce, C, Grey, L., Mereau, A., and Heath, J. K. (1994) Exp. Cell Res. 213, 340 –347. 11. Thoma, B., Bird, T. A., Friend, D. J., Gearing, D. P., and Dower, S. K. (1994) J. Biol. Chem. 269, 6215– 6222. 12. Gearing, D. P., Comeau, M. R., Friend, D. J., Gimpel, S. D., Thut, C. J., McGourty, J., Brasher, K. K., King, J. A., Mosley, B., Ziegler, S. F., and Cosman, D. (1992) Science 255, 1434 –1437. 13. Heymann, D., Godard, A., Raher, S., Bentouimou, N., Blanchard, F., Che´rel, M., Hallet, M. M., and Jacques, Y. (1996) Cytokine 8, 197–205.
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