Effects of fibronectin on hydroxyapatite formation

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Journal of Inorganic Biochemistry73 (1999) 129-136

Effects of fibronectin on hydroxyapatite formation Denis Couchourel a, C~line Escoffier a, Ramin Rohanizadeh a,b, Sylvain Bohic a, Guy Daculsi a, Yannick Fortun a, Marc Padrines a,, aLaboratoire de Recherche sur les Matdriaux d'lntdr~t Biologique, UPRES EA 2159, Facultd de Chirurgie Dentaire, I place Alexis Ricordeau, BP 84215, 44042 Nantes Cedex 01, France h New York University, Department of Dental Materials, Calcium Phosphate Research Laboratory, 345 East 24th Street, New York, N Y 10010-4086, USA

Received 4 May 1998; received in revised form 4 January 1999; accepted 7 January 1999

Abstract

There is increasing evidence that noncollagenous matrix proteins initiate bone mineralization in vivo. Fibronectin, which is present during the early phases of mineralization, may contribute to this process in bone tissues. In this context, the mineralization potential of fibronectin was tested in an agarose gel precipitation system and a metastable calcium phosphate solution. The protein inhibited the precipitation of calcium phosphate crystals in solution but had no apparent effect in gel. Conversely, fibronectin stimulated crystal formation when apatite powder was used to seed crystal growth in gel. Although these results in vitro do not clearly indicate that fibronectin is involved in the mineralization process, they are consistent with in vivo events. Free fibronectin (e.g. in biological fluids) could inhibit crystal growth but might also activate the mineralization process when absorbed on apatite powder in a bone environment and areas of ectopic mineralization. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Fibronectin; Biomineralization; Crystal growth; Hydroxyapatite

1. Introduction

The organic matrix, an important bone component, plays a major role in the ossification process [ 1-3] by guiding mineral deposition and favouring mineralization [ 4 - 6 ] . Although type I collagen was the first matrix substance shown to support mineral deposition [ 7 ], several studies have demonstrated that collagen itself cannot induce mineralization in vitro [ 8,9]. Moreover, a recent work indicated that insoluble dentin collagen and not bone collagen induces apatite formation in a metastable solution [ 10]. Bone collagen had an even lower organic phosphate content and did not induce mineral formation. These results suggest that collagen provides an oriented matrix for mineral deposition. The noncollagenous matrix substances, that can bind calcium ions, modified the C a / P ratio and thus influence crystal formation and growth [ 11 ]. Phosphophoryn, the major phosphoprotein of dentin, inhibits in vitro the formation of calcium phosphate (CAP) crystals at high concentrations but activates it at low concentrations [ 12]. Likewise, osteocalcin, a bone specific protein, is inhibitory in solution [ 13] and activating when fixed to a support [ 14]. Osteonectin is also inhibitory in * Corresponding author. Tel.: + 33-240-412-916; Fax: + 33-240-083-712; E-mail: marc'padrines@sante'univ-nantes'fr

solution [ 15] and activating in vitro with gel precipitation systems in denatured gelatin [9]. Conversely, bone sialoprotein under the same conditions induces crystal nucleation in solution or in agar or gelatin gel [ 16,17], whereas recombinant human bone sialoprotein, which lacks post-translational modifications, in solution acts as an inhibitor of crystal growth [ 18]. These different results indicate that the course of subsequent crystal growth reaction is markedly dependent on the protein present as well as the level of the supersaturation ratio of the solution and the local concentrations of coprecipitating ions. Various systems and conditions have been used in vitro to assess the factors which promote and suppress the formation of biologically relevant CaPs in vivo. The systems include synthetic solutions, biological fluids, inorganic (silica) or organic gels, and liposomes; and the conditions involve variations in pH and temperature, constant pH and composition, varying levels of saturation, spontaneous precipitation, seeded growth crystals, and the presence of inorganic or organic substances [ 19-27 ]. Although these studies have improved our knowledge of the mineralization process, various points remain unclear. For instance, it is uncertain which essential matrix components regulate the growth and formation of biological hydroxyapa-

0162-0134/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved. I'll S0162-0134(99)00006-9

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D. Couchourel et al. / Journal of Inorganic Biochemistry 73 (1999) 129-136

tite (HA) crystals. Many noncollagenous proteins have been extensively characterized in recent years, but in some cases their effects on the growth of CaP mineral are poorly known or undetermined. Fibronectin is distributed in all extracellular matrices and particularly in bone since it is synthesized by osteoblasts [ 28 ] and mesenchymal cells of bone and cartilage [29]. It is present quite early in developing bone [30], and the interaction of fibronectin with CaP crystals could play a significant role within bone tissue. This high molecular weight dimeric protein (500 kDa) can bind to HA crystal [ 31 ] and certain faces of brushite crystals (CaHPO4.2H20) [32]. It has several domains for binding to other proteins and an RGD sequence [33] which allows binding to several integrins present on the cell membrane. Each of these domains is itself formed by differentiated modular protein units on the base of its primary structure [ 34 ] and its three-dimensional conformation [ 35 ]. The present study considers the effect in vitro of fibronectin on the growth of CaP crystals. Precipitation trials were performed in an agarose gel matrix and a pH-controlled solution to determine whether fibronectin could induce de novo formation of CaP crystals in an adequate ionic environment and to define its potential role in the growth of newly formed crystals. Concomitantly, albumin, poly(L-glutamic acid) (polyGlu) and type I collagen, which have known effects on CaP precipitation [9,36-38], were tested to determine the specificity of the fibronectin effect on crystal growth.

2. Materials and methods 2.1. In vitro study o f CaP precipitation within an agarose gel matrix

The effect of proteins on the formation of apatite crystals was studied according to Fujisawa et al. [39]. All solutions used were prepared with reagent-grade chemicals. The in vitro precipitation system employed required the use of a 96well microplate (Nunc, Roskild, Denmark). The agarose gel (150 mM NaCI, 10 mM Na2HPO4, 50 mM Hepes (N-2( hydroxyethyl) -piperazine-N'-2-ethanesulfonic acid ), 0.5 % agarose, pH 7.40) was brought to a boil and then maintained at 45°C in a water bath. Four proteins were tested in this study: bovine serum albumin, type I collagen from calf skin, fibronectin of human plasma and polyGlu (P-4886:51 300 Da), all obtained from Sigma (which was also the supplier of agarose). These proteins were successively mixed with gels at different concentrations and immediately distributed in microplate wells ( 100/xl per well). After gelling, 150 Ixl of calcium solution were added (150 mM NaCI, 50 mM Hepes, 10 mM CaCI2, pH 7.40). The preparation was then incubated for 4 h at 37°C. Absorbance was measured at 405 nm every 30 min on a microplate reader (MRX, Dynatech Laboratories). This measurement is not quantitative but corresponds to the turbidity induced by CaP in the gel. Negative controls were performed by replacing the calcium solution

with 150 txl of bidistilled water with or without protein. Each incubation was carried out under sterile conditions at 37°C in triplicate and was repeated five times. A control was performed to ensure that a temperature of 45°C did not induce protein denaturation. Each protein was analysed by native-gel electrophoresis (10% acrylamide, without SDS and 13-mercaptoethanol) after 1 h incubation at 45 or 25°C. Hydroxyapatite powder [40] was then mixed with the gel to determine the effect of fibronectin in the presence of preformed seeds. The powder was obtained by alkaline hydrolysis of dihydrated dicalcic phosphate (CaHPO4 • 2H20), with a Ca/P atomic ratio of 1.6, a specific area of 47.8 mZ/g and a particle size of around 3 p,m. The specific area of HA powder was measured by the Brunauer-Emett-Teller (BET) method on a Quantasorb Jr. apparatus. Particle size distribution was determined by scanning electron microscopy (JSM 6300, Jeol, Tokyo, Japan) coupled with a semiautomatic image analyser (Quantimet 500, Leica, Cambridge, UK), after gold-palladium coating (Emoscope AE 1230, Ashford, Kent, UK). Several powder concentrations (0.1 to 0.5 mg/ml) were tested in the presence or absence of proteins. The crystals were not pre-incubated with the protein. For every concentration of apatite, negative controls were performed by replacing the calcium solution with 150 txl of bidistilled water. The absorbance values of the control were subtracted from those of the corresponding calcium-containing wells. Each incubation was performed under sterile conditions at 37°C in triplicate and was repeated five times. Results are expressed as the mean+SD. Comparative study of means was performed using the ANOVA statistical test. Results were considered significantly different when p < 0.05. 2.1.1. Microcharacterization

The gels were melted by heating and the precipitate was collected and analysed by: (1) Wide angle X-ray diffraction (Kristalloflex D5000 diffractometer, Siemens, Germany) using Oa Kct radiation (Fig. 1 (a)). ( 2 ) Fourier transform infrared (FT-IR) spectroscopy. The data were recorded with a Magna 550 spectrometer (Nicolet, Trappes, France) equipped with an IR-plan infrared microscope (Spectra-Tech Inc., Stamford, CT, USA) fitted with a narrow-band mercury--cadmium-telluride detector (MCTA) cooled to 77 K. A 15 × Cassegrain objective and condenser, a motorized stage (dx, dy, 1 Ixm) and two rectangular apertures were also used for data acquisition. The samples were placed between two infrared BaF2 windows in a compression cell. The resultant thin film was allowed to dry at room temperature. The microcrystal aggregates that precipitated in the gel were isolated through the microscope in visible mode using a visible polarizer (Spectra-Tech Inc., Stamford, CT, USA) and analysed (40 × 40 Ixm area) by FT-IR microspectroscopy. The spectral range was 4000-800 cm- ~. Spec-

D. Couchourel et aL /Journal of Inorganic Biochemistry 73 (1999) 129-136

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trode and adjusted to pH 7.40 by addition of 10 mM NaOH using a pH-stat assembly. The solution was maintained at pH 7.40 during crystal growth, and the volume of NaOH added was recorded continuously. Following each run, all components in contact with calcifying solutions were washed with 0.2 M HCI and then rinsed with bidistilled water. The titrator was recalibrated prior to each run. The volume of NaOH added after 300 min (V3oo) was plotted against the logarithm of the protein concentration. The protein concentration giving half-maximal inhibition of apatite formation (IC5o or 50% inhibition) was determined by nonlinear regression analysis using the 'Enzyme kinetics' program (Trinity software). All experiments were repeated three times. After experiments, samples were filtered on 0.22 ~m MFMillipore filters and the retained residues were frozen in liquid N2 for lyophilization before X-ray and FT-IR analysis. The amount of the precipitated solid phase formed was too small to allow reliable analytical characterization. Moreover, the phase detected in these experiments was of apatitic nature (results not shown).

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Fig. 1. (a) XRD pattern of crystals precipitated in the gel system display the main peak characteristic of an apatitic phase [40]. (b) FT-IR spectra obtained by FT-IR microspectroscopy of crystals isolated from the gel through the infra.red microscope (40 × 40 p~m area). FT-IR spectrum of ( 1) HA; (2) crystal aggregates precipitated in the gel; (3) same spectrum as in (2) but the residual contribution of the agarose was mathematically subtracted. In spectrum (2) or ( 3 ): peaks at 1028 cm - ' and shoulder at around 1080 c m - ~were assigned to the E~ PO 4 mode and those at 963 cm Lto the vt PO4 mode. Note the prominent shoulder at 1120 cm ~ also found in young poorly crystallized apatite and a broad band centred at 1190 cm ] with shoulders at 1220 and 1150 cm ', probably due to phosphate or acid phosphate ions in the gel. A weak broad band between 890 and 850 c m ( P--OH of HPO4) was also observed. The inset shows the infrared absorption band of O H (around 3570 cm ~). It was clearly observed in HA and was barely discernable in spectra of crystal aggregates precipitated from the agarose gel system.

tra were acquired with a 4 c m - J resolution using 256 scans and a Happ-Genzel apodization function. The total absorbance measured from a thin section was below 2.0, thus preserving the linear response of the detector (Fig. 1( b ) ) . The only phase found in these experiments was a poorly crystalline apatite.

2.2. Apatite formation in solution This study was performed according to Hunter et al. [41]. All solutions used for apatite formation from metastable CaP were prepared with reagent-grade chemicals using distilled water. All were filtered through an 0.2 p~m Millipore filter before use. 50 c m 3 of solutions supersaturated with respect to HA were prepared from stock solutions and mixed to final concentrations of 2.5 mM CaC12, 1.5 mM sodium phosphate (pH 7.40) and 150 mM NaCI. The metastable CaP solutions were maintained at 37°C with stirring and then purged with nitrogen to exclude carbon dioxide. The pH of the reaction solution was measured in situ with a combination glass elec-

3.Results 3.1. Effect of proteins on CaP.formation within an agarose gel matrix Increased absorbance corresponded to the appearance of CaP precipitates, although the relation between these events was not linear. Absorbance was measured at 405 nm in an agarose gel containing phosphate incubated with a calcium solution with or without proteins. This turbidity measurement enabled us to study the appearance of CaP crystals within the gel. Protein in an agarose gel without calcium added did not affect turbidity. This study required that agarose be maintained at 45°C when the proteins were added in order to prevent premature gelation. A polyacrylamide gel electrophoresis control of the four proteins displayed the same migration band at 45 and 25°C (results not shown), which suggests minimal or no denaturation of the four proteins. When the albumin concentration (Fig. 2(a) ) increased in the agarose matrix, absorbance decreased and was significantly different from that of the control ( p < 0 . 0 5 ) . This reduction in gel turbidity was dependent on the protein concentration and indicated that the quantity of CaP precipitates had decreased. Conversely, polyGlu (Fig. 2 ( b ) ) induced an increase in gel absorbance, indicating a greater amount of CaP precipitates. The effects of fibronectin and type I collagen on the growth of CaP crystals were less apparent than the other two proteins (Fig. 2 ( c ) - ( d ) ) . There was no significant variation in the formation of CaP crystals as the concentration of fibronectin or type I collagen increased. On the basis of the above results, fibronectin was tested in the same precipitation system, but with addition to the gel of

D. Couchourel et al. / Journal of lnorganic Biochemistry 73 (1999) 129-136

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Fig. 2. Effect of protein on CaP precipitation in the agarose gel (0.5% agarose, 100 ~1) containing phosphate ions and albumin (a), polyGlu (b), fibronectin (c) or type I collagen (d). Buffer ( 150 ill) containing calcium ions was placed on top of the gel in wells of microtiter plates. CaP precipitated in the gel was detected by measurement of absorption at 405 nm. Results are the mean + SD of five independent experiments performed in triplicate. Asterisk: p < 0.05 compared to the control.

calcium-deficient HA powder at a concentration of 0.5 mg/ ml (Fig. 3). Under these conditions, absorbance increased as the dose of fibronectin was augmented. This result may be compared with that of Fig. 2 (c) (fibronectin alone in the gel) and that of Fig. 4 (HA powder alone in the gel). Thus, the activity of fibronectin in the formation of CaP crystals depended on the presence of HA powder.

3.2. Effect of proteins on apatite formation in solution The formation of apatite crystals in a metastable CaP solution was obtained by incubation of solutions containing 2.5

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Fig. 3. Effect of fibronectin on in vitro mineralization in the gel in the presence of 0.5 m g / m l of apatite crystals. Precipitated CaP was detected by measurement of absorption at 405 nm. Results are the mean + SD of three independent experiments performed in triplicate. Asterisk: p < 0.05 compared to the control.

mM CaCI2, 1.5 mM sodium phosphate and 150 mM NaCI. Solution pH was measured in situ and adjusted to 7.40 by addition of 10 mM NaOH. These experiments sought to characterize CaP formation in solution by studying the release of H 3 0 + ions into the medium at the time of the precipitation reaction. By maintaining pH at 7.40 throughout the addition of NaOH, it was possible to evaluate apatite precipitation in the presence of different proteins. Fig. 5 shows a series of titration curves obtained in the presence of different protein concentrations. The increase in protein concentration induced a decrease in apatite formation. These experiments showed that the quantity of NaOH added after 300 min of incubation (V3oo) was a reproducible measurement of the amount of apatite formed. An inhibition curve was plotted representing V3ooas a function of the protein concentration (Fig. 6). The fibronectin concentration required to achieve 50% inhibition of apatite formation was 18.2__ 1.4 Ixg/ml. To determine the specificity of the fibronectin effect on the inhibition of apatite formation, we tested two other proteins (albumin and polyGlu) known for their inhibitory effects in solution. Notable differences were observed in the protein concentrations required to inhibit apatite formation. Fibronectin induced inhibition at a much lower concentration than polyGlu and albumin, as corroborated by a lower IC5o ( 18.2 Ixg/ml compared to 200 Ixg/ml and 3.2 mg/ml). When expressed in micromoles, this difference in inhibition potential was even more marked (0.04 IxM compared to 3.9 and 48.5 jxM) (Table 1). At a concentration of 50 ~g/ml of fibronectin, activation was observed in gel in the presence of apatite crystals, and

D. Couchourel et al. / Journal of Inorganic Biochemistry 73 (1999) 129-136

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more than 50% inhibition occurred in solution in the absence of crystals. The formation of CaP crystals was analysed in a metastable solution in the presence of 50 I~g/ml of fibronectin and 0.5 mg/ml of HA powder not initially incubated with protein. Under these conditions, fibronectin showed no effect on the formation of CaP crystals in solution, giving results comparable to those obtained in the absence of proteins (Fig. 7).

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Time(nan) Fig. 5. Titration curves for apatite formation in the presence of fibronectin (a), polyGlu (b) or albumin (c). The protein was added to metastable calcium phosphate solutions at 37°C. Apatite formation was determined by addition of l0 mM NaOH.

4. Discussion

Although many noncollagenous proteins have been extensively characterized in recent years, their effects on CaP rain-

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D. Couchourel et al. / Journal of Inorganic Biochemistry. 73 (1999) 129-136

eral growth are still poorly understood or undetermined. For fibronectin, our results clearly show an enhancement or inhibition of CaP precipitation with two indirect methods: a change of pH in a metastable solution and a variation of absorbance in an agarose gel precipitation system. During the two experiments, the solution conditions can change. In solution, the fibronectin can bind the calcium ions, and thereby modify the Ca/P ratio and thus influence the crystal growth. In gel, the fibronectin can adsorb on the crystal and so favorize the crystal growth. Our results for the CaP precipitation process, in accordance with the study model used, are in agreement with reports in the literature [ 8,9,36-38,41-43 ] indicating that the activity of a protein can vary according to the system used, which tends to confirm the validity of our observations for fibronectin. In addition, X-ray diffraction or FT-IR analysis confirmed the apatite nature of all these precipitates, as in other studies [ 16,25,42,44,45 ]. In solution, polyGlu inhibited apatite formation, whereas in an agarose gel matrix it became activating. However, albumin inhibited crystal growth regardless of the conditions used, and type I collagen had no effect. The spontaneous precipitation of CaP from supersaturated solutions has been extensively studied [25,38,41 ]. The critical precipitation point is usually determined by mixing solutions containing calcium and phosphate ions and observing the first appearance of the solid phase. The disadvantage of this procedure is that a considerable amount of precipitation has already taken place before the precipitate is visible in the solution. Yet such experiments are highly reproducible, allowing studies of the effects of factors such as supersaturation level, seed morphology and, as here, the presence of foreign substances. In our work, pH was constant and initial calcium and phosphate concentrations were identical for all experiments. A comparison of data obtained from different proteins in the same supersaturated solution showed that fibronectin inhibited precipitation as a function of the concentration. Comparison of the inhibitory activities of each protein gave an order of magnitude of the inhibitory power of fibronectin in solution. Under these conditions, fibronectin was 10 times as inhibitory as polyGlu and 1000 times as inhibitory as albumin. Nonetheless, other proteins are far more inhibitory than fibronectin, such as osteopontin [38] which is 20 times as powerful (IC5o=0.002 IxM). It would seem likely that fibronectin, which possesses 28 more or less weak binding sites for calcium [46], deprives the reaction medium of one of its substrates, so that the quantity of precipitated CaP is reduced. This mechanism may also account for the inhibitory action of CaP crystal growth mediated by some proteoglycans [44], whereby the addition of phosphate to solutions containing protein non-we-equilibrated in calcium resulted in complete inhibition of apatite formation. Thus, the apatite formation yield is decreased, but induction time may be constant, suggesting that nucleation is not altered but that growth is altered. The mechanism by which fibronectin prevents apatite formation seems to be a growth inhibition of apatite crystals, as in the case of osteopontin [41 ].

Unfortunately, the amount of newly formed crystals obtained under our conditions was not sufficient to determine whether fibronectin causes changes in crystal size or influences crystal proliferation. The effects of bone matrix protein on apatite formation in an agarose or gelatin gel system have been extensively studied [9,12,14,16,39]. In our experiments, pH was constant and the initial concentrations of phosphate in gel and of calcium in buffer were always identical. The initial buffer concentration was high, but that at the diffusion front was reduced [ 39,47 ]. Calcium ions diffuse slowly into gel and precipitate as CaP when they encounter phosphate ions. The effects of protein on in vitro mineralization provide indirect evidence of their interactions with mineral crystals. PolyGlu had positive effects on in vitro mineralization in gel (Fig. 2 ( b ) ) , as previously reported [37,39], indicating that this system is able to detect the effects of these proteins. In our gel phase, fibronectin apparently lost all its activity. Although our experimental conditions may not have altered the potential of fibronectin in the formation of CaP crystals, they could have been responsible for its apparent inactivity in the gel precipitation system. A larger amount of the fibronectin would probably be required to induce an inhibitory activity in gel, as had been observed for albumin. Subsequent to these experiments performed in the absence of seed crystal, the same procedures were repeated with addition of an apatite powder in the reaction medium. HA could serve as a seed for newly formed apatite crystals or/and as a support for fixation of the fibronectin molecule. The results were different when HA powder was integrated into the gel. Apatite alone in the agarose gel lost all its nucleation activity and could no longer mask the activity of fibronectin which, by itself, induced neither activation nor inhibition of crystal formation. CaP precipitation increased only when the HA-fibronectin mixture was present in the gel and was not observed with another studied protein (albumin, type I collagen). Thus, fibronectin would appear to serve as an activator only in the presence of HA in gel. Calcium was slowly diffused in gel and precipitated instantaneously in all experiments. The activator role of fibronectin could have been due to a local increase in calcium concentration. The protein could interact with Ca 2 + ions to create a microenvironment rich in calcium ions in the immediate vicinity of HA particles, thereby inducing a local alteration of the molar Ca/P ratio. It may be supposed that fibronectin initially deprives the gel of one of its substrates, reducing the quantity of precipitated CaP, so that the resulting formation of a calcium-rich microenvironment leads to mineral deposition. This hypothesis could account for the contradictory results obtained with our agarose gel system. As activation of apatite formation was not observed in the absence of HA seeds, fibronectin-induced reduction of calcium in the gel could have been equilibrated by initiation of mineral formation in the microenvironment. Since HA has a high affinity for fibronectin [31] and HA crystals generally proliferate by a process of secondary nucleation [48] in which new mineral forms on the surface of

D. Couchourel et al. / Journal of Inorganic Biochemistry 73 (1999) 129-136

existing crystals, the presence o f H A seeds could c o m p e n s a t e for c a l c i u m reduction in gel, and interaction o f H A and fibronectin could f a v o u r the activation o f apatite formation. A second hypothesis c o u l d be possible. It is likely that the c o m plexation o f calcium f r o m the seeds during the gel preparation dissolves partly the seeds and induces an increase o f phosphate concentration in the gel and, thus, an increase o f the supersaturation and o f the precipitation ability. The inhibitory effect o f fibronectin was no longer o b s e r v e d in solution in the presence of H A , contrary to the results o f T a k e u c h i et al. [49] w h o pre-incubated fibronectin with H A . This c o m p o u n d activated the formation o f C a P crystals in a very distinct m a n n e r since the amount o f N a O H needed to titrate the H 3 0 ÷ ions appearing in the solution during precipitation o f crystals was three times as great in its presence. As fibronectin may bind to H A particles, its intrinsic activity m a y or may not be modified. Thus, the possible effect o f the protein on precipitation o f crystals cannot be d e t e r m i n e d since in solution these properties w o u l d be m a s k e d by free HA. A l t h o u g h these data obtained in vitro do not p r o v e that fibronectin is i n v o l v e d in the mineralization process, they are consistent with in v i v o events. Fibronectin, which is present in plasma [ 50], could thus be part o f the matrix protein pool required for bone formation. Fibronectin, in the presence o f H A in gel, favours the formation o f apatite crystals. This potential cannot be expressed in a non-osseous e n v i r o n m e n t such as biological fluids (plasma, intercellular domain, etc.) or cell m e m b r a n e s , but could be expressed w h e n fibronectin is in the presence o f apatite crystals. In conclusion, this study demonstrates that fibronectin inhibited CaP crystal formation in a metastable calcium phosphate solution but tended to activate it in agarose gel in the presence of H A seed.

5. Abbreviations

HA CaP polyGlu

hydroxyapatite calcium phosphate poly (L-glutamic acid)

Acknowledgements W e are grateful to F. Renaudineau for technical assistance.

References [ 1] [2] [3] [41

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