The modulation of collagen on crystal morphology of calcium carbonate

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1 Journal of Crystal Growth 242 (2002) The modulation of collagen on crystal morphology of calcium carbonate F.H. Shen a, Q.L. Feng a, *, C.M. Wang b a Department of Materials Science and Engineering, Tsinghua University, Beijing , People s Republic of China b Beijing Technology and Business University, Beijing , People s Republic of China Received 7 April 2002; accepted 10 April 2002 Communicated by M. Schieber Abstract The modulation function of proteins was studied during biomineralization in vitro. Morphological development of calcium carbonate precipitation in collagen solutions at different concentrations has been examined using scanning electron microscope. X-ray diffraction is used to determine the polymorph of calcium carbonate crystals. Its data indicate that only calcite crystals are formed. The experimental results also show that the calcite growth is more and more inhibited as collagen is increasing in concentration. A simple crystal model is employed to explain these phenomena. Different crystal planes with different affinities to collagen play a crucial role in changing the morphology of calcite crystals. r 2002 Elsevier Science B.V. All rights reserved. PACS: P; 81.10; A Keywords: A1. Biomineralization; B1. Calcium compounds; B1. Collagen 1. Introduction In biomineralization processes, the formation of inorganic crystals is controlled by organic biomolecules such as proteins [1,2]. In order to study the mechanisms of the biomineralization, biomacromolecules were used to induce the nucleation and growth of inorganic crystals. It has been reported that the use of natural components such as extracted proteins or mantles *Corresponding author. Tel.: ; fax: address: biomater@mail.tsinghua.edu.cn (Q.L. Feng). of shells shows significant effects on the control of CaCO 3 crystallization [3,4]. Weiner and Addadi [5] studied the macromolecules in calcium carbonatecontaining biological materials and classified them into three groups. They also studied the crystallographic orientation of calcite, and took it as the nucleation model of mollusks. Manoli et al. induced the calcium carbonate crystals by chitin and elastin, and obtained calcite crystals in both cases [6,7]. Investigations show that proteins can modulate calcium carbonates not only on morphology but also on polymorph [8 11]. In spite of the above studies, collagen has not been reported to be used to modulate calcium carbonate crystals. The current understanding of /02/$ - see front matter r 2002 Elsevier Science B.V. All rights reserved. PII: S (02)

2 240 F.H. Shen et al. / Journal of Crystal Growth 242 (2002) the mechanisms of biomineralization is far from complete. Now, calcium carbonates are considered as bone scaffolding materials inevitably interacting with collagen. This paper mainly discusses the modulation of collagen on calcium carbonate crystals. The aim of these experiments is to understand how the adsorbed proteins influence the nucleation and growth of calcium carbonate crystals, in order to provide theoretical evidences for synthesizing biomimetic materials. 2. Experimental procedure desiccator CaCl 2 solution and collagen RT 2.1. Preparation of collagen Acid-soluble collagen type I was obtained from rat-tail tendon using a protocol identical to that described by Pins et al. [12]. The rat-tail tendon was dissolved in 10 mm HCl at room temperature for 7 h. The solution was centrifuged at 15,000 rpm at 41C for 45 min, and then filtered. 0.7 M NaCl was added to induce precipitation, then the precipitate was collected by centrifugation (15,000 rpm, 41C, 30 min) and redissolved in 10 mm HCl. Acid-soluble mixture was gathered by dialysis against an aqueous phosphate buffer (20 mm disodium hydrogen phosphate, ph 7.4) at 41C for 12 h. The precipitated collagen was collected by centrifugation (15,000 rpm, 41C, 45 min), and then dissolved into a concentrated solution by dialyzing the pellets against a large volume of 10 mm HCl at room temperature for 12 h. The collagen solution was stored at 41C Method of synthesizing crystals In our experiment, the method of synthesizing crystals was according to the report of Aizenberg et al. [13]. The crystals were grown by slow diffusion (about 7 days) of NH 4 HCO 3 vapor into cell-culture dishes containing 1 ml of 10 mm CaCl 2 (ph 7) solution in a closed desiccator. The cover glass-slips were put on the bottoms of the culture dishes for crystal precipitation. Collagen solutions at different concentrations were added into different dishes, respectively (Fig. 1). After crystallization the cover glass-slips were air-dried. Cover glass-slip 2.3. Characterization of synthesized crystals X-ray diffraction (XRD), using a D/max-RB X-ray diffractometer with 40 kev Cu Ka radiation, was employed to analyze the crystal structure and orientation. The morphologies of the crystals were observed under a Hitachi S-450 scanning electron microscope (SEM) after the crystal had grown for 7 days and deposited with gold sputtering. 3. Results and discussion NH 4 HCO 3 (s) Fig. 1. Experimental setting of sediment of calcium carbonates modulated by collagen. From XRD it can be seen that only calcite crystals are formed on the glass matrix in collagen solutions at different concentrations (Fig. 2). It means that collagen does not change the polymorph of calcium carbonates. The effect of the different concentrations on the calcite morphology is obvious, incremental as collagen is at increasing concentration. The crystals grown in absence of protein (control samples) are always perfect rhombohedral about 60 mm in size after 24 h growth (Fig. 3a). At low collagen concentration (o0.1 g/l), well faceted rhombohedral crystals are also formed with little disfigurement (Fig. 3b). At

3 F.H. Shen et al. / Journal of Crystal Growth 242 (2002) (104) X-ray intensity Fig. 2. XRD data for calcium carbonate crystals modulated by collagen. 2θ the collagen concentration of g/l, the edges of {1 0 4} planes, which are parallel to the c-axis, are inhibited, and new planes appear. These new planes belong to the {1 1 0} crystal plane of calcite (Fig. 3c). At an intermediate collagen concentration (5 10 g/l), multiple layer calcite crystals appear with thinner layer thickness (Fig. 3d). The higher the collagen concentration, the thinner the layer thickness. As the collagen concentration increases from 5 g/l to 10 g/l, the layer thickness decreases from 0.5 mm to 0.1 mm. In this condition, some planes with flower-like patterns can be seen (Fig. 3e). At higher collagen concentration (>10 g/l), aggregates are formed, and they become spherulitic as the concentration further increases (Fig. 3f). As expected, both the number and size of calcite crystals vary with the change of collagen concentration. The number of crystals mounts up, while size diminishes with the increase of the protein concentration. A simple crystal model is used to explain the changes of crystal morphologies. In this crystal model, the planes of the crystals are classified into three groups, F (flat), S (stepped) and K (kinked) planes, as described by Hartman and Markov [14,15] (Fig. 4). The K and S planes can be considered as full of kinks and steps, respectively. It is obvious that the highest growth rate will be achieved at the K plane, then the S plane, and finally the F plane [14]. Therefore, the K and S planes of original crystals shape the morphology of subsequent crystals. These final crystals have no new K and S planes. The experimental results above show that new planes started to appear at the edges of rhombohedral calcite crystals when collagen was present in the solution. It seems as if the initial stage of new planes developed at the corners (K planes) and edges (S planes) of calcite seed {1 0 4} planes. Paquette and Reeder [16] suggested that there are geometrically different types of plane sites on the original planes of the calcite seeds. Collagen has a higher affinity for some of these sites, the K and S planes in the experiment, and adsorbs on them during incorporation, that preferentially slows down growth in corresponding directions. As a consequence of inhibition by collagen, new crystal planes will be developed at the corners and edges, and these planes have a higher affinity to collagen, eventually creating the morphology changes. The expression of new planes on the calcite crystals results from the preferential direction of the planes, in such a way that the morphology of the crystal is modified by the expression of the newly stabilized planes. The common feature of crystal planes preferentially interacting with acidic proteins is the orientation of carbonate groups perpendicular to the crystal plane, such as crystal planes running parallel to the c-axis of calcite, i.e. (1 1 0) and (1 0 0) [2]. Although it is known that

4 242 F.H. Shen et al. / Journal of Crystal Growth 242 (2002) (a) (b) (c) (d) (e) (f) Fig. 3. SEM morphologies of the calcite crystals precipitated in the solution. (a) Rhombohedral calcite crystal grown in the solution without collagen. (b) Rhombohedral calcite crystal with little disfigurement, collagen concentration: o0.1 g/l. (c) Overgrown calcite crystal with new planes, collagen concentration: g/l. (d) Multiple layer calcite crystal with thinner layer thickness, collagen concentration: 5 10 g/l. (e) Some planes with flower-like pattern, collagen concentration: 5 10 g/l. (f) Spherulitic calcite aggregates at higher collagen concentration: >10 g/l.

5 F.H. Shen et al. / Journal of Crystal Growth 242 (2002) collagen density on the entire calcite plane area will gradually increase, and the overall calcite growth rate on the calcite crystal planes at a constant supersaturation and ratio of [collagen]/ [Ca 2+ ] will gradually decrease [19]. At lower concentration, collagen adsorbs on the K and S planes preferentially, resulting in multiple layer morphology. With the increasing collagen, these planes cannot burden this amount of proteins, which adsorbon the F plane and accumulate at the interspace of several calcite crystals, then, spherulitic aggregates are formed. Fig. 4. A schematic representation of a crystal illustrating F, S and K planes depending on whether they are parallel to two or one most dense rows of atoms, or are not parallel to any of the most dense rows of atoms, respectively. some planes are preferential to collagen, their exact index is not known. The adsorption of protein from solution onto a solid plane is determined by the stability of its structure. Collagen, having a high structural stability, behaves as colloidal particles and only adsorbs to ionic planes, i.e., calcite, if electrostatically attracted [17]. It must be an important feature influencing the biomineralization process. The negatively charged proteins adsorbon cover glass-slips form a layer of calcium ions. This layer promotes the oriented nucleation of calcite on its (0 0 1) plane, consisting of alternated layers of calcium and carbonate groups [18]. This orientation of calcite, with the c-axis perpendicular to the substrate, is the most common crystal orientation found in biological mineralization. One reason to explain what can cause the growth rate to slow down on the K and S planes is that the collagen density on the newly developed planes is higher than that on the original planes of the calcite seeds. As a high collagen density gives a lower growth rate, the relative growth rate on the F plane can be higher than those on the K and S planes. As the crystal morphology is gradually changing from low-collagen-content plane dominant to high-collagen-content plane dominant, the average 4. Conclusion The present paper shows that collagen has a morphological effect on calcite growth-inhibition of specific crystal planes, furthermore, the effect becomes more pronounced with increased concentration. The inhibition of calcite growth by collagen is caused because collagen is non-uniformly incorporated into the calcite crystal planes and develops new crystal planes having a higher collagen density and lower growth rate than that of the original calcite seed planes. These slower growing planes dominate the morphology of the growing calcite crystals. Acknowledgements This work was supported by the National Natural Science Foundation of China, Grant No References [1] S. Mann, in: D.W. Bruce, D. O Hare (Ed.), Inorganic Materials, 2nd ed., Wiley, Chichester, 1996, p [2] L. Addadi, S. Weiner, Proc. Natl. Acad. Sci. USA 82 (1985) [3] A.M. Belcher, X.H. Wu, R.J. Christensen, P.K. Hansma, G.D. Stucky, D.E. Morse, Nature 381 (1996) 56. [4] M. Fritz, A.M. Belcher, M. Radmacher, D.A. Walters, P.K. Hansma, G.D. Stucky, D.E. Morse, S. Mann, Nature 371 (1994) 49. [5] S. Weineranna, L. Addadi, J. Mater. Chem. 7 (1997) 689.

6 244 F.H. Shen et al. / Journal of Crystal Growth 242 (2002) [6] F. Manoli, S. Koutsopoulos, E. Dalas, J. Crystal Growth 182 (1997) 116. [7] F. Manoli, E. Dalas, J. Crystal Growth 201 (1999) 369. [8] T. Kato, T. Suzuki, T. Amamiya, N. Komiyama, Supramolecular Science 5 (1998) 411. [9] T. Kato, T. Amamiya, Chem. Lett. 3 (1999) 199. [10] T. Kato, T. Suzuki, T. Irie, Chem. Lett. 2 (2000) 186. [11] M. Vucak, J. Peric, M.N. Pons, S. Chanel, Power Technol. 101 (1999) 1. [12] G.D. Pins, D.L. Chirstiansen, R. Patel, F.H. Silver, Biophys. J. 73 (1997) [13] J. Aizenberg, J. Hanson, T.F. Koetzle, S. Weiner, L. Addadi, J. Am. Chem. Soc. 119 (1997) 881. [14] P. Hartman, in: P. Hartman (Ed.), Crystal Growth: An Introduction, Amsterdam North-Holland Publ., Amsterdam (North-Holland series in Crystal Growth), 1973, p. 1. [15] I.V. Markov, Crystal Growth for Beginners Fundamentals of Nucleation, Crystal Growth and Epitaxy, World Scientific, Singapore. [16] J. Paquette, R.J. Reeder, Geochim. Cosmochim. Acta 59 (1995) 735. [17] T. Arai, W. Norde, Colloids Surf. 51 (1990) 1. [18] L. Addadi, J. Moradian, E. Shay, N.G. Maroudas, S. Weiner, Proc. Natl. Acad. Sci. USA 84 (1987) [19] Y. Zhang, R.A. Dawe, Chem. Geol. 163 (2000) 129.