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Thin film of low-crystalline calcium phosphate apatite formed at low temperature
Biomaterials 21 (2000) 1129}1134
Thin "lm of low-crystalline calcium phosphate apatite formed at low temperature夽 Hyun-Man Kim *, Yoonji Kim , Su-Jin Park , Christian Rey, HyunMi Lee , Melvin J. Glimcher, Jea Seung Ko Laboratory of Hard Tissue Engineering, Department of Oral Anatomy and Dental Research Institute, College of Dentistry, Seoul National University, 28-22, YeonKun-Dong, ChongRo-Ku, Seoul 110-749, South Korea Laboratoire de Physico-Chimie des Solides, Institut Polytechnique de Toulouse, C.N.R.S. 38, rue des 36 Ponts, Toulouse 31400, France Laboratory for the Study of Skeletal Disease and Rehabilitation and Department of Orthopedic Surgery, Children's Hospital and Harvard Medical School, 300 Longwood Ave., Boston, MA 02115, USA Received 24 September 1999; accepted 3 December 1999
Abstract Surface modi"cation of biomaterials to improve biocompatibility without changing their bulk properties is desired for many clinical applications and has become an emerging technology in biomaterial research and industry. In the present study, a simple method of coating the solid surfaces of metals, organic tissue matrices, glasses, inorganic ceramics as well as organic polymers with a thin "lm of low-crystalline apatite crystals (LCA) was developed. Acidic solution containing calcium and phosphate ions was neutralized with alkaline solution to form calcium phosphate precipitates at low temperature. Precipitates of solid calcium phosphate particles were, then, removed by "ltration. Concentration of free ions in the "ltered ion solution which were not involved in the formation of calcium phosphate precipitate was high enough to induce the heterogeneous nucleation on the solid surfaces at low temperature. Thin layers of calcium phosphate crystals were formed on the surfaces of metals, glasses, inorganic ceramics, organic polymers including hydrophobic ones, and biological tissue matrices with this solution. The thin layer of crystals consisted of poorly crystalline calcium phosphate apatite crystals which contain high amount of labile ions like bone crystals and did not dissolve in the physiologic solutions. Various cells attached to this crystal layer and proliferated well. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Low-crystalline apatite; Thin "lm; Biomaterial; Low temperature; Cell culture
1. Introduction Calcium phosphate (Ca-P) apatite has been well known to have good properties as a biomaterial for bone repair, augmentation, substitution, and surface coating [1}3]. Coating the surfaces of dental and orthopedic materials with biocompatible calcium phosphate apatite can elicit favorable biological and chemical responses on the surfaces and this can enable us to mimic the reactions occurring in the natural calci"ed tissues without losing bulk properties of materials such as durability and inertness.
夽 Part of this paper was presented at the sixth International Conference of the Chemistry and Biology of Mineralized Tissues, Vittel, France, 1}6 November 1998. * Corresponding author.
Calcium phosphate apatite is the only type of calcium phosphate crystal in calci"ed tissues such as bones, teeth, calci"ed cartilage, and cultured matrix produced by osteoblasts [4}8]. It has biocompatibility with most cell types such as osteoblasts, osteoclasts, "broblasts, and periodontal ligament cells found in the calci"ed tissues [9}13] and has osteoconductivity allowing the formation of bone on its surface by attachment, migration, proliferation, and di!erentiation of bone-forming cells [14}16]. All apatites, however, are not similar in their crystallographic properties. Pure hydroxy apatite crystals consisting of calcium, phosphate, and hydroxyl ions are stoichiometric crystals of rods with high crystallinity [17], while biocrystals isolated from bone or calci"ed cartilage are non-stoichiometric apatites of low crystallinity [6}8]. Biocrystals are small thin plates [6}8] in shape of extensive speci"c surface that makes them
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metabolically active with high surface reactivity [18]. Their surfaces are assumed to be covered with non-apatitic highly reactive labile ions of CO\, PO\, HPO\, where organic constituents or cells can be attached to form a cell}material interface [19,20]. These properties are not expected from highly crystalline apatite crystals widely used clinically so far, but only from bone-like low-crystalline apatite crystals (LCAs). Apatite coatings formed at high temperature by plasma spraying have been shown to be heterogeneous and to contain large oxy}hydroxy apatite crystals [21] as well as a high proportion of amorphous phases [22]. Thus, there is a demand to develop a method to form an apatite layer with properties similar to those of bone crystals on solid surfaces such as metals, polymers, and ceramics to enhance bioreactivity as well as biocompatibility when engineering implant materials. In the present study, a simple method to get a thin coat of LCAs was developed. Apatite crystals were induced to form and grow directly on solid surfaces including the surfaces of low interfacial energy by increasing the solution supersaturation in the solution. The crystallographic analysis of the apatite crystals in the thin coat showed a close similarity to that of bone crystals in their crystal habit, crystallinity, and the amount of labile domains of surface ions.
2. Materials and methods 2.1. Preparation of thin xlm of apatite In the classical Ostwald's nucleation theory, the free energy for nucleation depends on the supersaturation of solution (S), the net interfacial energy for nucleation (p), the temperature (¹), and the particle surface area (A): *G"!R¹ ln S#pA. This nucleation theory indicates that increasing the solution supersaturation S and reducing the net interfacial energy p can induce the heterogeneous nucleation. In the present study, the solution supersaturation S was greatly increased to reduce the free energy for nucleation *G. This increase was high enough to induce heterogeneous nucleation even on the solid surface of low surface energy without any treatment to decrease the net interfacial energy p. Homogeneous precipitation occurring in highly supersaturated solutions was prevented by using a lowtemperature system. Following this strategy, highly supersaturated stable calcium and phosphate ion solution was prepared at low temperature. Synthetic calcium phosphate apatite crystals [23] were dissolved in 0.2 N HCl (1 mg/ml). 1 ml of this acidic ion solution was mixed with 1.35 ml of 0.2 M tris[hydroxymethyl]amino methane (Tris) solution to form Ca-P precipitate at 43C. Then, Ca-P precipitate was
removed by "ltration (pore size: 0.22 lM) to get a metastable calcium and phosphate ion solution (pH 7.3). Materials to be coated were left in this Ca-P ion solution at 83C for 24 h to induce the nucleation of Ca-P on the surfaces, then the temperature was maintained or increased (up to 603C) to form a thin "lm coat of LCAs by the growth of apatite crystals. Thicker thin "lm was obtained by increasing the time of growth phase. This method was used to coat the surfaces of the following materials; metals, glasses, inorganic ceramics, organic polymers including hydrophobic organic polymers such as poly lactic-gluconic acid co-polymer sponge (PLGA), polystyrene, polypropylene, silicone, polytetra#uorethylene, and organic biological tissue matrix like decalci"ed membranes of crab (consisting of chitin), collagens, and "bers of silk. To exclude the possibility of gravitational precipitates of amorphous calcium phosphates (ACPs) formed by homogeneous nucleation in the solution as the source of crystals on the surfaces, the surfaces of materials such as disks of titanium or polypropylene were positioned vertically and the crystals formed on the lateral surfaces were examined. 2.2. Analysis of the thin xlm of apatites 2.2.1. Electron microscopic analysis The phase and habit of Ca-P crystals were examined in the thin "lm formed on carbon-coated formvarreinforced grids using transmission electron microscopy (TEM) (1200 IIX, JEOL, Japan), or on a solid surface using scanning electron microscopy (SEM) (840A, JEOL, Japan) after sputter-coating with gold}palladium. The electron di!raction patterns were also obtained. 2.2.2. X-ray diwraction analysis The long-range orders of crystals which were scraped o! from the surface of culture dishes were determined by powder X-ray di!raction [24] using di!ractometer (MXP 18, Mac Science, Japan) at 100 mA and 50 kV. Bone crystals isolated from bovine femur [7] were also analyzed for comparison. 2.2.3. Fourier-transformed infrared spectroscopy (FTIR) analysis The FTIR spectra of crystals were recorded on a Perkin Elmer Fourier transform spectrometer 1700, after embedding the crystals removed from the culture dishes and bone crystals isolated from bovine femur [7] in KBr pellets. Resolution-enhanced deconvolution of the FTIR spectra was performed with constructor software. The sensitivity coe$cients and the bandwidths are reported in the "gure legends. Integral intensities of deconvoluted spectra in the l4 PO\ were calculated [25].
H.-M. Kim et al. / Biomaterials 21 (2000) 1129}1134
2.2.4. Calcium and phosphate analysis The concentration of calcium was determined by atomic absorption spectroscopy [26] (Perkin Elmer, USA). Concentration of phosphate was determined by a colorimetric method [27]. 2.3. Culture of cells L929 "broblasts (ATCC NCTC clone 929) and MC3T3 osteoblasts were cultured on thin "lms of apatite formed on various substrates such as culture plates (Corning, USA), without any other treatment of the surfaces. Cells were cultured in CO incubator supplied with 95% air and 5% CO at pH 7.4 in a-MEM supplemented with 10% fetal bovine serum (FBS). Attachment and proliferation of cells were examined directly under phase contrast microscope and/or SEM.
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3. Results With this simple method, thin "lm layers of LCAs were formed on the surfaces of metals, glasses, inorganic ceramics, organic polymers including hydrophobic organic polymers such as PLGA, polystylene, polypropylene, silicone, polytetra#uorethylene, and organic biological tissue matrix like decalci"ed membranes of crab (consisting of chitin), collagens, and "bers of silk (Fig. 1). The "lms of LCAs formed on both hydrophilic and hydrophobic surfaces were not disintegrated or detached from the surface when immersed in the physiological solution for over 1 month. The "rst Ca-P formed on the surface comprised particles of nano-size, which was studied by examining the "rst precipitation formed on the carbon-coated formvar "lm using TEM (Fig. 2a). Formation of "lms of LCAs on
Fig. 1. Sequential observation of the formation of thin layer of LCAs on the surface of the culture dishes. (a) Non-discrete particles are scattered in the initial stage. (b}c) Then, discrete crystals form and grow on the surface, and proliferate and interconnect to cover all surfaces. (d) Apatite coating of LCAs is covering porous PLGA co-polymer sponge. Scanning electron microscope (SEM), scale bar: (a}c) 1 lm, (d) 10 lm.
Fig. 2. TEM observation of the formation of thin layer of LCAs. Non-stained examination. (a) The "rst Ca-P depositions on the carbon-coated formvar "lm under TEM do not give any di!raction maxima of Ca-P crystals (inset), which indicates ACP as a seeding substance of heterogeneous nucleation on the surface. Scale bar: 500 nm. Electron di!raction: 100 kV, 80 cm. (b) Cross-sectional view of the thin "lm shows closely packed nano-sized crystals of thin plates in the thin "lm. Scale bar: 100 nm.
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the vertically positioned solid surface or the underneath surface excluded the possibility of gravitational precipitation of particles from the ion solution as the source of calcium phosphate particles on the surface. Particles of ACPs on the surface were transformed to apatite, then a thin "lm layer was formed by multiplication of more Table 1 XRD of LCAs in thin "lm coating compared to bone crystals hkl
2h
111 002 102 211 112 300
22.70 25.98 27.84 31.72 32.16 33.14
(22.82) (25.86) (28.16) (31.84) (32.65) (33.97)
d (As )
FWHM
3.91 3.43 3.20 2.82 2.78 2.70
0.58 0.44 0.32 0.38 0.44 0.20
(3.89) (3.44) (3.17) (2.81) (2.74) (2.63)
(0.53) (0.33) (0.39) (0.86) (0.42) (0.45)
Values in parentheses are data of bone crystals isolated from bovine femur. FWHM: full-width at half-maximum value.
crystals of thin plates through secondary crystallization [28] around pre-formed apatite crystals. By varying the time of growth, "lms of di!erent thicknesses from a few nm up to about a few lm were formed and this was determined by examining the cross section of thin "lm using TEM (Fig. 2b). XRD showed that the thin "lm of LCA consisted of poorly crystalline calcium phosphate apatite like bone crystals (Table 1). FTIR analysis (Fig. 3a) also indicated their poor crystallinity. Resolution enhancement [25] of l2 CO\ (Fig. 3b) and l4 PO\ (Fig. 3c) showed peaks of non-apatitic labile domains of PO\, HPO\, and CO\, and type B CO\ substituting PO\ in addition to apatitic ions which were also found in bone crystals (Table 2). Fibroblasts attached and proliferated well on the thin "lm of LCAs formed on polyglactin 910 (vicryl) "bers (Fig. 4a). Osteoblasts also adhered well to the substrate of Table 2 The relative intensities in the l4 PO\ domains of LCAs in thin "lm compared to bone crystals IR band (cm\) Domains [31] LCAs Bone crystals
Fig. 3. FTIR spectrum of thin layer of LCAs. Crystals were obtained by scraping the surface of polypropylene o! thin layer of LCAs. (a) l4 PO\, l3 PO\, l2 CO\, and l4 CO\ are seen. (b) Deconvoluted FTIR spectrum in the l2 CO\ domain shows that carbonate ions are located in anionic sites of the apatitic structure indicating type B carbonate}apatite (CO\ substituted for PO\ groups). In addition, a la bile non-apatitic CO\ environment is also present. Deconvolution parameters: bandwidth 10 cm\; sensitivity coe$cient 2.25. (c) Deconvoluted FTIR spectrum in the l4 PO\ domain shows that labile nonapatitic environment is present in addition to apatitic PO\ envi ronment. Deconvolution parameters: bandwidth 18 cm\; sensitivity coe$cient 2.25.
534 Labile HPO 8.17 7.74
550 HPO 2.50 1.65
560 575 600 617 PO\ PO\ PO\ Labile PO 8.89 9.27 8.66 3.24 9.34 9.16 9.28 3.1
Fig. 4. Cells cultured on thin "lm of LCAs. Scale bar: 10 lm. (a) SEM of MC3T3 osteoblasts cultured on thin "lm substrate of LCAs formed on PGLA for 2 d. (b) SEM of L929 "broblasts cultured on thin "lm of apatite formed on polygalactin 910 (vicryl) "bers for 3 d. They attach and proliferate well on the "lm.
H.-M. Kim et al. / Biomaterials 21 (2000) 1129}1134
"lm formed on PLGA (Fig. 4b). Cellular behavior on the substrate of the "lm could be clearly observed under phase contrast microscope due to the transparency of the "lm.
4. Discussion Application of low temperature was considered to be the only condition in this system for obtaining the thin "lm of LCAs. Less calcium and phosphate ions might be involved in the formation of calcium phosphate precipitate in the neutralized ion solution due to the low temperature. Then free ions in the "ltered solution high enough to overcome the energy barrier for heterogeneous nucleation on the surface were considered to be provided to the surfaces. The low temperature again might preclude a homogeneous precipitation within this solution. Direct observation of the formation of a thin layer of LCAs under TEM has con"rmed that this system follows the general sequence for the heterogeneous nucleation and growth of crystals from a supersaturated solution [28,29]. Nanosized ACPs were formed on the solid surface as the "rst phase of Ca-P. This can be ascribed to their low surface energy which requires less net energy for their de novo formation [28,29]. The transformation of ACPs into LCAs was followed. Then, a thin layered coat was formed by multiplication and growth of LCAs. However, further study is required to explain the exact mechanism of the formation of the thin "lm in this system. Crystallographic properties of the crystals in this "lm, such as thin plate shape, low crystallinity, and high amount of labile ions indicate that they are similar to the crystals of bones [7]. The shape of the thin plates of the crystals grown by this method can provide a high speci"c area [18], which in turn enables them to be potentially highly interactive in cellular or organic environment. In addition, crystals were rich in non-apatitic labile domain of PO\, HPO\, and CO\ abundant in bone crystals [30,31]. It was suggested that these labile ions cover the surfaces of LCA and be highly reactive to organic constituents to form an organic}inorganic interface [19,20]. Technically, the present method has an important advantage over the previously reported methods in accessibility of coating site. Ions of calcium and phosphate can reach into the hidden surfaces inside the bulk of materials because they are provided in a solution. The most probable candidates for the application of this method are the porous polymers like polyglycolic acid, polylactic acid, PLGA co-polymers which have a great potential for drug and cell delivery as well as giving a sca!old for bone ingrowth. In addition, application of low temperature in this method will be advantageous in reducing the chance of losing the activity of drugs for the drug delivery system. This technology can also be applied to make the
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non-biocompatibile materials with high mechanical properties to be more biocompatible. There has been much e!ort to form highly bioactive bone-like Ca-P crystals on various substrates with a simulated body #uid or some low supersaturated Ca-P solution [32}37]. However, in such a low supersaturated concentration, inducing the crystal formation was limited only to speci"c solid surfaces like the strictly manipulated one by chemical treatment and has not been possible on hydrophobic surfaces. In addition, those coating procedures needed a relatively long time and high temperature which might limit the control of the crystallographic nature of Ca-P apatite crystals. In the present study, a low-temperature system was used to delay the homogeneous nucleation of solid Ca-P in the ionic solution thereby leaving the concentration of free ions high (up to [Ca>];[PO\]"20 mM). This overcame the energy barrier in inducing heterogeneous nucleation on the surfaces such as hydrophobic materials whose surface enery is very low. In summary, we developed a method to form a thin apatite "lm on various materials at a low temperature. Their crystallographic property is similar to that of the bone crystals and cells attached and proliferated well on this "lm. This indicates that this apatite crystal "lm can be used as a coating material over various substrates and can modify the biocompatibility and bioreactivity of substrates without losing their own bulk properties. Acknowledgements The authors thank Dr. Izumi Asahina (Tokyo Medical and Dental University, Tokyo, Japan) for providing them with porous PLGA co-polymers sponge, Ms. SeongHyun Park and Ms. Su-Jin Kim for their assistance with TEM and SEM, Mr. Chong-Hee Cho for XRD, and Ms. Sa-Im Park for assistance with the preparation of this manuscript. This work was partly supported by Grant for Basic Medical Research from Korea Research Foundation (H.-M. Kim). References [1] Cornell CN, Lane JM. Current understanding of osteoconduction in bone regeneration. Clin Orthop 1998;355(Suppl):S267}73. [2] Lace"eld WR. Current status of ceramic coatings for dental implants. Implant Dent 1998;7:315}22. [3] Soballe K, Overgaard S, Hansen ES, Brokstedt-Rasmussen H, Lind M, Bunger C. A review of ceramic coatings for implant "xation. J Long Term E!ects Med Implants 1999;9:131}51. [4] LeGeros RZ. Calcium phosphates in enamel, dentin and bone. In: Calcium phosphates in oral biology and medicine. Basel: Karger, 1991. p. 108}29. [5] Rey C, Kim H-M, Gerstenfeld L, Glimcher MJ. Structural, chemical characteristics, and maturation of the calcium-phosphate crystals formed during the calci"cation of the organic matrix synthesized by chicken osteoblasts in cell culture. J Bone Miner Res 1995;10:1577}88.
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[6] Kim H-M, Rey C, Glimcher MJ. Isolation of calcium-phosphate crystals of mature bovine bone by reaction with hydrazine at low temperature. In: Brown PW, Constantz B, editors. Hydroxyapatite and related materials. Boca Raton: CRC Press, 1994. p. 331}8. [7] Kim H-M, Rey C, Glimcher MJ. Isolation of calcium-phosphate crystals of bone by non-aqueous methods at low temperature. J Bone Miner Res 1995;10:1589}601. [8] Kim H-M, Rey C, Glimcher MJ. X-ray di!raction, electron microscopy, and Fourier transform infrared spectroscopy of apatite crystals isolated from chicken and bovine calci"ed cartilage. Calcif Tissue Int 1996;59:58}63. [9] Gregorie M, Orly I. Menanteau. The in#uence of calcium phosphate biomaterials on human bone cell activities: an in vitro approach. J Biomed Mater Res 1990;24:163}77. [10] Bagambisa FB, Joos U, Schilli W. The interaction of osteogenic cells with hydroxylapatite implant materials in vitro and in vivo. Int Oral Maxillofac Implants 1990;5:217}26. [11] Cheung HS, Haak MH. Growth of osteoblasts on porous calcium phosphate ceramic: an in vitro model for biocompatibility study. Biomaterials 1988;10:63}7. [12] Davies JE, Baldan N. Scanning electron microscopy of the bone}bioactive implant interface. J Biomed Mater Res 1997; 15:429}40. [13] Nery E, LeGeros RZ, Daculsi G. Tissue response to biphasic calcium phosphate ceramic with di!erent ratios of biphasic calcium phosphate ceramic with di!erent ratios of HA/TCP in periodontal osseous defects. J Periodont 1992;63:729}35. [14] Galgut PN, Waite IM, Tinkler SMB. Histological investigation of the tissue response to hydroxyapatite used as an implant material in periodontal treatment. Clin Mater 1990;6:105}21. [15] Amler MH. Osteogenic potential of non-vital tissues and synthetic implant materials. J Periodont 1988;58:759}61. [16] Kuhn-Spearing L, Rey C, Kim H-M, Glimcher MJ. Carbonate apatite nanocrystals from bone. Proceedings of the Annual Meeting of the Minerals, Metals & Matrials Society, Anaheim, California, USA, 1996. [17] Elliott JC. Hydroxyapatite and nonstoichiometric apatites. In: Structure and chemistry of the apatites and other calcium orthophosphates, studies in inorganic chemistry, vol. 18. Amsterdam: Elsevier, 1994. p. 111}90. [18] Posner AS, Betts F, Blumenthal NC. In: Simons DJ, Kunin AS, editors. Skeletal research: an experimental approach. New York: Academic Press, 1979. p. 167}92. [19] Lees SL, Glonek T, Glimcher MJ. P nuclear magnetic resonance spectroscopic evidence for ternary complex formation of fetal phosphoprotein with calcium and inorganic orthophosphate ions. Calcif Tissue Int 1983;35:815}8. [20] Rey C, Glimcher MJ. In: Slavkin H, Price P, editors. Chemistry and biology of mineralized tissues. North Holland: Elsevier, 1992. p. 5}18. [21] Ellies LG, Nelson DG, Featherstone JD. Crystallographic changes in calcium phosphates during plasma-spraying. Biomaterials 1992;13:313}6.
[22] Weng J, Weng J, Liu X, et al. Further studies on the plasmasprayed amorphous phase in hydroxyapatite coatings and its deamorphization. Biomaterials 1993;14:578}82. [23] Rey C, Collins B, Goehl T, Dickson IR, Glimcher MJ. The carbonate environment in bone mineral: a resolution-enhanced Fourier infrared spectroscopy study. Calcif Tissue Int 1989; 45:157}64. [24] Klug HP, Alexander LE. X-ray di!raction procedures for polycrystalline and amorphous materials, 2nd ed. New York: Wiley (Interscience), 1974. [25] Rey C, Shimizu M, Collins B, Glimcher MJ. Resolution-enhanced Fourier transform infrared spectroscopy study of the environment of phosphate ions in the early deposits of a solid phase of calcium-phosphate in bone and enamel, and their evolution with age. I: investigations in l4 PO domains. Calcif Tissue Int 1990; 46:384}94. [26] Willis JB. The determination of metals in blood serum by atomic absorption spectroscopy. I: calcium. Spectrochim Acta 1960; 16:259}72. [27] Bartlett GD. Phosphorus assay in column chromatography. J Biol Chem 1959;234:466}8. [28] Eanes ED. In: Bonucci E, editor. Calci"cation in biological systems. Boca Raton: CRC Press, 1992. p. 1}17. [29] Eanes ED, Termine JD, Nylen MU. An electron microscopic study of the formation of amorphous calcium phosphate and its transformation to crystalline apatite. Calcif Tissue Res 1973; 12:143}58. [30] Rey C, Renugopalakrishnan B, Collins B, Glimcher MJ. Fourier transform infrared spectroscopic study of the carbonate ions in bone mineral during aging. Calcif Tissue Int 1991;49:251}8. [31] Rey C, Kim H-M, Glimcher MJ. Maturation of poorly crystalline synthetic and biological apatites. In: Brown P, Constanz B, editors. Hydroxyapatite and related materials. Boca Raton: CRC Press, 1994. p. 181}8. [32] Kim H-M, Miyaji F, Kokubo T, Nakamura T. Preparation of bioactive Ti and its alloys via simple chemical surface treatment. J Biomed Mater Res 1997;32:409}17. [33] Li P, Bakker D, van Blitterswijk CA. The bone-bonding polymer Polyactive 80/20 induces hydroxycarbonate apatite formation in vitro. J Biomed Mater Res 1997;34:79}86. [34] Wen HB, de Wijn JR, Cui FZ, de Groot K. Preparation of calcium phosphate coatings on titanium implant materials by simple chemistry. J Biomed Mater Res 1998;41:227}36. [35] Matsuoka H. Osteoclastic resorption of bone-like apatite formed on a plastic disk as an in vitro assay system. J Biomed Mater Res 1998;42:278}85. [36] Rhee SH, Tanaka J. Hydroxyapatite coating on a collagen membrane by a biomimetic method. J Am Ceram Soc 1998; 81:3029}31. [37] Kim HM, Kishimoto K, Miyaji F, et al. Composition and structure of the apatite formed on PET substrates in SBF modi"ed with various ionic activity products. J Biomed Mater Res 1999; 46(2):228}35.
Thin "lm of low-crystalline calcium phosphate apatite formed at low temperature夽 Hyun-Man Kim *, Yoonji Kim , Su-Jin Park , Christian Rey, HyunMi Lee , Melvin J. Glimcher, Jea Seung Ko Laboratory of Hard Tissue Engineering, Department of Oral Anatomy and Dental Research Institute, College of Dentistry, Seoul National University, 28-22, YeonKun-Dong, ChongRo-Ku, Seoul 110-749, South Korea Laboratoire de Physico-Chimie des Solides, Institut Polytechnique de Toulouse, C.N.R.S. 38, rue des 36 Ponts, Toulouse 31400, France Laboratory for the Study of Skeletal Disease and Rehabilitation and Department of Orthopedic Surgery, Children's Hospital and Harvard Medical School, 300 Longwood Ave., Boston, MA 02115, USA Received 24 September 1999; accepted 3 December 1999
Abstract Surface modi"cation of biomaterials to improve biocompatibility without changing their bulk properties is desired for many clinical applications and has become an emerging technology in biomaterial research and industry. In the present study, a simple method of coating the solid surfaces of metals, organic tissue matrices, glasses, inorganic ceramics as well as organic polymers with a thin "lm of low-crystalline apatite crystals (LCA) was developed. Acidic solution containing calcium and phosphate ions was neutralized with alkaline solution to form calcium phosphate precipitates at low temperature. Precipitates of solid calcium phosphate particles were, then, removed by "ltration. Concentration of free ions in the "ltered ion solution which were not involved in the formation of calcium phosphate precipitate was high enough to induce the heterogeneous nucleation on the solid surfaces at low temperature. Thin layers of calcium phosphate crystals were formed on the surfaces of metals, glasses, inorganic ceramics, organic polymers including hydrophobic ones, and biological tissue matrices with this solution. The thin layer of crystals consisted of poorly crystalline calcium phosphate apatite crystals which contain high amount of labile ions like bone crystals and did not dissolve in the physiologic solutions. Various cells attached to this crystal layer and proliferated well. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Low-crystalline apatite; Thin "lm; Biomaterial; Low temperature; Cell culture
1. Introduction Calcium phosphate (Ca-P) apatite has been well known to have good properties as a biomaterial for bone repair, augmentation, substitution, and surface coating [1}3]. Coating the surfaces of dental and orthopedic materials with biocompatible calcium phosphate apatite can elicit favorable biological and chemical responses on the surfaces and this can enable us to mimic the reactions occurring in the natural calci"ed tissues without losing bulk properties of materials such as durability and inertness.
夽 Part of this paper was presented at the sixth International Conference of the Chemistry and Biology of Mineralized Tissues, Vittel, France, 1}6 November 1998. * Corresponding author.
Calcium phosphate apatite is the only type of calcium phosphate crystal in calci"ed tissues such as bones, teeth, calci"ed cartilage, and cultured matrix produced by osteoblasts [4}8]. It has biocompatibility with most cell types such as osteoblasts, osteoclasts, "broblasts, and periodontal ligament cells found in the calci"ed tissues [9}13] and has osteoconductivity allowing the formation of bone on its surface by attachment, migration, proliferation, and di!erentiation of bone-forming cells [14}16]. All apatites, however, are not similar in their crystallographic properties. Pure hydroxy apatite crystals consisting of calcium, phosphate, and hydroxyl ions are stoichiometric crystals of rods with high crystallinity [17], while biocrystals isolated from bone or calci"ed cartilage are non-stoichiometric apatites of low crystallinity [6}8]. Biocrystals are small thin plates [6}8] in shape of extensive speci"c surface that makes them
0142-9612/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 9 9 ) 0 0 2 6 5 - 3
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metabolically active with high surface reactivity [18]. Their surfaces are assumed to be covered with non-apatitic highly reactive labile ions of CO\, PO\, HPO\, where organic constituents or cells can be attached to form a cell}material interface [19,20]. These properties are not expected from highly crystalline apatite crystals widely used clinically so far, but only from bone-like low-crystalline apatite crystals (LCAs). Apatite coatings formed at high temperature by plasma spraying have been shown to be heterogeneous and to contain large oxy}hydroxy apatite crystals [21] as well as a high proportion of amorphous phases [22]. Thus, there is a demand to develop a method to form an apatite layer with properties similar to those of bone crystals on solid surfaces such as metals, polymers, and ceramics to enhance bioreactivity as well as biocompatibility when engineering implant materials. In the present study, a simple method to get a thin coat of LCAs was developed. Apatite crystals were induced to form and grow directly on solid surfaces including the surfaces of low interfacial energy by increasing the solution supersaturation in the solution. The crystallographic analysis of the apatite crystals in the thin coat showed a close similarity to that of bone crystals in their crystal habit, crystallinity, and the amount of labile domains of surface ions.
2. Materials and methods 2.1. Preparation of thin xlm of apatite In the classical Ostwald's nucleation theory, the free energy for nucleation depends on the supersaturation of solution (S), the net interfacial energy for nucleation (p), the temperature (¹), and the particle surface area (A): *G"!R¹ ln S#pA. This nucleation theory indicates that increasing the solution supersaturation S and reducing the net interfacial energy p can induce the heterogeneous nucleation. In the present study, the solution supersaturation S was greatly increased to reduce the free energy for nucleation *G. This increase was high enough to induce heterogeneous nucleation even on the solid surface of low surface energy without any treatment to decrease the net interfacial energy p. Homogeneous precipitation occurring in highly supersaturated solutions was prevented by using a lowtemperature system. Following this strategy, highly supersaturated stable calcium and phosphate ion solution was prepared at low temperature. Synthetic calcium phosphate apatite crystals [23] were dissolved in 0.2 N HCl (1 mg/ml). 1 ml of this acidic ion solution was mixed with 1.35 ml of 0.2 M tris[hydroxymethyl]amino methane (Tris) solution to form Ca-P precipitate at 43C. Then, Ca-P precipitate was
removed by "ltration (pore size: 0.22 lM) to get a metastable calcium and phosphate ion solution (pH 7.3). Materials to be coated were left in this Ca-P ion solution at 83C for 24 h to induce the nucleation of Ca-P on the surfaces, then the temperature was maintained or increased (up to 603C) to form a thin "lm coat of LCAs by the growth of apatite crystals. Thicker thin "lm was obtained by increasing the time of growth phase. This method was used to coat the surfaces of the following materials; metals, glasses, inorganic ceramics, organic polymers including hydrophobic organic polymers such as poly lactic-gluconic acid co-polymer sponge (PLGA), polystyrene, polypropylene, silicone, polytetra#uorethylene, and organic biological tissue matrix like decalci"ed membranes of crab (consisting of chitin), collagens, and "bers of silk. To exclude the possibility of gravitational precipitates of amorphous calcium phosphates (ACPs) formed by homogeneous nucleation in the solution as the source of crystals on the surfaces, the surfaces of materials such as disks of titanium or polypropylene were positioned vertically and the crystals formed on the lateral surfaces were examined. 2.2. Analysis of the thin xlm of apatites 2.2.1. Electron microscopic analysis The phase and habit of Ca-P crystals were examined in the thin "lm formed on carbon-coated formvarreinforced grids using transmission electron microscopy (TEM) (1200 IIX, JEOL, Japan), or on a solid surface using scanning electron microscopy (SEM) (840A, JEOL, Japan) after sputter-coating with gold}palladium. The electron di!raction patterns were also obtained. 2.2.2. X-ray diwraction analysis The long-range orders of crystals which were scraped o! from the surface of culture dishes were determined by powder X-ray di!raction [24] using di!ractometer (MXP 18, Mac Science, Japan) at 100 mA and 50 kV. Bone crystals isolated from bovine femur [7] were also analyzed for comparison. 2.2.3. Fourier-transformed infrared spectroscopy (FTIR) analysis The FTIR spectra of crystals were recorded on a Perkin Elmer Fourier transform spectrometer 1700, after embedding the crystals removed from the culture dishes and bone crystals isolated from bovine femur [7] in KBr pellets. Resolution-enhanced deconvolution of the FTIR spectra was performed with constructor software. The sensitivity coe$cients and the bandwidths are reported in the "gure legends. Integral intensities of deconvoluted spectra in the l4 PO\ were calculated [25].
H.-M. Kim et al. / Biomaterials 21 (2000) 1129}1134
2.2.4. Calcium and phosphate analysis The concentration of calcium was determined by atomic absorption spectroscopy [26] (Perkin Elmer, USA). Concentration of phosphate was determined by a colorimetric method [27]. 2.3. Culture of cells L929 "broblasts (ATCC NCTC clone 929) and MC3T3 osteoblasts were cultured on thin "lms of apatite formed on various substrates such as culture plates (Corning, USA), without any other treatment of the surfaces. Cells were cultured in CO incubator supplied with 95% air and 5% CO at pH 7.4 in a-MEM supplemented with 10% fetal bovine serum (FBS). Attachment and proliferation of cells were examined directly under phase contrast microscope and/or SEM.
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3. Results With this simple method, thin "lm layers of LCAs were formed on the surfaces of metals, glasses, inorganic ceramics, organic polymers including hydrophobic organic polymers such as PLGA, polystylene, polypropylene, silicone, polytetra#uorethylene, and organic biological tissue matrix like decalci"ed membranes of crab (consisting of chitin), collagens, and "bers of silk (Fig. 1). The "lms of LCAs formed on both hydrophilic and hydrophobic surfaces were not disintegrated or detached from the surface when immersed in the physiological solution for over 1 month. The "rst Ca-P formed on the surface comprised particles of nano-size, which was studied by examining the "rst precipitation formed on the carbon-coated formvar "lm using TEM (Fig. 2a). Formation of "lms of LCAs on
Fig. 1. Sequential observation of the formation of thin layer of LCAs on the surface of the culture dishes. (a) Non-discrete particles are scattered in the initial stage. (b}c) Then, discrete crystals form and grow on the surface, and proliferate and interconnect to cover all surfaces. (d) Apatite coating of LCAs is covering porous PLGA co-polymer sponge. Scanning electron microscope (SEM), scale bar: (a}c) 1 lm, (d) 10 lm.
Fig. 2. TEM observation of the formation of thin layer of LCAs. Non-stained examination. (a) The "rst Ca-P depositions on the carbon-coated formvar "lm under TEM do not give any di!raction maxima of Ca-P crystals (inset), which indicates ACP as a seeding substance of heterogeneous nucleation on the surface. Scale bar: 500 nm. Electron di!raction: 100 kV, 80 cm. (b) Cross-sectional view of the thin "lm shows closely packed nano-sized crystals of thin plates in the thin "lm. Scale bar: 100 nm.
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the vertically positioned solid surface or the underneath surface excluded the possibility of gravitational precipitation of particles from the ion solution as the source of calcium phosphate particles on the surface. Particles of ACPs on the surface were transformed to apatite, then a thin "lm layer was formed by multiplication of more Table 1 XRD of LCAs in thin "lm coating compared to bone crystals hkl
2h
111 002 102 211 112 300
22.70 25.98 27.84 31.72 32.16 33.14
(22.82) (25.86) (28.16) (31.84) (32.65) (33.97)
d (As )
FWHM
3.91 3.43 3.20 2.82 2.78 2.70
0.58 0.44 0.32 0.38 0.44 0.20
(3.89) (3.44) (3.17) (2.81) (2.74) (2.63)
(0.53) (0.33) (0.39) (0.86) (0.42) (0.45)
Values in parentheses are data of bone crystals isolated from bovine femur. FWHM: full-width at half-maximum value.
crystals of thin plates through secondary crystallization [28] around pre-formed apatite crystals. By varying the time of growth, "lms of di!erent thicknesses from a few nm up to about a few lm were formed and this was determined by examining the cross section of thin "lm using TEM (Fig. 2b). XRD showed that the thin "lm of LCA consisted of poorly crystalline calcium phosphate apatite like bone crystals (Table 1). FTIR analysis (Fig. 3a) also indicated their poor crystallinity. Resolution enhancement [25] of l2 CO\ (Fig. 3b) and l4 PO\ (Fig. 3c) showed peaks of non-apatitic labile domains of PO\, HPO\, and CO\, and type B CO\ substituting PO\ in addition to apatitic ions which were also found in bone crystals (Table 2). Fibroblasts attached and proliferated well on the thin "lm of LCAs formed on polyglactin 910 (vicryl) "bers (Fig. 4a). Osteoblasts also adhered well to the substrate of Table 2 The relative intensities in the l4 PO\ domains of LCAs in thin "lm compared to bone crystals IR band (cm\) Domains [31] LCAs Bone crystals
Fig. 3. FTIR spectrum of thin layer of LCAs. Crystals were obtained by scraping the surface of polypropylene o! thin layer of LCAs. (a) l4 PO\, l3 PO\, l2 CO\, and l4 CO\ are seen. (b) Deconvoluted FTIR spectrum in the l2 CO\ domain shows that carbonate ions are located in anionic sites of the apatitic structure indicating type B carbonate}apatite (CO\ substituted for PO\ groups). In addition, a la bile non-apatitic CO\ environment is also present. Deconvolution parameters: bandwidth 10 cm\; sensitivity coe$cient 2.25. (c) Deconvoluted FTIR spectrum in the l4 PO\ domain shows that labile nonapatitic environment is present in addition to apatitic PO\ envi ronment. Deconvolution parameters: bandwidth 18 cm\; sensitivity coe$cient 2.25.
534 Labile HPO 8.17 7.74
550 HPO 2.50 1.65
560 575 600 617 PO\ PO\ PO\ Labile PO 8.89 9.27 8.66 3.24 9.34 9.16 9.28 3.1
Fig. 4. Cells cultured on thin "lm of LCAs. Scale bar: 10 lm. (a) SEM of MC3T3 osteoblasts cultured on thin "lm substrate of LCAs formed on PGLA for 2 d. (b) SEM of L929 "broblasts cultured on thin "lm of apatite formed on polygalactin 910 (vicryl) "bers for 3 d. They attach and proliferate well on the "lm.
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"lm formed on PLGA (Fig. 4b). Cellular behavior on the substrate of the "lm could be clearly observed under phase contrast microscope due to the transparency of the "lm.
4. Discussion Application of low temperature was considered to be the only condition in this system for obtaining the thin "lm of LCAs. Less calcium and phosphate ions might be involved in the formation of calcium phosphate precipitate in the neutralized ion solution due to the low temperature. Then free ions in the "ltered solution high enough to overcome the energy barrier for heterogeneous nucleation on the surface were considered to be provided to the surfaces. The low temperature again might preclude a homogeneous precipitation within this solution. Direct observation of the formation of a thin layer of LCAs under TEM has con"rmed that this system follows the general sequence for the heterogeneous nucleation and growth of crystals from a supersaturated solution [28,29]. Nanosized ACPs were formed on the solid surface as the "rst phase of Ca-P. This can be ascribed to their low surface energy which requires less net energy for their de novo formation [28,29]. The transformation of ACPs into LCAs was followed. Then, a thin layered coat was formed by multiplication and growth of LCAs. However, further study is required to explain the exact mechanism of the formation of the thin "lm in this system. Crystallographic properties of the crystals in this "lm, such as thin plate shape, low crystallinity, and high amount of labile ions indicate that they are similar to the crystals of bones [7]. The shape of the thin plates of the crystals grown by this method can provide a high speci"c area [18], which in turn enables them to be potentially highly interactive in cellular or organic environment. In addition, crystals were rich in non-apatitic labile domain of PO\, HPO\, and CO\ abundant in bone crystals [30,31]. It was suggested that these labile ions cover the surfaces of LCA and be highly reactive to organic constituents to form an organic}inorganic interface [19,20]. Technically, the present method has an important advantage over the previously reported methods in accessibility of coating site. Ions of calcium and phosphate can reach into the hidden surfaces inside the bulk of materials because they are provided in a solution. The most probable candidates for the application of this method are the porous polymers like polyglycolic acid, polylactic acid, PLGA co-polymers which have a great potential for drug and cell delivery as well as giving a sca!old for bone ingrowth. In addition, application of low temperature in this method will be advantageous in reducing the chance of losing the activity of drugs for the drug delivery system. This technology can also be applied to make the
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non-biocompatibile materials with high mechanical properties to be more biocompatible. There has been much e!ort to form highly bioactive bone-like Ca-P crystals on various substrates with a simulated body #uid or some low supersaturated Ca-P solution [32}37]. However, in such a low supersaturated concentration, inducing the crystal formation was limited only to speci"c solid surfaces like the strictly manipulated one by chemical treatment and has not been possible on hydrophobic surfaces. In addition, those coating procedures needed a relatively long time and high temperature which might limit the control of the crystallographic nature of Ca-P apatite crystals. In the present study, a low-temperature system was used to delay the homogeneous nucleation of solid Ca-P in the ionic solution thereby leaving the concentration of free ions high (up to [Ca>];[PO\]"20 mM). This overcame the energy barrier in inducing heterogeneous nucleation on the surfaces such as hydrophobic materials whose surface enery is very low. In summary, we developed a method to form a thin apatite "lm on various materials at a low temperature. Their crystallographic property is similar to that of the bone crystals and cells attached and proliferated well on this "lm. This indicates that this apatite crystal "lm can be used as a coating material over various substrates and can modify the biocompatibility and bioreactivity of substrates without losing their own bulk properties. Acknowledgements The authors thank Dr. Izumi Asahina (Tokyo Medical and Dental University, Tokyo, Japan) for providing them with porous PLGA co-polymers sponge, Ms. SeongHyun Park and Ms. Su-Jin Kim for their assistance with TEM and SEM, Mr. Chong-Hee Cho for XRD, and Ms. Sa-Im Park for assistance with the preparation of this manuscript. This work was partly supported by Grant for Basic Medical Research from Korea Research Foundation (H.-M. Kim). References [1] Cornell CN, Lane JM. Current understanding of osteoconduction in bone regeneration. Clin Orthop 1998;355(Suppl):S267}73. [2] Lace"eld WR. Current status of ceramic coatings for dental implants. Implant Dent 1998;7:315}22. [3] Soballe K, Overgaard S, Hansen ES, Brokstedt-Rasmussen H, Lind M, Bunger C. A review of ceramic coatings for implant "xation. J Long Term E!ects Med Implants 1999;9:131}51. [4] LeGeros RZ. Calcium phosphates in enamel, dentin and bone. In: Calcium phosphates in oral biology and medicine. Basel: Karger, 1991. p. 108}29. [5] Rey C, Kim H-M, Gerstenfeld L, Glimcher MJ. Structural, chemical characteristics, and maturation of the calcium-phosphate crystals formed during the calci"cation of the organic matrix synthesized by chicken osteoblasts in cell culture. J Bone Miner Res 1995;10:1577}88.
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[6] Kim H-M, Rey C, Glimcher MJ. Isolation of calcium-phosphate crystals of mature bovine bone by reaction with hydrazine at low temperature. In: Brown PW, Constantz B, editors. Hydroxyapatite and related materials. Boca Raton: CRC Press, 1994. p. 331}8. [7] Kim H-M, Rey C, Glimcher MJ. Isolation of calcium-phosphate crystals of bone by non-aqueous methods at low temperature. J Bone Miner Res 1995;10:1589}601. [8] Kim H-M, Rey C, Glimcher MJ. X-ray di!raction, electron microscopy, and Fourier transform infrared spectroscopy of apatite crystals isolated from chicken and bovine calci"ed cartilage. Calcif Tissue Int 1996;59:58}63. [9] Gregorie M, Orly I. Menanteau. The in#uence of calcium phosphate biomaterials on human bone cell activities: an in vitro approach. J Biomed Mater Res 1990;24:163}77. [10] Bagambisa FB, Joos U, Schilli W. The interaction of osteogenic cells with hydroxylapatite implant materials in vitro and in vivo. Int Oral Maxillofac Implants 1990;5:217}26. [11] Cheung HS, Haak MH. Growth of osteoblasts on porous calcium phosphate ceramic: an in vitro model for biocompatibility study. Biomaterials 1988;10:63}7. [12] Davies JE, Baldan N. Scanning electron microscopy of the bone}bioactive implant interface. J Biomed Mater Res 1997; 15:429}40. [13] Nery E, LeGeros RZ, Daculsi G. Tissue response to biphasic calcium phosphate ceramic with di!erent ratios of biphasic calcium phosphate ceramic with di!erent ratios of HA/TCP in periodontal osseous defects. J Periodont 1992;63:729}35. [14] Galgut PN, Waite IM, Tinkler SMB. Histological investigation of the tissue response to hydroxyapatite used as an implant material in periodontal treatment. Clin Mater 1990;6:105}21. [15] Amler MH. Osteogenic potential of non-vital tissues and synthetic implant materials. J Periodont 1988;58:759}61. [16] Kuhn-Spearing L, Rey C, Kim H-M, Glimcher MJ. Carbonate apatite nanocrystals from bone. Proceedings of the Annual Meeting of the Minerals, Metals & Matrials Society, Anaheim, California, USA, 1996. [17] Elliott JC. Hydroxyapatite and nonstoichiometric apatites. In: Structure and chemistry of the apatites and other calcium orthophosphates, studies in inorganic chemistry, vol. 18. Amsterdam: Elsevier, 1994. p. 111}90. [18] Posner AS, Betts F, Blumenthal NC. In: Simons DJ, Kunin AS, editors. Skeletal research: an experimental approach. New York: Academic Press, 1979. p. 167}92. [19] Lees SL, Glonek T, Glimcher MJ. P nuclear magnetic resonance spectroscopic evidence for ternary complex formation of fetal phosphoprotein with calcium and inorganic orthophosphate ions. Calcif Tissue Int 1983;35:815}8. [20] Rey C, Glimcher MJ. In: Slavkin H, Price P, editors. Chemistry and biology of mineralized tissues. North Holland: Elsevier, 1992. p. 5}18. [21] Ellies LG, Nelson DG, Featherstone JD. Crystallographic changes in calcium phosphates during plasma-spraying. Biomaterials 1992;13:313}6.
[22] Weng J, Weng J, Liu X, et al. Further studies on the plasmasprayed amorphous phase in hydroxyapatite coatings and its deamorphization. Biomaterials 1993;14:578}82. [23] Rey C, Collins B, Goehl T, Dickson IR, Glimcher MJ. The carbonate environment in bone mineral: a resolution-enhanced Fourier infrared spectroscopy study. Calcif Tissue Int 1989; 45:157}64. [24] Klug HP, Alexander LE. X-ray di!raction procedures for polycrystalline and amorphous materials, 2nd ed. New York: Wiley (Interscience), 1974. [25] Rey C, Shimizu M, Collins B, Glimcher MJ. Resolution-enhanced Fourier transform infrared spectroscopy study of the environment of phosphate ions in the early deposits of a solid phase of calcium-phosphate in bone and enamel, and their evolution with age. I: investigations in l4 PO domains. Calcif Tissue Int 1990; 46:384}94. [26] Willis JB. The determination of metals in blood serum by atomic absorption spectroscopy. I: calcium. Spectrochim Acta 1960; 16:259}72. [27] Bartlett GD. Phosphorus assay in column chromatography. J Biol Chem 1959;234:466}8. [28] Eanes ED. In: Bonucci E, editor. Calci"cation in biological systems. Boca Raton: CRC Press, 1992. p. 1}17. [29] Eanes ED, Termine JD, Nylen MU. An electron microscopic study of the formation of amorphous calcium phosphate and its transformation to crystalline apatite. Calcif Tissue Res 1973; 12:143}58. [30] Rey C, Renugopalakrishnan B, Collins B, Glimcher MJ. Fourier transform infrared spectroscopic study of the carbonate ions in bone mineral during aging. Calcif Tissue Int 1991;49:251}8. [31] Rey C, Kim H-M, Glimcher MJ. Maturation of poorly crystalline synthetic and biological apatites. In: Brown P, Constanz B, editors. Hydroxyapatite and related materials. Boca Raton: CRC Press, 1994. p. 181}8. [32] Kim H-M, Miyaji F, Kokubo T, Nakamura T. Preparation of bioactive Ti and its alloys via simple chemical surface treatment. J Biomed Mater Res 1997;32:409}17. [33] Li P, Bakker D, van Blitterswijk CA. The bone-bonding polymer Polyactive 80/20 induces hydroxycarbonate apatite formation in vitro. J Biomed Mater Res 1997;34:79}86. [34] Wen HB, de Wijn JR, Cui FZ, de Groot K. Preparation of calcium phosphate coatings on titanium implant materials by simple chemistry. J Biomed Mater Res 1998;41:227}36. [35] Matsuoka H. Osteoclastic resorption of bone-like apatite formed on a plastic disk as an in vitro assay system. J Biomed Mater Res 1998;42:278}85. [36] Rhee SH, Tanaka J. Hydroxyapatite coating on a collagen membrane by a biomimetic method. J Am Ceram Soc 1998; 81:3029}31. [37] Kim HM, Kishimoto K, Miyaji F, et al. Composition and structure of the apatite formed on PET substrates in SBF modi"ed with various ionic activity products. J Biomed Mater Res 1999; 46(2):228}35.