Materials Science and Engineering B 169 (2010) 151158

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Materials Science and Engineering B

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Composite biocompatible hydroxyapatitesilk broin coatings for medical implants obtained by Matrix Assisted Pulsed Laser Evaporation
F.M. Miroiu a, , G. Socol a , A. Visan a , N. Stefan a , D. Craciun a , V. Craciun a , G. Dorcioman a , I.N. Mihailescu a , L.E. Sima b , S.M. Petrescu b , A. Andronie c , I. Stamatin c , S. Moga d , C. Ducu d
a

National Institute for Lasers, Plasma, and Radiation Physics, 409 Atomistilor Street, RO-77125, MG-36, Magurele-Ilfov, Romania Institute of Biochemistry, Romanian Academy, 296 Splaiul Independentei, 060031 Bucharest, Romania 3Nano-SAE Alternative Energy Sources-University of Bucharest, Faculty of Physics, 409 Atomistilor Street, RO-77125, Magurele-Ilfov, Romania d University of Pitesti, Targul din Vale Str, no. 1, 110040 Pitesti, Romania
b c

a r t i c l e

i n f o

a b s t r a c t
The aim of this study was to obtain biomimetic inorganicorganic thin lms as coatings for metallic medical implants. These contain hydroxyapatite, the inorganic component of the bony tissues, and a natural biopolymer silk broin added in view to induce the surface functionalization. Hydroxyapatite (HA), silk broin (FIB) and composite HAFIB lms were obtained by Matrix Assisted Pulsed Laser Evaporation (MAPLE) in order to compare their physical and biological performances as coatings on metallic prostheses. We used an excimer laser source (KrF*, = 248 nm, = 25 ns) operated at 10 Hz repetition rate. Coatings were deposited on quartz, Si and Ti substrates and then subjected to physical (FTIR, XRD, AFM, SEM) analyses, correlated with the results of the cytocompatibility in vitro tests. The hybrid lms were synthesized from frozen targets of aqueous suspensions with 3:2 or 3:4 weight ratio of HA:FIB. An appropriate stoichiometric and functional transfer was obtained for 0.40.5 J/cm2 laser uence. FTIR spectra of FIB and HAFIB lms exhibited distinctive absorption maxima, in specic positions of FIB random coil form: 1540 cm1 amide II, 1654 cm1 amide I, 1243 cm1 amide III, while the peak from 1027 cm1 appeared only for HA and composite lms. Osteosarcoma SaOs2 cells cultured 72 h on FIB and HAFIB lms showed increased viability, good spreading and normal cell morphology. The well-elongated, attened cells are a sign of an appropriate interaction with the MAPLE FIB and composite HAFIB coatings. 2009 Elsevier B.V. All rights reserved.

Article history: Received 10 June 2009 Received in revised form 5 October 2009 Accepted 7 October 2009 Keywords: Biomimetic Thin lms Hydroxyapatite Fibroin Implants Matrix Assisted Pulsed Laser Evaporation (MAPLE)

1. Introduction It is already known that a composite hydroxyapatitebroin material should meet bioafnity, enhanced osteoinductivity, adequate mechanical strength and exibility for use as implant material. Hydroxyapatite (HA), with the chemical formula (Ca10 (OH)2 (PO4 )6 ), constitutes the majority mineral part of natural bones and teeth, providing innate excellent biocompatibility, osteoconductivity and bioactivity [1,2]. Therefore, it is largely studied and used in different applications like bone llers, maxillofacial reconstructions, dental applications or biomedical ceramic coatings [36]. On the other hand, silk broin (FIB), the main constituent of the natural silk, is a natural biopolymer, a protein spun in bers by some lepidoptera larvae, as silkworms or spiders [7]. It shows three different conformations of peptide chains and a crystalline dimorphism: amorphous random coil, crystalline -sheet and crystalline

Corresponding author. Tel.: +40 21 4574491; fax: +40 21 4574243. E-mail address: marimona.miroiu@inpr.ro (F.M. Miroiu). 0921-5107/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mseb.2009.10.004

-helix forms [8]. The silk broin is known and proved to be biocompatible, biodegradable, having unusual high tensile strength and elasticity. It has applications in scaffolds aimed to manipulate the osseous growth in the desired forms, sutures, articial skin, articial tendons, and substrate for cell culture, bone tissue engineering, coatings [913]. Silk broinhydroxyapatite is a quite new composite particularly studied as 3D scaffolds, nanocomposites or thick coatings [1418]. In our studies, silk broin is added to the hydroxyapatite in view to induce the surface functionalization of the coating and to improve the mechanical properties (elasticity). The HApolymer composites mimic the natural bone, formed by the inorganic phase (nanometric biological HA) and organic compounds (mainly collagen). The intimate synergy between inorganic and organic phase provide the hard tissues with the providential features as high fracture toughness, exibility and strength. The nanometric dimension of the inorganic element, of high specic surface, similar to the one in the bony apatite is important from the point of view of the mechanical and biological properties [19,20]. The aim of our work was to obtain biomimetic ceramicpolymer composite coatings for metallic medical implants. We have cho-

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Table 1 Selected samples of hydroxyapatite, broin and composite thin lms deposited by MAPLE. Code FIB5 HA5 FIBNaOH FIBNaCl HA3FIB2 HA3FIB4 C Sample type Fibroin (5 wt% in suspension) Hydroxyapatite (5 wt%) Fibroin (5% suspension) with NaOH added Fibroin (5% suspension) with NaCl added Hydroxyapatite (3 wt%)broin (2 wt%) Hydroxyapatite (3 wt%)broin (4 wt%) Controlborosilicate glass

sen to synthesize the hybrid hydroxyapatitepolymer coatings and simple hydroxyapatite and simple polymer coatings, respectively on titanium substrates (as basis of the orthopedical implants) by a novel fabrication method for this composite, Matrix Assisted Pulsed Laser Deposition (MAPLE), as being the most appropriate for synthesis of large organic molecule thin lms [21]. 2. Experimental procedure: methods, materials and analyses 2.1. Matrix Assisted Pulsed Laser Evaporation (MAPLE) method MAPLE is an advanced laser technique, based on a cryogenic approach, which has been developed since 1998, to produce a pro-

tected accurate transfer of organic and polymeric materials in form of thin lms [22,23]. The material subjected to the laser irradiation which is called target is a frozen composite. It is obtained by freezing following the dissolution of the active biomaterial (up to 5 wt%) in an appropriate volatile solvent, highly absorbing the laser wavelength, but not reacting under laser exposure. The laser pulses intensities are adjusted to avoid the biomolecules damage. The structural and functional delity is preserved inducing a non-direct lasermaterial interaction in a vacuum chamber. Due to the low concentration of biomolecules in the frozen target, the laser photons preponderantly interact with the matrix (the solvent), which is vaporized. The complex biomolecules are released undamaged and, by means of the collisions with the other molecules, moved toward the substrate, where they form a uniform thin lm. In the same time, the volatile solvent is pumped away by the vacuum system. During the deposition process the target is kept at low temperature by a cooler. As its precursor, Pulsed Laser Deposition, MAPLE is a layer-bylayer growing method, which can deposit thin lms with dopants and may also be applied with masks, in order to induce a controlled distribution of the local structure parameters by the nature and type of the coating. 2.2. Materials, set-up and MAPLE experiments We used in our experiments commercial 2 m granulation polymer powder of degummed (i.e. sericin-free) Bombyx mori broin (Wuxi Allied Technologies, Inc., China), and commercial Merck hydroxyapatite powder. For MAPLE experiments we prepared different broin and hybrid solutions or suspensions. According to literature, the water-insoluble broin can be solubilised in organic substances (N-methyl morpholine N-oxide, MMNO N-dimethyl acetamide, copper-ethylendiamine, Ca(NO3 )2 /MeOH) or in saturated solutions of calcium chloride or lithium bromide, simple or with ethylic alcohol [2426]. Thus, a number of lms were grown from broin subjected rstly to a solubilisation process, and then to one of separation. In view to homogenise them, the calcium chloride and lithium bromide solutions were heated at 95 C [27] and 60 C [28] and kept at these temperatures for 8 and 4 h, respectively. Subsequently, to separate the broin from salt, the solutions were subjected to a chemical dialysis process in bidistilled water, for 4 days. In the end,

Fig. 1. FTIR spectra of (a) HA, FIB and hybrid HAFIB lms on silicon, compared to those of broin and HA powders; (b) broin thin lms (from different broin solutions) on silicon compared with the original powder.

Fig. 2. XRD spectra of the broin and HAbroin thin lms on silicon substrates deposited by MAPLE.

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Fig. 3. AFM images of broin and HAbroin MAPLE thin lms on quartz substrates (up: FIB, HA3FIB4, HA3FIB2 samples, 15 m 15 m; down: HA3FIB4, HA3FIB2 samples, 60 m 60 m).

the dialysis membranes kept only the broin molecules larger than 10,000 Da, i.e. the aqueous broin solutions. Alternatively, a quantity of salty-broin solutions were separated by centrifugation with a Universal 320R Hettich Zentrifugen, at 4 C and 6000 rpm, for 30 min. Supplementary, the lithium bromide solution was centrifuged 20 min at 9000 rpm, obtaining a pure solubilised broin. The calcium chloride solution was additionally centrifuged for 30 min at 9000 rpm, however without being separated. The third method to get the polymer (broin) frozen target for MAPLE technique was to obtain broin aqueous suspensions, of 25 broin wt%. The very homogenous aspect of the suspension was kept by using a mechanical agitator. For the hybrid HAbroin lms, we used polymer solutions, adding the hydroxyapatite up to a xed HA/polymer weight ratio, 3/2 or 3/4. The solutions were mechanically stirred, in view of the homogenisation. In order to reach the optimum, neutral pH 7.4, preferred by cells, in some samples a few drops of NaOH or NaCl were added, neutralizing the slightly acid broin suspensions. By comparison, HA coatings were deposited by MAPLE from broin (5 wt%) aqueous suspension, using the same experimental parameters as for the broin-containing coatings. The codes of the samples are mentioned in the Table 1. In all cases, according to MAPLE technology, the solid targets were obtained by solutions/suspensions frozen in liquid nitrogen and their maintenance, for the period of the deposition process, under the melting point, by means of a cooler device, fed also with liquid nitrogen. An excimer laser source KrF*, = 248 nm, FWHM = 25 ns, with 1015 Hz pulses repetition rate was used in our MAPLE experi-

ments. The laser uence incident on the sample was set between 0.4 and 0.5 J/cm2 and the pressure inside the vacuum chamber kept at 7 Pa. The deposition substrates medical grade etched titanium, quartz or double polished Si (1 1 1) were placed at 4 cm distance, parallel to the target and maintained at 30 C. A series of 20,000 or 50,000 laser pulses was applied for each deposited lm. The coatings have been estimated to have about 900 nm thickness for 50,000 subsequent pulses, as resulted from the UVvis transmission spectra by measurements of the interference fringes. 2.3. Analyses The stoichiometry of the polymer and of polymerhydroxyapatite coatings was assessed by Fourier transform infrared spectroscopy and X-ray diffraction analyses, and the morphology by atomic force microscopy and scanning electron microscopy investigations, respectively. Following the physico-chemical characterization, all coatings were subjected to in vitro biological characterizations, in order to evaluate both the optimal behaviour and their most favourable deposition regime. Fourier transform infrared spectroscopy (FTIR) analyses were obtained by means of a FTIR Shimadzu 8400S working in 7800350 cm1 in transmission mode (samples on double-side polished Si). Atomic force microscopy (AFM) records were performed by an Integrated Platform SPMNTegra model Prima microscope, in semicontact mode and the scanning electron microscopy (SEM) images were taken with a SEM JEOL 6400 apparatus. X-ray diffraction (XRD) spectra were recorded with a Rigaku Ultima IV diffractometer, using Cu K radiation. The acquisition conditions were the following: 2 conguration and parallel beam optics, high voltage applied on the X-ray tube U = 40 kV, current intensity I = 30 mA, angular 2 range of 860 , (2 ) = 0.05 , xed angle of incidence = 1 . MTS assay is a colorimetric method to determine cytotoxicity or the number of viable proliferating cells. In vitro tests are performed by adding a small amount of reagent directly to cul-

Table 2 Roughness values of MAPLE broin and HAbroin coatings deposited on quartz, estimated by AFM. Sample/roughness, 15 m 15 m Peak-to-peak (nm) Average (nm) Fibroin 106 26 HA3FIB4 220 95 HA3FIB2 397 171

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Fig. 4. SEM micrographs of hydroxyapatite, broin and HAbroin MAPLE thin lms on Si (1 1 1) double polished substrates.

ture wells, incubating for 14 h and then recording absorbance at 490 nm. The reductions of the MTS salt takes place, in the presence of phenazine methosulfate, only when reductase enzymes are active. Therefore the quantity of formazan measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture. One hundred thousand osteosarcoma SaOs2 cells were seeded on each sample and cultured for 72 h on the six types of coated titanium surfaces and on the control borosilicate cover glass control, respectively. MTS assay was then performed in view to compare the viability of cells grown on the samples surface.

Results of the 490 nm absorbance measurements are represented in the diagram hereunder. Samples were tested in duplicate and for each duplicate sample the reaction was quantied in triplicate. The rst in vitro uorescence microscopy experiments were performed with the ER-Tracker dye. Identical samples to those tested before were incubated in a 1:1000 dye dilution in complete media at 37 C for 30 min, to allow its uptake and distribution into the endoplasmic reticulum. This experimental approach allows the live analysis of the cell morphology as it does not require cells xation and the uorescently marked organelle forms a network

Fig. 5. MTS assay on SaOs2 cells grown on HA/Ti, FIB/Ti and HAFIB/Ti MAPLE deposited structures. C is the standard control borosilicate cover glass.

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distributed in almost all the cytoplasm volume. Titanium and HAPLD samples were tested using primary human osteoblasts cells cultured as described before [29]. Then SaOs2 cells were analysed for adhesion molecules expression and localization by immunouorescence. First, cells were cultured on the biomaterial surface and on the standard control sample of borosilicate cover glass, xed and permeabilized according to a previously described protocol [30,31].

3. Results and discussion 3.1. Physico-chemical characterization For the broin powder (Fig. 1a), the following absorption FTIR maxima, specic to the broin beta-form, are evidenced: 1265 (1260) cm1 , 1520 cm1 , 1625 (1630) cm1 , and 1698 (1703 sau 1695) cm1 [24,3236], as well as an absorption band at

Fig. 6. Fluorescence microscopy: (A) ER-Tracker staining of SaOs2 cells grown on MAPLE deposited broin/Ti, HA/Ti and HAFIB/Ti coatings; (B) ER-Tracker staining of primary osteoblast cells grown on Ti and PLD-deposited HA coatings.

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700 cm1 (amide V) [24]. At 612 cm1 a rather small maximum of poly (l-alanine) in beta-sheet form is observed [37]. Another maximum at 1235 cm1 , which is attributed to the random coil form of broin is also present [38]. The hydroxyapatite powder spectrum (Fig. 1a) exhibits maxima at: 564 cm1 (570 cm1 ) and 601 cm1 , assigned to the asymmetric bending PO4 , 961 (954) cm1 for the symmetrical PO4 stretching vibration, a sharp maximum at 1031 cm1 of the asymmetrical PO4 stretching vibration and two shoulders at 630 cm1 (631 cm1 ) and 1081 cm1 , respectively, belonging to

PO4 group, as well as another maximum at 3566 (3562) cm1 , attributable to OH [39]. FTIR analysis provided fairly similar spectra for all broin lms, independently of the provenience solution (Fig. 1b). It is known that thin lms with low degree of crystallinity or stoichiometry give broadening, shifting or diminishing of the bands intensity. In this respect, the coatings mostly resembling to the broin powder are those of the suspension with 5 wt% broin concentration in water. This result suggests that MAPLE target preparation can be made by the easiest method, of the aqueous broin suspen-

Fig. 7. Immunouorescence microscopy: adhesion of SaOs2 cells stained for actin (red) and vinculin (green) markers; best results on HA3FIB4/Ti sample in comparison to titanium, FIB5 and HA5 samples.

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sion, without the previous salty saturated solutions and without the corresponding complicated and costly techniques to separate broin from these solutions. Hence, even if all the samples showed relatively the same positive behaviour of biocompatibility (data of MTS assay not shown herewith), the next series of experiments resumed only to samples made by simple broin suspensions and, respectively, of hybrid hydroxyapatitebroin aqueous suspensions. The FTIR spectra of the simple HA, FIB polymer and HAFIB, respectively, revealed that the lms derived from the suspensions HA3FIB2 or HA3FIB4 have maxima specic to both materials composing the hybrid (Fig. 1a). The broin lms present distinctive amides absorption maxima, in specic positions of broin random coil form, e.g. at 1540 cm1 amide II, 1654 cm1 (1660) amide I, 1243 cm1 (1240) amide III, and strong absorption band 3309 cm1 , due to NH stretching. The skeletal stretching region (1100900 cm1 ) includes a band at 1015 cm1 and another twin band at 998 and 975 cm1 , from the (gly-gly)- and (gly-ala)periodic sequences of broin [24]. As the FIB and the HA lms, respectively, the composite lms show absorption for amide (I, II, III) broin random coil at 1654, 1540, 1243 cm1 , the NH stretching vibration at 3290 (3309) cm1 and the PO4 stretching vibration 10271030 cm1 . The maximum attributable to OH, at 3566 (3562) cm1 , appears in all the coatings, but has an intensity hardly visible. The HA lm exhibited most visibly the sharp maximum around 1031 cm1 of the PO4 stretching vibration (Fig. 1a). As HA phase is known as amorphous when deposited by Pulsed Laser Deposition (PLD) at room temperature [4042], there are no HA peaks in the XRD spectrum of the composite lm. The XRD spectra of broin and HAbroin coatings (HA3FIB4) show a diffraction peak at 20.5 , specic to -sheet crystalline form of broin [32,37], over a broad hallo centred around 21.5 , specic also to the silk protein, possible to -helix form [37] (Fig. 2). The very weak emerging peak at 12 is attributed also to -helix broin [32,37]. However the data suggest a predominantly amorphous content of broin. The AFM images (Fig. 3) put in evidence morphology specic to MAPLE lms: homogeneous, but with a certain degree of roughness, which is gradually decreasing when the silk particles compose the lms (Table 2). We mention that the topography with extended active surface area is favourable to the cell growth and adhesion [43]. The SEM images of hydroxyapatite, broin and HAbroin lms on Si (1 1 1) double polished substrates reveal a topography conrming that of the AFM analysis, where the HA presence enhances the surface prole (Fig. 4). 3.2. Biological in vitro assays Cell viability does not present major variations between assayed samples. All tested samples are non-toxic during the 72 h of the test (Fig. 5). However, the samples which most resemble to the standard control sample are FIB5, HA3FIB2 and FIBNaOH. The sample generating the most reduced viability is FIBNaCl. The data were conrmed by the morphology analysis of uorescence microscopy. A uniform cell distribution is observed in Fig. 6 for FIB5 and HA3FIB2 samples. Bone cells are very well spread on the substrate, elongated and attened, indicating a good interaction with the deposited lms. A good interaction with the surface, which conrms the viability data, is also exhibited by FIBNaOH lms. On the contrary, on the HA5 and FIBNaCl samples cells adopt a heterogeneous morphology, predominantly spherical, as a sign of the lack of compatibility between their physical-chemical properties and cell adhesion. We might therefore conclude that for these MAPLE deposition parameters the pure HA lms are less favourable to cell interaction and that the droplets of NaCl solution added to

change the pH of the broin suspension are not favourable. When using PLD HA thin lms on titanium, growth of primary human osteoblasts was not impaired as shown in Fig. 6B. The inset depicts a magnication to prove the optimal dye distribution in the ER. As an intermediary behaviour, cells grown on HA3FIB4 samples show a signicant spreading on the lms surface, but their distribution is less uniform and they are fewer, compared to FIB5, FIBNaOH and HA3FIB2 samples. Bone cells attached to titanium but their proliferation was less efcient then on HA-containing coatings (Fig. 6B). In view to evaluate adhesion of osteoblasts cultured in direct contact with the broin/Ti and HAbroin/Ti surfaces, SaOs2 cells were stained for actin (red) and vinculin (green) markers and analysed by immunouorescence microscopy. The most favourable surface proved to be HA3FIB4/Ti, where actin and vinculin patterns are similar to the cells grown in standard conditions and on bare Ti (Fig. 7). FIB5 alone generates poor vinculin expression and a decrease in the total number of contact foci. Actin laments are less stretched and cells spread less uniformly. Also, no specic staining was observed on HA5 only coatings, which emit autouorescent signals. This behaviour indicates optimal cell adhesion of osteoblasts on HAbroin hybrid thin lm. 4. Conclusions The FTIR analyses provide similar spectra for all frozen targets obtained from solutions of polymers, suggesting that preparation can be done by the simplest method, an aqueous suspension of broin without prior achievement of saturated salt solutions and subsequent techniques, like complicated separation of broin of these solutions. Compared to the simple broin or HA thin lms, AFM and SEM analyses show an intermediary topography of the composite HAbroin coatings, with micronic droplets which gives an extended surface, favourable to bone cells anchorage and proliferation. FTIR and XRD analyses, both for simple polymer lms, as well as for the HApolymer hybrids, indicate a cvasistoichiometric transfer of these compounds from the solid target in the form of thin lms. The presence of the main FTIR absorption maxima, specic to the basic substances, validates that the transfer method chosen, MAPLE, is correct. The broin content in the lms is a mixture of preponderantly random coil with crystalline forms, -sheet and helix. In vitro viability tests prove appropriate bone cell behaviour for the interaction with the selected MAPLE broin and HAbroin coatings: non-toxicity, good spreading and normal cell morphology. The best performances, in terms of physico-chemical and biological properties (viability, adhesion), were proved for the composite samples of HA3FIB4 type. Before validation for further advanced in vivo tests, in view of the orthopedical applications as hybrid biomimetic coatings, supplementary in vitro studies on osteoblast expression markers will be performed. Acknowledgements This work was supported under the Contract PN2 TD287/2007 nanced by the Romanian Ministry of Education, Research and Youth. F.M. Miroiu acknowledges with thanks the kind help of Mrs. Cristina Hlevca, Eleonora Gheorghiu and Vasilica Z ah ar achescu from the National Institute for Chemical Pharmaceutical Research and Development ICCF Bucharest. We thank Dr. Karine Anselme for the kind gift of providing the SaOs2 cell line. References
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