Bioactive CaO−SiO2−PDMS Coatings on Ti6Al4V Substrates

Chem. Mater. 2005, 17, 1591-1596

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Bioactive CaO-SiO2-PDMS Coatings on Ti6Al4V Substrates Natalia Hijo´n, Miguel Manzano, Antonio J. Salinas, and Marı´a Vallet-Regı´* Departamento de Quı´mica Inorga´ nica y Bioinorga´ nica, Facultad de Farmacia, UniVersidad Complutense, 28040-Madrid, Spain ReceiVed July 29, 2004. ReVised Manuscript ReceiVed January 19, 2005

Bioactive CaO-SiO2-poly(dimethylsiloxane) (PDMS) organic-inorganic hybrid coatings over Ti6Al4V substrates have been prepared for the first time by using a sol-gel dip-coating method. The influence of the sol viscosity upon the film formation was investigated. When the new CaO-SiO2-PDMS coatings were soaked in a fluid mimicking human plasma for 7 days, a new nanocrystalline phase over all the investigated films was formed. The use of several complementary characterization techniques makes it possible to identify the material formed over the coating surfaces as a nanocrystalline apatite-like phase. These hybrid coatings can be of interest in the orthopedic and dentistry industries because of their bioactive behavior.

Introduction Several metallic alloys are widely used in orthopedic surgery and dentistry mainly due to their mechanical properties. However, under human body conditions metals release ions that are toxic for human cells up to certain concentration values.1,2 Furthermore, the metal-bone interface plays a key role in the use of metals as implants; as a consequence of the weak linkage between metals and bone tissues, the micromovements in the interface can grow, leading in many cases to prosthesis failure. To overcome these drawbacks, metal implants are frequently coated with a thin film layer of a bioactive material.3 This layer hinders the ion release to the body, and promotes the formation of a mechanically strong bond between the bioactive coating and the living bone. Therefore, the desirable mechanical properties of metals are maintained while the characteristics of their surfaces are improved. Metals usually employed in implants include stainless steels (mainly AISI 316L used as osteoarticular biomaterial), Co-Cr alloys (still used in dentistry and hip prosthesis stems), and Ti-based alloys, with a higher elastic modulus than that of living bone but lower than that of other metallic biomaterials.4,5 Thus, Ti-based alloys such as Ti6Al4V close the gap between mechanical properties of natural bone and artificial metallic implants. In recent years, organic-inorganic hybrid research has become an important subject for materials and medical researchers.6 For clinical applications, the preparation of bioactive hybrids able to bond to living tissues is an important task. Thus, bioactive organic-inorganic hybrid materials * To whom correspondence should be addressed. E-mail: [email protected].

(1) Brown, S. A.; Farnsworth, L.; Merrit, K.; Crowe, T. D. J. Biomed. Mater. Res. 1988, 22, 321. (2) Long, M.; Rack, H. J. Biomaterials 1998, 19, 1621. (3) Hijo´n, N.; Caban˜as, M. V.; Izquierdo-Barba, I.; Vallet-Regı´, M. Chem. Mater. 2004, 16, 1451. (4) Pilliar, M. R.; Weatherly, G. C. Developments in implant alloys. CRC Crit. ReV. Biocompat. 1984, 1, 371. (5) Brunski, J. B. In Biomaterials Science; Ratner, B. D., Ed.; Academic Press: San Diego, CA, 1996; p 37.

have been produced in bulk, and some compositions could be thought to be suitable for metal implant coatings due to their mechanical properties and bioactivity.7 Thus, the CaO-SiO2-poly(dimethylsiloxane) (PDMS) system combines in a single material the excellent bioactivity of the inorganic component,8 CaO-SiO2, and the rubber-like mechanical properties induced by the organic constituent, PDMS.9 The dip-coating method, based on the sol-gel process, allows the deposition of organic-inorganic hybrids at soft temperatures onto metallic substrates. These substrates are immersed into the aqueous multicomponent sols and then thermally treated. However, as far as we know, no bioactive coatings of CaO-SiO2-PDMS organic-inorganic hybrids on Ti6Al4V substrates have been reported. This coating method would allow (i) corrosion of the metallic substrate to be avoided because of the formation of a barrier to ion release from the implant to the body, (ii) the formation of the apatite layer on the surface to be favored (fixing the implant), and (iii) a material intermediate between bone and the implant to be produced, damping their different mechanical properties. The aim of this work was the preparation of CaO-SiO2PDMS bioactive thin films, using the sol-gel process for sol production, and dipping Ti6Al4V substrates into it. The hybrid composition was selected because it presented in vitro bioactivitysi.e., an apatite-like layer was formed after the CaO-SiO2-PDMS thin films were soaked in a solution mimicking human plasmaswhen produced in bulk.10 Optimal processing conditions to obtain homogeneous films were also targeted. (6) Livage, J.; Coradin, T.; Roux, C. In Functional Hybrid Materials; Go´mez-Romero, P., Sa´nchez, C., Eds.; Wiley-VCH: Weinheim, Germany, 2004; p 387. (7) Tsuru, J.; Ohtsuki, C.; Osaka, A.; Iwamoto, T.; Mackenzie, J. D. J. Mater. Sci.: Mater. Med. 1997, 8, 157. (8) Martı´nez, A.; Izquierdo-Barba, I.; Vallet-Regı´, M. Chem. Mater. 2000, 12, 3080. (9) Mackenzie, J. D.; Huang, Q.; Iwamoto, T. J. Sol-Gel Sci. Technol. 1996, 7, 151. (10) Salinas, A. J.; Merino, J. M.; Hijo´n, N.; Martı´n, A. I.; Vallet-Regı´, M. Key Eng. Mater. 2004, 254-256, 481.

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Table 1. Nomenclature of the Hybrid Coated (Hc) Samples coated sample

Hc-2

Hc-4

Hc-7

Hc-10

Hc-16

aging time (h)

2

4

7

10

16

Experimental Section Hybrid Coating Synthesis. CaO-SiO2-PDMS organicinorganic hybrid coatings were deposited onto Ti6Al4V substrates (kindly supplied by Industrias Quiru´rgicas de Levante S.L., Biomet. Merck) by the dip-coating method. This method is based on the immersion of a metallic substrate into a previously prepared sol and the subsequent extraction at a constant withdrawal rate, followed by different thermal treatments. Sols were prepared using a composition that was previously reported as bioactive when applied to obtain organic-inorganic hybrid materials in bulk.10 The molar ratio was TEOS:PDMS:H2O:Ca(NO3)2‚4H2O ) 1:0.29:2:0.1. Solution A, containing 96 mmol of tetraethyl orthosilicate (TEOS; Aldrich) and 27.4 mmol of hydroxy-terminated PDMS (Aldrich, Mn ≈ 550) in 20 mL of reagent grade isopropyl alcohol (IPA; Aldrich) as solvent, was mixed with solution B, which was prepared with 10 mmol of calcium nitrate tetrahydrate (Ca(NO3)2‚4H2O; Aldrich), 2.3 mL of distilled water, and 10 mmol of nitric acid (HNO3; Aldrich). The mixed solution was stirred for 2 h at room temperature. Then, the sol was cast at room temperature for different periods of time ranging from 2 to 16 h (Table 1) before the dipping process was carried out. The viscosity variation of the sol with time was measured at 25 °C using a Haaker ReoStress RS75 rheometer at a shear rate ranging from 1 to 300 s-1. Coatings were deposited by dipping polished disk substrates of Ti6Al4V (13 mm in diameter, 1 mm thick) into the sol at a constant withdrawal rate of 2500 µm/s, which was selected from our previous studies.11 The produced coatings were kept at room temperature for 24 h for gelation and aged at 60 °C for 48 h. Finally, drying was done at 150 °C for 24 h (Figure 1). The thickness of the coatings was ca. 0.5 µm, and it was increased to ca. 1 µm when the aging of the sol was longer. Before thin film deposition, metal substrates were polished with SiC of grit 320 and 9-3-1 µm diamond paste, and successively washed for 5 min in an ultrasound bath with distilled water, alcohol, and acetone.11 In Vitro Bioactivity. The in vitro behavior of the coatings was evaluated using the simulated body fluid (SBF) proposed by Kokubo et al.12 SBF is an acellular, aqueous solution with an inorganic ionic composition that closely resembles that of human plasma, buffered to physiological pH (7.25-7.40) at 37 °C with tris(hydroxymethyl)aminomethane/HCl. The coated samples were soaked for up to 7 days in 6 mL of SBF at 37 °C buffered at pH 7.4 in sterile polyethylene containers. To avoid microorganism contamination, the SBF solution was previously filtered with a 0.22 µm Millipore system, and all operations/manipulations of these pieces and SBF were done in a laminar flux cabinet Telstar AV-100. After being soaked, the pieces were rinsed with deionized water and acetone and dried in air at room temperature. The variation of Ca2+ concentration in solution was determined with an ILyte Na+, K+, Ca2+, pH analyzer (Instrumentation Laboratories). Coating Characterization. Hybrid coatings, before and after being soaked in SBF, were characterized by X-ray diffraction (XRD) with a Philips X-Pert MP diffractometer equipped with a thin film (grazing incidence) attachment and using Cu KR radiation. (11) Izquierdo-Barba, I.; Asenjo, A.; Esquivias, L.; Vallet-Regı´, M. Eur. J. Inorg. Chem. 2003, 1608. (12) Kokubo, T.; Kushitani, H.; Sakka, S.; Kitsugi, T.; Yamamura, Y. J. Biomed. Mater. Res. 1990, 24, 721.

Fourier transform infrared (FTIR) spectroscopy was performed with a Nicolet Nexus spectrometer using a Golden Gate attenuated total reflectance (ATR) device. The morphology of the samples was examined by scanning electron microscopy (SEM) with a JEOL 6400 instrument, and the elemental composition of the coatings was determined by energy dispersive X-ray spectrometry (EDS) with a LINK AN 10000 system coupled to the microscope. Finally, the crystals formed over the surface of the coatings after the in vitro assays were analyzed by electron diffraction (ED) and transmission electron microscopy (TEM) with a JEOL 2000 FX electron microscope working at 200 kV coupled with an Oxford Pentafet Super A/W (EDS) analyzer microscope system to find the composition of the crystals.

Results and Discussion Rheology of the CaO-SiO2-PDMS Sol. The viscosity of the sol together with the withdrawal speed of the substrate plays a key role in the final properties of the films. The withdrawal speed of the coatings was kept constant at 2500 µm/s, to reduce the variables of the process, leaving the solution viscosity as the main parameter to take into account. Consequently, a better control over the final properties of the films was targeted. The viscoelastic behavior of a gel as a function of shear rate is commonly used for the identification of the gel point of a system.13 Viscosity measurements vs shear rate indicate a Newtonian behavior (shear-rate-independent viscosity) in all cases excluding the 24 h sol (see Figure 2a). This could be explained because at shorter periods of time the low molecular weight of the clusters being formed caused the sol viscosity to be unaffected by cluster-cluster interactions. Thus, during aging times equal or lower than 16 h, the viscosity was observed independent of the shear rate, indicative of a Newtonian flow behavior (Figure 2a). As aging time is increased to 24 h, the clusters within the sol grow to a stage that they can be broken by the rotating cylinder of the viscosimeter, so the sol becomes shear thinning.13 At this point, the viscosity decreases with increasing rate because the aggregates break down, releasing immobilized liquid embedded within the aggregates and, thus, reducing the viscosity (non-Newtonian behavior because viscosity depends on the shear rate). Also further aging leads to extensive network formation because some segments of the gel network can still move close enough together to allow further condensation. This corresponds to thixotropic flow behavior,13 where the viscosity shows an irregular behavior (increasing and decreasing), as observed after 24 h of gelation (Figure 2a). Viscosity vs time measurements are normally used to identify the aging conditions of a determined system.13 An increase in the sol viscosity was observed when the time that the sol was kept at room temperature was increased (Figure 2b). After 24 h of aging, the viscosity of the sol tends to increase abruptly, indicating that the gel point is close to this time. Nevertheless, the exact determination of the gel point of this gel lies beyond the scope of our investigation. (13) Brinker, C. J.; Scherer, G. W. The Physics and Chemistry of Sol-Gel Processing; Academic Press Inc.: San Diego, 1990.

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Figure 1. Scheme of the preparation of CaO-SiO2-PDMS thin films on Ti6Al4V substrates. Steps 2-5 were kept constant, and the time in step 1 was modified from 2 to 16 h.

Figure 2. Viscosity vs shear rate (a) and viscosity vs aging time (b) of the CaO-SiO2-PDMS sols.

Figure 3. FTIR spectrum (a) and XRD pattern (b) of the Hc-7 coating before the in vitro assay.

Characterization of Hybrid Coatings. All CaO-SiO2PDMS hybrid coatings presented similar FTIR spectra and XRD patterns independently of the time that the sol was kept at room temperature (parts a and b of Figure 3, respectively). In the FTIR spectrum of Hc-7, the most intense band at 1010 cm-1 corresponds to the Si-O-Si asymmetric stretching

vibrations. The absorption bands at 2963 and 2904 cm-1 are due to the asymmetric and symmetric C-H stretching vibrations from CH3, the band at 1258 cm-1 corresponds to the Si-CH3 symmetric deformation, and the absorption band at 788 cm-1 is due to the Si-CH3 bending vibrations (Figure 3a). The XRD patterns of all coatings were also analogous. As an example, in Figure 3b the diffractogram of Hc-7 is presented. Only three maxima attributable to (100), (002), and (101) reflections of the R hexagonal phase of titanium14 were observed. This result is indicative of the amorphous nature of the deposited coatings. The morphology of the hybrid coatings obtained at different aging times was evaluated using SEM, and their elemental composition was assessed by EDS analyses (Figure 4). For comparative purposes, the SEM-EDS results of the uncoated metallic substrate are included in Figure 4. Hc-2 and Hc-4 samples showed certain heterogeneity at the coating surfaces probably due to an incomplete hydrolysis and condensation processes (Figure 4). On the other hand, when aging time increased (Hc-7 and Hc-10), the material seemed more homogeneous than at lower aging times. Thus, these aging conditions (between 7 and 10 h) were selected as the more appropriate to obtain homogeneous coatings within this system. When a larger aging time was applied (Hc-16), a kind of porosity was observed, probably due to the evapora(14) JCPDS No. 44-1294 Database sets 1-49 plus 70-86, International Center for Diffraction Data, Newton Square, PA, 1999.

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Figure 4. SEM images and EDS analyses of the Ti6Al4V substrate and the hybrid films deposited at different sol times. Table 2. Variation of Calcium Content (mmol‚L-1) with Soaking Time in SBF

Hc-2 Hc-4 Hc-7 Hc-10 Hc-16

[Ca2+](t ) 0)

[Ca2+](t ) 3 days)

[Ca2+](t ) 7 days)

2.57 2.57 2.57 2.57 2.57

3.64 3.23 3.02 3.12 3.04

2.32 2.12 2.10 2.07 2.33

tion of the solvent during the drying process. The porosity caused by the stress of the drying process can now be observed because the film is thicker (longer aging times lead to thicker films) than in other samples. The deposition of a hybrid layer onto the substrates was confirmed by EDS analyses because of the presence of calcium and silicon peaks (Figure 4 insets). At low gelation times (Hc-2 and Hc-4), the percentage of Ti is bigger than at higher times, which could be explained considering that the deposited film is thinner (ca. 0.5 µm) than at long aging times (ca. 1 µm). When the time increased (Hc-7 and Hc-10), the amount of Ti observed by EDS was smaller than in previous cases, and that could be attributed to a thicker hybrid layer deposition. The EDS analysis of the uncoated Ti6Al4V substrate showed the peaks of its components (Ti, Al, and V). In Vitro Bioactivity. The in vitro bioactivity of a material is normally assessed by monitoring the formation of an apatite-like layer on its surface when soaked in SBF. In all the samples under study, an increase of the calcium concentration in solution was observed during the first 3 days of assay (Table 2) as a consequence of the hydrolysis of the Si-O-Ca groups. For longer periods of assay, the calcium concentration in SBF decreased. This could be considered as a first indication of the growth of an apatite-like layer on the hybrid surface. It should be considered that, once the apatite nuclei are formed, they grow spontaneously by

Figure 5. FTIR spectra of the surface of the coatings before and after 7 days of soaking in SBF.

consuming the calcium ions from the surrounding fluid. Assuming that the soaking process of CaO-SiO2-PDMS hybrids in SBF gives rise to the release of calcium ions to the fluid, an exchange with protons of the solution will be induced, leading to an increase in silanol groups (Si-OH) on the hybrid surface. According to the literature,15 the silanol groups can induce the apatite nucleation. (15) Hench, L. L. J. Am. Ceram. Soc. 1991, 74, 1487.

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Figure 6. (a) SEM images, (b) EDS analyses, and (c) XRD patterns of Hc-7 coatings after 7 days in SBF. (d) Proposed model of calcium phosphate layer growth over the hybrid coating.

FTIR spectra of the surface of the coatings before and after 7 days of soaking in SBF are shown in Figure 5. The presence of phosphate groups on the hybrid surface after soaking was confirmed with the presence of the typical bands at 560 and 601 cm-1 usually assigned to the P-O bending mode in a crystalline environment.16,17 The existence of such bands (marked in Figure 5) is also in agreement with the formation of an apatite-like layer on the hybrid surface. The most intense absorptions of these types of bands were observed in Hc-4 and Hc-7 (Figure 5). The surface morphology (SEM micrograph), elemental composition (EDS analysis), and phase characterization (XRD) of Hc-7 after 7 days of soaking in SBF are shown in Figure 6. The formation of a new layered material of flakeshaped particles joined together in pseudospherical agglomerates over the CaO-SiO2-PDMS films was observed by SEM (Figure 6a). EDS studies reveal the inclusion of phosphorus in the composition of the newly formed layer. This phosphorus comes from the SBF solution. This analysis also shows an increase in the calcium content with respect to the hybrid coatings before they were soaked in SBF, although the Si peak from the hybrid is still the most intense. A small amount of Ti from the metallic substrate was also detected (Figure 6b). The Ca:P molar ratio (1.62) of the new layer formed, determined by EDS on the surface of the samples after 7 days of assay, was found to be close to the characteristic value of the calcium hydroxyapatite (1.67). The XRD pattern of Hc-7 after it was soaked in SBF for 7 days is plotted in Figure 6c. Maxima at 2θ ) 35.5, 38.5, and 40° of the Ti6Al4V substrate can be seen. These maxima were also observed in the XRD pattern of the coatings before they were soaked in SBF (Figure 3b). In addition, two new diffraction peaks at 2θ ) 26 and 32° are present which could be assigned to (002) and (211) reflections of an apatite-like phase. The low intensity and broadness of the (211) maximum could be attributed to the presence of crystals at the nanoscopic domain, with sizes larger than 15 nm, which is the detection limit of a (16) Elliot, J. C. Structure and Chemistry of the Apatites and other Calcium Orthophosphates; Studies in Inorganic Chemistry 18; Elsevier: Amsterdam, 1994. (17) LeGeros, R. Z. In Calcium phosphate in oral biology and medicine; Myers, H. M., Ed.; Monographs in Oral Science; Karger: Zurich, Switzerland, 1991.

Figure 7. TEM micrographs, ED patterns, and EDS spectra of two different kinds of crystals (a and b) grown on Hc-7 after 7 days in SBF.

hydroxyapatite crystal by XRD.18 On the other hand, the high intensity of the (002) reflection could be due to an anisotropic growth of the crystals favored in the c axis direction. However, the identification of hydroxyapatite only with these XRD diffraction maxima is far from being conclusive. Analogous SEM-EDS and XRD results were obtained for the rest of the hybrid coatings investigated after they were soaked in SBF for 7 days. All these results could lead to the ideal model proposed in Figure 6d where the calcium phosphate layer grows over the organic-inorganic hybrid coating. To confirm the composition and structure of this new layer, TEM-EDS and ED studies were carried out. Parts a and b of Figure 7 show, respectively, the TEM micrographs with the correspondent ED patterns and EDS spectra of the two types of crystals obtained from scratching (18) Vallet-Regı´ M.; Izquierdo-Barba I.; Salinas A. J. J. Biomed. Mater. Res. 1999, 46, 560.

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the surface of Hc-7 after 7 days in SBF. Both ED patterns show diffuse diffraction rings that can be indexed to the (002), (211), (310), (222), and (213) spacings of an apatitelike phase. However, the ED diagram in Figure 7b also includes diffraction maxima which are indicative of a higher crystallinity. Regarding the EDS spectra, in that of the low crystallinity sample (Figure 7a) a complex elemental composition was found, Ca, P, and O being the basic components, plus Si from the hybrid and Mg from SBF. The Ca:P molar ratio of this kind of crystal was found to be between 1.60 and 1.79; such a wide range could indicate that several crystals with different Ca:P molar ratios were averaged. On the other hand, the EDS spectrum of the one with high crystallinity (Figure 7b) showed a simpler composition with only Ca, P, and O. In this case, the Ca:P molar ratio was found to be 1.65, which is very close to that of hydroxyapatite (1.67). The material formed over the coating surfaces when they were soaked in SBF was nanocrystalline, and as a consequence of this, it is difficult to characterize. Nevertheless, the use of several complementary techniques such as FT-IR, SEM-EDS, XRD, TEM-EDS, and ED allow us to reach partial findings that lead to the conclusion that the

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material formed over the coating surfaces was a nanocrystalline apatite-like phase. Conclusions The sol-gel dip-coating method has been successfully used for the preparation of CaO-SiO2-PDMS organicinorganic hybrid films deposited onto Ti6Al4V substrates. The rheology of the system (viscosity vs shear rate and vs aging time) before dip-coating was investigated. The aging time of the sol before deposition has been optimized to obtain homogeneous coatings within this system. The use of several complementary characterization techniques confirmed the bioactive behavior of the coatings because a nanocrystalline apatite-like layer was formed over the film surface when soaked in SBF for 7 days. Acknowledgment. The financial support of CICYT, Spain, through Research Projects MAT 2002-0025 and MAT 20011445-C02-01 is acknowledged. We also thank Dr. F Conde (CAI Difraccio´n de rayos X, UCM) and A. Rodrı´guez and A. Go´mez (CAI Centro de Microscopia Electro´nica Luis Bru, UCM). CM048755I