Composites: Part A 36 (2005) 721–727 www.elsevier.com/locate/compositesa

Fabrication, characterisation and assessment of bioactivity of poly(D,L lactid acid) (PDLLA)/TiO2 films A.R. Boccaccinia,*, L.-C. Gerhardta,b, S. Rebelinga, J.J. Blakera

aDepartment of Materials, Imperial College London, Prince Consort Road, London SW7 2BP, UK bCentral institute for Medical Engineering ZIMT, TU Munich, Boltzmannstrasse 11, D-85748 Garching bei Mu¨nchen, Germany

Received 7 September 2004; revised 31 October 2004; accepted 7 November 2004

Abstract

This study deals with the fabrication and characterisation of matrix composites containing titanium dioxide (TiO2) . Poly(D,L lactid acid) (PDLLA) films incorporated with different percentages (0, 5, 20 wt%) of TiO2 nanoparticles were prepared by solvent casting and characterised by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The in vitro bioactive properties of the films were assessed after immersion in simulated body fluid (SBF) for up to 21 days. No hydroxyapatite (HA) formation was observed on the surfaces, neither for pure PDLLA samples nor for PDLLA samples filled with TiO2 nanoparticles. This confirms that under simulated physiological conditions, TiO2 nanoparticles do not impart bioactivity to the PDLLA matrix. The present study provides an analytical method for the assessment of the suitability of titanium dioxide nanoparticles to be used as filler in resorbable polymer matrices for biomedical applications. q 2004 Elsevier Ltd. All rights reserved.

Keywords: PDLLA; A. Nano-structures

1. Introduction efficacy [4]. Nanostructured composites are considered promising materials in bone tissue engineering applications Tissue engineering can be defined as the ‘science of as they simulate the nanometre surface roughness found in persuading the body to heal by its intrinsic repair natural osseous tissue [5–10]. At nanometre dimensions, mechanisms’ [1]. Tissue engineering requires suitable tissue engineering materials can be controlled on the atomic biocompatible materials which can be used as scaffolds or molecular level, thereby improving cell/material surface for the seeding with cells for the growth of new tissue. interactions [7]. Several investigations have suggested the Whereas so-called second-generation biomaterials were advantages of nanoparticles such as alumina, designed to be either resorbable or bioactive, the next titanium dioxide and hydroxyapatite in comparison to generation of biomaterials for tissue engineering is conventional micrometric ceramic particle sizes in terms combining these two properties, with the aim of developing of cellular behaviour [8–10]. Enhanced cellular functions biocompatible scaffolds that, once implanted, will induce such as greater osteoblast adhesion, greater alkaline tissue regeneration [2]. Nanostructured composites on the phosphatase (ALP) synthesis (biochemical marker for basis of bioresorbable and ceramic nanoparticles bone metabolism) as well as enhanced concentration of (grain sizes !100 nm, [3]) may possess the ability to calcium in the extracellular matrix were observed when simulate the surface and/or chemical properties of bone, using ceramic nanoparticles either as bulk material or in allowing for exciting alternatives in the design of prosthesis polymer matrices. It has been reported that composites as well as tissue engineering scaffolds with greater made of polylactic/glycolic acid (PLGA) containing titanium dioxide nanoparticles have a higher cytocompat- ibility than those made using conventional (micrometer) * Corresponding author. E-mail address: [email protected] (A.R. Boccaccini). TiO2 incorporated into the polymer, i.e. the adhesion of

1359-835X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.compositesa.2004.11.002 722 A.R. Boccaccini et al. / Composites: Part A 36 (2005) 721–727 osteoblasts and chondrocytes is much higher with nano- view, the nanoparticles consist of approximately 80% particle additions [4]. These findings imply that TiO2 anatase and 20% rutile [25]. nanoparticles could be a potentially improved substitution for other bioceramic (microsized) particles presently used as 2.2. Sample preparation fillers in bioresorbable polymer scaffolds such as hydro- xyapatite or bioactive glass particles [11–13]. A polymer stock suspension was prepared in three Bioactive properties of materials used for tissue engin- separated centrifuge tubes and subsequently composite eering scaffolds are generally examined by in vitro soaking films were processed by solvent casting using chloroform procedures in simulated body fluid (SBF), which provide a (CHCl3) (Sigma, Steinheim, Germany). The PDLLA relevant assessment of their expected bioactive behaviour in granules were dissolved in chloroform to produce an initial vivo [14,15]. Although TiO2 has been traditionally con- polymer weight to solvent ratio of 4% (w/v). The polymer sidered to be a ‘bioinert’ ceramic [16,17], several recent suspension was then magnetically stirred until complete studies have suggested that some forms of TiO2 might act as dissolution. For the composites, appropriate amounts of a bioactive material [18–23] in the sense that the material titanium dioxide nanoparticles previously calculated to leads to an ‘interfacial bonding to tissue by means of the obtain final TiO2 proportions of 5 and 20 wt%, referring to formation of a biologically active hydroxyapatite (HA) the initial polymeric weight, were added to the tubes. layer on the implant surface’, as originally defined by Hench Subsequently, the mixture was sonicated for 10–15 min at [14,24]. Especially sol–gel-derived titania coatings have 250 W (Ultrawave JP200, Ultrawave, Cardiff, UK) in a demonstrated a high degree of bioactivity in in vitro water bath to improve the dispersion of TiO2 particles in the experiments [18–23]. polymer solution and to desegregate possible titanium Both, the advantageous characteristics of ceramic dioxide agglomerations. Borosilicate glass cover slides nanoparticles in general as well as the reported bioactive (CoverGlass, BDH, Dorset, UK), used as substrate for the behaviour of TiO2, were motivation to examining TiO2 films, were degreased and washed with chromic acid and nanoparticles for their use as filler in bioresorbable PDLLA acetone. An aliquot (1 mL) of the polymer solution was then matrices in this study. To the authors’ knowledge there has casted and spread onto the glass substrate (average diameter been no previous investigation regarding polymer–ceramic 13 mm) using a pipette. Three different film compositions combinations consisting of poly(D,L lactic acid) (PDLLA) containing 0, 5 or 20 wt% TiO2 nanoparticles were and titanium dioxide (TiO2). Although PLGA/TiO2 compo- fabricated. sites have been already studied [4], due to the different crystallinity as well as in vitro and in vivo degradation 2.3. Bioacitvity experiment in simulated body fluid behaviour of PDLLA and PLGA, the addition of TiO2 nanoparticles is expected to affect differently the overall In vitro bioactivity studies were carried out using behaviour of these biodegradable matrices, hence the simulated body fluid (SBF) based on the formulation and interest in investigating PDLLA based composites here. method developed by Kokubo et al. [15]. Briefly, the SBF The overall objective of this study was thus to investigate which has inorganic ion concentrations similar to those of TiO2 nanoparticles with regard to their use as filler for human extracellular fluid, was prepared by dissolving PDLLA-based composites. The bioactivity, characterised respective amounts of reagent chemicals (all purchased by formation of hydroxyapatite upon immersion of the from Sigma, Steinheim, Germany) of NaCl, NaHCO3, KCl, material in SBF, was investigated both for the TiO2 powder K2HPO4$3H2O, MgCl2$6H2O, CaCl2$2H2O, and Na2SO4 and for PDLLA films containing different percentages (0, 5 into distilled water. The SBF was adjusted to physiological and 20 wt%) of titanium dioxide nanoparticles. pH (pH 7.25) by HCl and buffered by tris(hydroxyl-methyl) aminomethane. After solvent evaporation overnight, the films attached to the cover glass were placed in 24-well plates using tweezers, and subsequently an aliquot (2 mL) of 2. Materials and methods SBF (37 8C) was added to the films. During the immersion period, the films were kept at 37 8C in a humidified 2.1. Poly(D,L lactic acid) (PDLLA) and titanium dioxide incubator, and the SBF was refreshed after 6 h of incubation (TiO2) followed by 24, 48 h and then every three days. The films were then collected after 1, 3, 7, 14 and 21 days of Polymer pellets of amorphous PDLLA with an inherent incubation. The samples were rinsed in distilled water three viscosity of 1.62 dL/g were obtained from Purac (Purac times, then vacuum dried for 3 h and finally stored in biochem, Gorinchem, The Netherlands) and served as matrix. dessicators for further examination. w Commercially available TiO2 nanopowder (Aeroxide P25, For the assessment of the bioactive characteristics of the Degussa, Frankfurt a. M., Germany) with a mean primary particles themselves, an amount of 100 mg of TiO2 powder particle size of 21 nm and a specific surface area of 50 m2/g was placed in a conical flask, immersed in 50 mL of was employed as filler material. From a crystallographic SBF and incubated for 3 weeks using an orbital shaker A.R. Boccaccini et al. / Composites: Part A 36 (2005) 721–727 723

(C24 Incubator Shaker, New Brunswick Scientific), which maintained a constant temperature of 37 8C and rotated at 175 rpm. At the point of extracting the powder from the SBF, a thin spoon was used and the wet powder was placed in a beaker. The powder was rinsed with acetone, dried under vacuum and stored in a dessicator awaiting further examination.

2.4. Film characterisation

Surface morphology, film microstructure homogeneity as well as uniformity of TiO2 particle distribution in the composites were characterised using scanning electron microscopy (SEM) (JEOL JSM) Small pieces of the films were mounted onto stubs using adhesive tapes and sputtered Fig. 1. XRD pattern of (a) titanium dioxide powder exposed to SBF for 3 with a gold layer. Accelerating voltages in the range of weeks. The XRD patterns of (b) as-received TiO2 powder (P25, Degussa) 6–18 kV were used for the observation of cross-sections and and of (c) hydroxyapatite (HA) [26] are also shown for comparison. surface topography. Additionally, powder X-ray diffraction (XRD) was performed using a Philips PW 1710 diffract- a ‘bioinert’ material, in agreement with earlier findings ometer in order to characterise the crystalline phases in the [16,17]. films and the possibly formed hydroxyapatite crystals after film exposure to SBF. The films were exposed to high energetic (Cu-K a, lZ1.5406 A˚ ) X-rays and the 2q angles 3.2. Characterisation of PDLLA/TiO2 nanocomposite films were recorded using a scintillation counter in the 2q interval between 5 and 758 at scan steps sizes of 0.048 and a SEM micrographs of the as-prepared PDLLA/TiO2 films detection time of 1.5 s per scan. with 0, 5 and to 20 wt% of TiO2 before immersion in SBF As-prepared and degraded films after immersion in SBF are shown in Fig. 2(a–c). The PDLLA film containing no were also characterised by Raman spectroscopy. titanium dioxide nanoparticles (Fig. 2a) has a smooth A Renishaw spectrometer (RM 2000, Renishaw, Glouces- surface with no evidence of surface topography observed at tershire, UK) connected with a Leica microscope (objective the magnification used during SEM observations. The magnification !20, NAZ0.4) was used. The samples were composite containing 5 wt% TiO2 (Fig. 2b) exhibits an illuminated with a 785 nm diode laser (w300 mW) and even distribution of TiO2 nanoparticles with few agglom- subsequently spectra were recorded for 20 s in the erates. Similarly, the composite filled with 20 wt% TiO2 wavenumber interval of 250–1900 cmK1. Three spectra (Fig. 2c) had an even distribution of nanoparticles but there per location were recorded. The surface roughness of are also some large agglomerates of titanium dioxide samples before and after immersion in SBF was character- present. It can be observed that an increasing concentration w ised by surface topography measurements using a Zygo of TiO2 nanoparticles in the composites results in rougher instrument. The bioactive behaviour of the ceramic powder, surfaces, which should lead to a greater surface area. This characterised by possible formation of hydroxyapatite upon can be qualitatively assessed when comparing the SEM immersion in SBF, was also examined using SEM and XRD micrographs of films containing 5 wt% TiO2 and 20 wt% analysis. TiO2. The SEM micrographs of the composite films in Fig. 2b and c also imply that at least some of the titanium dioxide nanoparticles are not exposed on the composite 3. Results and discussion surface but rather are embedded in the PDLLA matrix. The results of surface roughness measurements using a w 3.1. Bioactive properties of TiO2 nanoparticles Zygo instrument on as-fabricated films with 0 and 20 wt% TiO2 nanoparticles are shown in Fig. 3a and b, respectively. The XRD pattern of the TiO2 powder immersed in SBF These images confirm that the addition of nanoparticles for 3 weeks shows no formation of crystalline hydroxyapa- increases the roughness of the surface, the typical RMS tite (Fig. 1). The peaks in the graph represent the anatase values being 2.71 and 4.70 for the surfaces in Fig. 3a and b, structure of titania powder and a smaller quantity of rutile, respectively. This indicates that films incorporating increas- which qualitatively agrees with the crystallographic com- ing amount of TiO2 particles will have increasing surface position (80% anatase, 20% rutile) of the as-received area, which should lead to enhanced cell adhesion provided powder [25]. The salt NaCl was also identified, which may the other surface characteristics remain unchanged. It has have precipitated from SBF. Fig. 1 thus proves that the as- been reported, for example, that bulk TiO2 substrates received commercial TiO2 nanopowder can be considered (circular discs) processed with nanoparticles have 724 A.R. Boccaccini et al. / Composites: Part A 36 (2005) 721–727

Fig. 3. Surface roughness values measured using a Zygow instrument for

(a) PDLLA matrix, (b) PDLLA/TiO2 composite with 20 wt% TiO2 particles in as-fabricated condition, and (c) PDLLA/TiO2 composite with 20 wt% TiO2 after 21 days in SBF. RMS values are: 2.71, 4.70 and 8.57, respectively.

with conventional micrometer-sized TiO2, osteoblast adhesion and chondrocyte adhesion were between 1.5 and 2 times greater on PLGA composites containing nanophase titanium dioxide [4]. Another way of assessing the distribution of nanoparti- cles in the films is to examine fracture surfaces and cross- sections. A fracture surface of an arbitrarily chosen composite film containing 20 wt% TiO2 is shown in Fig. 2. SEM micrographs of (a) the as-prepared PDLLA film without TiO2 Fig. 4, confirming the large extent of TiO2 particle nanoparticles, a smooth surface can be observed, (b) as-prepared PDLLA agglomeration at this relatively high concentration. How- film containing 5 wt% TiO2 nanoparticles; the film exhibits a fairly ever, relatively well distributed titanium dioxide clusters are homogeneous particle distribution (small white spots) and a rougher surface compared to the as-prepared PDLLA film, and (c) the composite film seen throughout the approximately 20 mm thick PDLLA containing 20 wt% TiO2 nanoparticles. A distinctive rough surface with film matrix. This shows that the method of incorporating some large TiO2 agglomerations is apparent due to the increased particle titanium dioxide nanoparticles into PDLLA matrices used concentration. (The white structure in the middle of the micrograph in (a) is here has been successful since there is no preferential an artefact due to dust or a scratch on the surface). segregation of nanoparticles in the polymer matrix. approximately 35% more surface area compared to their However the agglomeration problem has not been solved equivalent conventional microstructured materials (fabri- for the composites with high concentration of TiO2. The cated with TiO2 of grain size O100 nm) leading to a 30% fracture surface exhibits also a rough appearance due to the greater osteoblast adhesion [10]. Furthermore, compared nanoparticles being pulled out of the remaining surface. A.R. Boccaccini et al. / Composites: Part A 36 (2005) 721–727 725

Fig. 4. SEM micrograph showing the fracture surface of a PDLLA film Fig. 5. SEM micrograph of a PDLLA film containing 20 wt% TiO2 containing 20 wt% TiO2, exhibiting TiO2 particle debonding and nanoparticles after immersion in SBF for 21 days. No formation of HA agglomeration. crystals is discernable, the films showed no macroscopic physical change compared to the as-prepared samples (compare with Fig. 2c). This indicates that in the present composites a relatively weak interface between TiO2 particles and the PDLLA There was no evidence of hydroxyapatite formation matrix exists. This should be taken in consideration when found in all tested samples (0, 5, 20 wt% TiO2) when the investigating the mechanical competence of biomedical films were examined using SEM after incubation periods of materials, e.g. porous scaffolds, made of this particular 1, 3, 7, 14 and 21 days. XRD analyses also led to negative polymer/ceramic combination. Usually, strong particle/ results regarding formation of crystalline HA on sample matrix interfacial bonding is desired to produce stiffer and surfaces. Fig. 6 shows for example the XRD pattern of a stronger composites, but a weak interfacial strength might PDLLA composite film containing 20 wt% TiO2 nanopar- be beneficial for enhanced toughness in the case of brittle ticles after immersion in SBF for 3 weeks. There is no matrix composites, leading to particle debonding [27], characteristic peak of hydroxyapatite present. Further which is apparently the behaviour of the present composites evidence supporting the results of SEM and XRD came (Fig. 4). from the Raman spectroscopy measurements. Fig. 7 shows Raman spectra for PDLLA and TiO2 nanoparticles in as- received condition and for PDLLA containing 20 wt% TiO2 3.3. Possible bioactivity of PDLLA/TiO2 nanoparticles after 3 weeks in SBF. Formation of stoichio- As it is common practice in the field of bioactive metric or non-stoichiometric hydroxyapatite should have materials [11,14,24], the bioactivity of the films was assessed by immersion of samples in SBF for different periods of time. SEM examination of samples after immersion indicated no changes in the appearance and morphology (microstructure) of the films during the entire incubation period in SBF (21 days) for all tested composite films. All films showed similar appearance to their respective as-prepared (no exposure to SBF) control samples. This is shown for the PDLLA film containing 20 wt% TiO2 particles in Fig. 5. The trends in distribution of particles and increase in surface roughness with increasing TiO2 particle content were found to be the same as for the control samples. However samples with the same concen- tration of TiO2 nanoparticles exhibited increased roughness with increasing immersion time in SBF, as can be observed comparing Fig. 3b and c for composites with 20 wt% TiO2. Fig. 6. XRD pattern of a PDLLA film containing 20 wt% TiO The RMS value characterising roughness measured using 2 w nanoparticles after immersion in SBF for 3 weeks. There is no evidence the Zygo instrument was 8.57 for the sample immersed of HA formation when comparing with the reference spectrum of HA. The in SBF for 3 weeks (Fig. 3c), as compared to 4.70 for the only peaks observed are those corresponding to the phases of the starting as-fabricated sample (Fig. 3b). TiO2 material (anatase and rutile). 726 A.R. Boccaccini et al. / Composites: Part A 36 (2005) 721–727

immersion in SBF investigated (up to 21 days), the presence of TiO2 nanoparticles does not seem to affect the degradation of PDLLA, according to a qualitative assess- ment by comparing SEM images of samples before and after degradation in SBF (e.g. Figs. 2c and 5). However the quantitative analysis of surface roughness, characterised by the RMS value measured using the Zygow instrument, indicates a roughness increase of w90%, as documented in Fig. 3c. As mentioned above and reported in the literature [4,10], this increase in roughness in in vitro conditions should lead to enhanced cell adhesion, well before degradation of the polymer matrix is significant, which should have implications regarding the use of the present composites as substrates or scaffolds in tissue engineering strategies. The present results thus indicate that the improved cell adhesion in polymer/TiO2 nanocomposites might be linked only to the changing nanotopography of the materials surfaces as the composites degrade in vitro, rather Fig. 7. Raman spectra of (a) as-prepared PDLLA film, (b) PDLLA film than to any chemical reactivity (bioactivity) induced by the containing 20 wt% TiO2 particles after 3 weeks immersion in SBF, (c) as- presence of the TiO2 nanoparticles. received TiO2 powder. There is no evidence of hydroxyapatite formation The long-term in vitro and in vivo behaviour of K due to absence of a peak at 961 cm 1 [28]. biocomposites made with ceramic nanoparticles remains

K1 to be investigated. In particular, the fate of TiO2 led to a Raman peak at wavenumber 960–963 cm [28], nanoparticles once the polymer matrix has degraded must which is absent, however, as seen in Fig. 7. Raman be known. Despite the large number of studies dealing with spectroscopy emerged thus no clear evidence of hydro- degradable polymer/ceramic composites for xyapatite formation in agreement with SEM and XRD biomedical applications [3–10], this aspect has not been results. extensively discussed. However cell culture studies have The investigation by SEM and XRD of TiO2 powders shown wear debris composed of nanophase TiO2 less which had been immersed in SBF for up to 21 days led to the adversely affects bone and cartilage cell function compared same negative conclusion regarding HA deposition (Fig. 1). to larger conventional ceramic wear particles [29]. Recent This confirms that the particular form of TiO used here 2 studies have considered the effect of TiO2 nanoparticles on (nanoparticles of combined anatase/rutile crystalline struc- endothelial cell function [30], hemocompatibility [31] and ture) is not bioactive in the sense of the definition used in the vascular response in an animal model [32], however more biomaterials literature [11,14,24], which links bioactivity to research is required to investigate the possible integration of the ability of a material to induce HA formation on its non-resorbable TiO2 nanoparticles used in scaffolds or surface upon immersion in SBF, confirming earlier results prostheses into newly formed bone tissue. [16,17]. Bioactivity of titania has been confirmed on TiO2 gels or sol–gel films, which are characterised by the presence of abundant Ti-OH groups on their surfaces 4. Conclusions [18–22]. These groups are considered to be responsible for the nucleation of apatite crystals upon immersion in SBF PDLLA films were incorporated with titanium dioxide [20,22]. Thus the lack of HA nucleation on the surfaces of nanoparticles and the bioactive behaviour of the compo- the nanoparticles used here can be explained by the lack of sites was investigated. Films were fabricated by solvent such Ti-OH groups on the nanoparticles surfaces. In fact the casting and a relatively homogeneous distribution of TiO2 nucleation of apatite on titania gels is not only dependent on nanoparticles in the PDLLA matrix was achieved. The the presence of Ti-OH groups but also on the crystalline increase in nanoparticles content increased the surface modification of the gel [20]. Anatase for example has been roughness which should improve the adhesion of cells, shown to provide atomic arrangements suitable for the osteblasts in particular. The macroscopic appearance of epitaxy of apatite crystals [22]. Since the nanoparticles used the films under SEM was independent of the length of here are mainly (80%) anatase modification, a suitable time that they were immersed in SBF (for up to 3 weeks) treatment of the surface to induce hydroxyl groups could however surface roughness increased notably with immer- render the particles bioactive, this being the focus of current sion time in SBF. There was no evidence of formation of research. hydroxyapatite on samples immersed in SBF for up to Another interesting result of the present experiments is 3 weeks, indicating that titanium dioxide nanopowder is the confirmation that at least for the time periods of not bioactive neither as a free powder nor as particulate A.R. Boccaccini et al. / Composites: Part A 36 (2005) 721–727 727

filler in PDLLA. However, this finding does not imply [14] Hench LL, West JK. Biological applications of bioactive glasses. Life that titanium dioxide nanoparticles when used in polymer Chem Rep 1996;13:187–241. composites are not convenient for enhancing adhesion of [15] Kokubo T, et al. Solutions able to reproduce in vivo surface-structure changes in bioactive glass–ceramic A-W. J Biomed Mater Res 1990; cells, such as osteoblasts and chondrocytes, where the 24(6):721–34. nanotopography might be the dominant effect. [16] Khalil TKK, Mu¨ller U, Ondracek G. Bioinert implant . 1. Technological relevant phase investigations in the Al2O3-SiO2- TiO2-system. Materialwiss Werkstofftech 1996;27(3):119–21. Acknowledgements [17] Fredel MC, Boccaccini AR. Processing and mechanical properties of biocompatible Al2O3 platelet reinforced TiO2. J Mater Sci 1996;31: 4375–80. We acknowledge the assistance of Ms Julia Olsen [18] Moritz N, et al. Local induction of calcium phosphate formation on with the preparation of the manuscript. The assistance of TiO2 coatings on titanium via surface treatment with a CO2 laser. Dr I. Notingher with the Raman spectroscopy measure- J Biomed Mater Res 2003;65A(1):9–16. ments is greatly appreciated. [19] Jokinen M, et al. Influence of sol and surface properties on in vitro bioactivity of sol–gel-derived TiO2 and TiO2–SiO2 films deposited by dip-coating method. J Biomed Mater Res 1998;42(2):295–302. [20] Uchida M, Kim H-M, Kokubo T, Fujibayashi S, Nakamura T. References Structural dependence of apatite formation on titania gels in a simulated body fluid. J Biomed Mater Res 2003;64A:164–70. [1] Agrawal CM, Ray RB. Biodegradable polymeric scaffolds for [21] Areva S, et al. Use of sol–gel-derived titania coating for direct soft musculoskeletal tissue engineering. J Biomed Mater Res 2001; tissue attachment. J Biomed Mater Res 2004;70A(2):169–78. 55(2):141–50. [22] Li P, et al. Bonelike hydroxyapatite induction by sol–gel derived [2] Hench LL, Polak JM. Third-generation biomedical materials. Science titania coating on a titanium substrate. J Am Ceram Soc 1994;77(5): 2002;295(5557):1014–7. 1307–12. [3] Siegel RW. Creating nanophase materials. Scientific Am 1996;275: [23] Viitala R, et al. In vitro bioactivity of aerosol–gel deposited TiO(2) 42–7. thin coatings. J Biomed Mater Res 2001;54(1):109–14. [4] Kay S, et al. Nanostructured polymer/nanophase ceramic composites [24] Hench LL. Introduction to bioceramics. World scientific. Singapore: enhance osteoblast and chondrocyte adhesion. Tissue Eng 2002;8(5): World Scientific; 1993. 753–61. [25] Ettlinger M. Fine particles. In Technical bulletin pigments, No. 80. [5] Savaiano JK, Webster TJ. Altered responses of chondrocytes to Degussa AG, Inorganic Chemical Products Division: Duesseldorf. p. nanophase PLGA/nanophase titania composites. Biomaterials 2004; 5. 25(7–8):1205–13. [26] ICDD. Powder diffraction file, inorganic volume: sets 5, 9, 21, I. [6] Kaplan FS, et al. Orthopaedic basic science. In: Simon SR, editor. International Centre for Diffraction Data, Editor. 1967 (set 9), 1980 Orthopaedic basic science. Columbus, OH: American Academy of (set 21): Swarthmore, PA, USA. p. 05–0628, 9–432, 21–1271, 21– Orthopaedic Surgeons; 1994. p. 127–85. 1276. [7] Webster TJ. Nanophase ceramics as improved bone tissue engineering [27] Matthews FL, Rawlings RD. Composite materials: engineering and materials. Am Ceram Soc Bull 2003;82(6):23–8. science. London: Chapman & Hall; 1994. [8] Webster TJ, et al. Specific proteins mediate enhanced osteoblast [28] Penel G, Leroy G, Rey C, Bres E. MicroRaman spectral study of the adhesion on nanophase ceramics. J Biomed Mater Res 2000;51(3): PO4 and CO3 vibrational modes in synthetic and biological apatites. 475–83. Calcif Tissue Int 1998;63:475–81. [9] Webster TJ, et al. Enhanced functions of osteoblasts on nanophase [29] Gutwein LG, Webster TJ. Osteoblast and chrondrocyte proliferation ceramics. Biomaterials 2000;21(17):1803–10. in the presence of alumina and titania nanoparticles. J Nanopart Res [10] Webster TJ, Siegel RW, Bizios R. Osteoblast adhesion on nanophase 2002;4:231–8. ceramics. Biomaterials 1999;20(13):1221–7. [30] Peters K, Unger RE, Kirkpatrick J, Gatti AM, Monari E. Effects of [11] Zhang RY, Ma PX. Poly(alpha-hydroxyl acids) hydroxyapatite porous nano-scaled particles on endothelial cell funtion in vitro: studies on composites for bone-tissue engineering. I. Preparation and mor- viability, proliferation and inflammation. J Mater Sci Mater Med phology. J Biomed Mater Res 1999;44:446–55. 2004;15:321–5. [12] Boccaccini AR, Maquet V. Bioresorbable and bioactive polymer/- [31] Huang N, Yang P, Leng YX, Chen JY, Sun H, Wang J, et al. Bioglass(R) composites with tailored pore structure for tissue Hemocompatibility of Titanium Oxide Films. Biomaterials 2003;24: engineering applications. Compos Sci Technol 2003;63(16):2417–29. 2177–87. [13] Blaker JJ, et al. In vitro evaluation of novel bioactive composites [32] Cehreli MC, Onur MA, Tas Z, Sahin S. Vascular response to titanium based on bioglass-filled polylactide foams for bone tissue engineering dioxide: a study on the rat carotid artery. J Biomed Mater Res Part B scaffolds. J Biomed Mater Res 2003;67A(4):1401–11. 2004;70B:348–53.