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Tio2 Nanocomposite Films 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 nanocomposite 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 polymer matrix composites containing titanium dioxide (TiO2) nanoparticles. 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 ceramic 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 polymers 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),
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