Effects of Substrate Morphology on Osteoblastic Response to Thin Film Calcium Phosphate Coatings F. Horgan 1, B.J. Meenan 1 1

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1 Key Engineering Materials Online: ISSN: , Vols , pp doi: / Journal Citation (to be inserted by the publisher ) Copyright 2003 Trans by Trans Tech Publications, Tech Publications Switzerland Effects of Substrate Morphology on Osteoblastic Response to Thin Film Calcium Phosphate Coatings F. Horgan 1, B.J. Meenan 1 1 NIBEC, University of Ulster at Jordanstown, Newtownabbey, Northern Ireland fergal@nibec-s1.nibec.ulst.ac.uk, bj.meenan@ulst.ac.uk Keywords: Sputtered Calcium Phosphate, tanium Interlayer, 6Al4V, Surface Roughness, Topography, Contact Angle, Osteoblast Response, Alkaline Phosphatase, MTT assay. Abstract. Calcium phosphate (CaP) thin films have been deposited on prepared 6Al4V substrates by RF magnetron sputtering. Coating morphology, composition and structure were determined by means of AFM, FTIR and SEM. CaP surfaces were observed to present differing morphologies and roughness (Ra) values that depended on either mechanical preparation or the use of a sputter deposited underlying titanium metal layer. Contact angle studies have highlighted the significant impact that post deposition thermal annealing and the introduction of a titanium interlayer can have on the hydrophobocity of the CaP coatings. Human osteosarcoma (HOS) cells have been used to assess the impact of substrate morphology on the biologically activity of the CaP films. Cells seeded onto the as-deposited coatings demonstrated the highest levels of attachment and spreading. These in vitro studies also showed cell response to be significantly affected by the presence of the titanium interlayer. Introduction Research in the field of biomaterials has identified the critical roles that surface topography and localised chemistry play in engendering a successful biological response in vivo [1]. Major efforts have been directed toward engineering devices with specific morphological, chemical and physical characteristics [2] to promote cell growth. For hard tissue applications, bioactive CaP coatings can be applied onto devices so as to engender bone apposition in vivo. In this context, thin film deposition techniques such as sputtering can produce CaP coatings with well-controlled substrate topography [3]. Bioactive CaP ceramic coatings are commonly used in biomaterials applications in order to provide localised fixation of a medical implant [4]. Employing RF magnetron sputtering to deposit coatings of sub-micron thickness implies that the underlying substrate topography is a critical factor in defining the nature of the deposited bioceramic layer. Hence, key coating properties, and thereby the resultant biofunctionality, may be significantly influenced by both the choice of sputtering conditions and the nature of the underlying substrate surface. By deliberately changing the morphology of the substrate surface, it is proposed that the structure and composition of the resultant coatings can also be dictated, and the attendant bio-response controlled. In addition, the overall functionality of the sputter deposited CaP coating must be considered in terms of its durability, as this is a key factor in estimating the effective performance of the coatings on implant surfaces in vivo. A typical sputter deposited coating is amorphous, which for ceramic materials normally confers lesser durability than those comprising more crystalline phases. Application of a post-deposition heat treatment (annealing) should provide thermal energy sufficient to produce a more crystalline coating [5]. As such, it is conceivable that annealing thin film CaP coatings may result in a bioactive surface condition that possesses the relative physical, chemical and morphological aspects necessary for a successful bioactive coating. All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, (ID: , Pennsylvania State University, University Park, USA-04/03/16,01:22:26)

2 434 Bioceramics 15 In this study, CaP thin films have been sputter deposited onto substrates with pre-defined surface topography. Key properties, such as surface roughness and contact angle have been determined and correlated with the condition of the underlying native substrate. Additionally, the nature of CaP coatings deposited onto sputtered titanium interlayers has been determined. Cell culture studies have been used to determine the potential of each of these systems to elicit specific responses to bone cells in vitro. Materials and Methods Calcium phosphate coatings were deposited by RF magnetron sputtering from hydroxyapatite powder (Merck KGaA, Germany) targets. Deposition occurred over 10 hours at a power of 150 W, argon gas pressure of 15 mtorr, and gas flow of 5 Sccm. Coatings were deposited onto 6Al4V substrates (15 x 15 x 1 mm), that had been abraded with 600 and 1200 grit SiC and titanium substrates coated with a sputtered titanium interlayer. The latter were prepared from a metal target (K.J. Lesker, %) at gas pressure 20 mtorr; power 400 W; gas flow 5 Sccm for 30 minutes duration. CaP coated samples were annealed post-deposition at 500 C (Lenton muffle furnace) for 2 hours at a ramp rate of 2 C/min. The following nomenclature summarizes coating types used in these experiments; a) abraded 6Al4V alloy (6Al4V), (b) as deposited CaP on 6Al4V (CaP AD), (c) annealed CaP on 6Al4V (CaP AN), (d) titanium interlayer on 6Al4V (6Al4V/), (e) as deposited CaP on titanium on 6Al4V (CaP AD ) and (f) annealed CaP on titanium on 6Al4V (CaP AN ). CaP coatings were evaluated by a variety of analytical techniques both before and after heating to 500 C. Fourier Transform Infrared (FTIR) spectra were used to confirm the presence of characteristic phosphate and hydroxyl groups. A Bio-Rad Excalibur (FTS300MX Series) Fourier Transform Infrared (FTIR) spectrometer with PIKE Easidiff DRIFTS accessory was used to investigate the chemical bonding in the cm -1 region. A total of 16 scans were taken to obtain each spectrum with a resolution of 4 cm -1 in absorbance mode. Scanning Electron Microscopy (SEM) at low vacuum (Hitachi S-3200N) was used to illustrate coating topography and Atomic Force Microscopy (AFM) (Burleigh Instruments) operating in contact mode was used to determine morphology and surface roughness values. Contact angles for the coatings were determined from sessile drop measurements made with doubly distilled water using a CAM 100 instrument (KSV Instruments). Human osteosarcoma (HOS) cells were cultured on 6Al4V substrates and CaP coatings (asdeposited and annealed) sputter deposited onto substrates with and without titanium interlayers. All samples were sterilised by autoclave at 120 C prior to seeding at a density of 1 x 10 5 cells ml -1. Samples were subsequently incubated at 37 C in DMEM supplemented with 10 % foetal calf serum, ascorbic acid phosphate, HEPES, non-essential amino acids, l-glutamine and penicillinstreptomycin in a humidified atmosphere of 5 % CO 2 / 95 % air for 4 and 24 hours. The attachment of cells was determined after enzymatic release and counted in a hemocytometer. An MTT assay (Sigma Aldrich) was used to determine cell viability/proliferation. Activity of alkaline phosphatase (ALP) was measured (Sigma ALP-10, St. Louis, MO) to determine changes in cell phenotype. SEM of fixed cultures was used to determine the morphology of the cells at the respective time points. Samples were fixed as follows: immersed in 2 % glutaraldehyde and rinsed with PBS followed by exposure to a 2 % osmium tetroxide solution and then graded ethanol solutions. Finally, hexamethyldisilazane (HMDS) was dropped onto each disc, prior to goldplatinum sputter coating (Polaron E5000). All biological assays were carried out in triplicate and repeated to confirm results.

3 Key Engineering Materials Vols Results FTIR analysis confirmed that the sputter deposited coatings were indeed CaP in nature. In all cases the degree of hydroxylation increased after heating to 500 C. Contact angle data indicates that the sputter deposited titanium layer has the highest surface energy when compared to that recorded for a CaP coating sputtered onto an abraded 6Al4V substrate or a CaP thin film on a titanium interlayer. This indicates that the sputtered titanium layer is more hydrophobic than the similarly deposited CaP coatings regardless of the substrate surface condition employed. Post deposition annealing was shown to increase the contact angle for CaP coatings on both the 6Al4V and titanium interlayer (Student t-test, p<0.005). 120 Contact Angle (Degrees) a b c d e f Figure 1. Contact angle data for a) 6Al4V, b) 6Al4V/, c) CaP AD, d). CaP AN, e) CaP AD, f) CaP AN Both the sputter deposited CaP coatings and the interlayers were determined to be 1 µm thick from AFM analysis. Localised (50 µm x 50 µm) surface roughness values for CaP coatings presented a statistically higher Ra value for CaP coatings upon thermal annealing (Student t-test, p<0.005). Therefore the differences reported in contact angle for these coatings indicate a significant contribution from the morphological surface conditions in addition to physico-chemical attributes. Table 1. Localised (50 µm x 50 µm) surface roughness values recorded for abraded 6Al4V, 6Al4V/ and CaP as deposited and annealed on both substrate types. Substrate 6Al4V CaP AD CaP AN Localised Surface Roughness (Ra) [nm] ± ± ± Al4V/ ± 1.02 CaP AD ± 0.38 CaP AN ± 0.98 The presence of the titanium interlayer (6Al4V/) has conferred a more ordered structure on the CaP coating resulting in a decrease in the Ra value recorded (Fig. 2 a - b). Significant in-filling of surface defects in the underlying 6Al4V substrate by the titanium interlayer may account for this significant decrease in localised roughness. This observation was further confirmed by SEM analysis. Osetoblastic activity increased with the presence of a CaP coating at both of the exposure intervals used. Moreover, the as-deposited coatings on the native 6Al4V substrate (CaP AD) and on the sputtered titanium interlayer (CaP AD ) exhibited the highest attachment (Fig. 3.) and cellular response, indicative of the optimal effects of a lower contact angle and a less hydroxylated CaP coating.

4 436 Bioceramics 15 (a) Ra nm (b) Ra nm Figure 2 (a - b): AFM images (50 µm x 50 µm) indicating localised surface morphology and roughness of; a) 6Al4V/, b) CaP AN Hours 24 Hours Number of Cells (1 x 10 4 ) a b c d e f Figure 3. Cell Attachment data for a) 6Al4V, b) 6Al4V/ c) CaP AD, d). CaP AN, e) CaP AD, f) CaP AN. Discussion and Conclusions The results presented here indicate that control of substrate surface topography has an important role to play in determining the function of CaP thin film overlayers. In particular, the presence of a sputtered titanium interlayer has been shown to provide a CaP layer with many of the features known to enhance biofunctionality in vivo. Hence, these data imply that a combination of controlled surface topological and localised chemistry can be used to optimise the cell-surface interactions of the type required for tissue engineering applications. Acknowledgments FH thanks the Dept. of Education for N. Ireland and UU for the award of postgraduate studentship. References [1] A.S.G. Curtis et al: J Biomater Sci: Polym Ed. Vol. 9 (1998), p [2] A. Ohl et al. Surface and Coatings Technology. Vol (1999), p [3] B.J. Meenan et al: Bioceramics. Vol. 12 (1999), p [4] J.G.C. Wolke et al: Bioceramics. Vol. 12 (1999), p [5] Lo et al: Jn. Biomed. Mat Res. Vol. 46 (1999), p

5 Bioceramics / Effects of Substrate Morphology on Osteoblastic Response to Thin Film Calcium Phosphate Coatings / DOI References [2] A. Ohl et al. Surface and Coatings Technology. Vol (1999), p /S (99)