TOWARDS AN IN VITRO MODEL OF TESTING OSTEOBLAST CELLULAR Towards An In Vitro Model of Testing Osteoblast Cellular Function In Contact With Various Surfaces

Ti is the material of choice for orthopaedic applications because it is biocompatible and encourages osteoblast ingrowth. It was shown that the biocompatibility of Ti metal is due to the presence of a thin native sub-stoichiometric Ti oxide layer which enhances the adsorption of mediating proteins on the surface (49). The present studies were devised to evaluate the adhesion, survival, and growth of cells on the surface of new engineered nano-crystal films of Ti and TiO2 and compare them with orthopaedic-grade Ti with micro-crystals. The engineered nano-crystal films with hydrophilic properties are produced by employing an IBAD technique. IBAD combines physical vapor deposition with concurrent ion beam bombardment in a high vacuum environment to produce films (with 3 to 70 nm grain size) with superior properties. These films are “stitched” to the artificial orthopaedic implant materials with characteristics that affect the wettability and mechanical properties of the coatings. To characterize the biocompatibility of these nano-engineered surfaces, we have studied osteoblast function including cell adhesion, growth, and differentiation on different nano-structured samples. Cell responses to surfaces were examined using SAOS-2 osteoblast-like cells. We also studied a correlation between the surface nano-structures and the cell growth by characterizing the SAOS-2 cells with immunofluorescence and measuring the amount alizarin red concentration produced after 7 and 14 days. The number of adherent cells was determined by means of nuclei quantification on the nano-crystalline Ti, TiO2, and micro-crystalline Ti and analysis were performed with Image J. Our experimental results indicated that nano-crystalline TiO2 is superior 37 to both nanoand micro-crystalline Ti in supporting growth, adhesion, and proliferation. Improving the quality of surface oxide, i.e., fabricating stoichiometric oxides as well as nano-engineering the surface topology, is crucial for increasing the biocompatibility of Ti implant materials. INTRODUCTION Osseointegration of orthopaedic implants is dependent upon different parameters of the implant surface such as surface properties and physicochemical properties including surface energy, charge, wettability, chemistry, and topography. Surface topography can impact cellular behavior including adhesion, migration, morphology, and orientation in addition to focal adhesion, the development of cytoskeleton, and differentiation (50). Surface modifications on implant surfaces can be manipulated by etching (51), plasma deposition (52), sintering powders (53), machining/micro-machining (54), and ion-beam assisted deposition (22). When an implant is placed in the body, a multi-step process occurs. First, serum/plasma proteins adsorb on the implant surface and cells attach to the protein layer by integrins which recognize presented extracellular ligands and mediate the initial interactions of cells and the implant material (55). After the cells adhere, there is a rearrangement of cytoskeleton proteins, formation of tight focal adhesion contacts, activation of focal adhesion kinase, and the induction of several intracellular signal transduction pathways, which leads to proliferation and differentiation of the cells (55, 56). Jager et al. and Stevens et al. have shown that roughened Ti surfaces can increase the focal contacts for cellular adhesion and they are able to guide membrane receptor organization and cytoskeletal assembly (57, 58). In vitro experiments have shown that rough implant surfaces promote the adsorption of FN and albumin (59, 60). In vivo osteointegration has also been improved on roughened surfaces compared to smoother surfaces, which may suggest that the surface modulates the response including osteoblast differentiation, ECM deposition, and calcification (61, 62). Hence, if there is an improvement in the symbiosis of the implant and osteointegration, it 38 should accelerate the healing time, increase the implant longevity, and reduce the necessity of revision surgery (55). In the present work, osteoblast functions including cell adhesion, growth, and differentiation on different nano-samples were investigated. Cell responses to surfaces were examined using SAOS-2 osteoblast-like cells. We studied a correlation between the surface topography and the cell growth by characterizing the SAOS-2 cells with immunofluorescence and measuring the alizarin red concentration produced after 7 and 14 days. MATERIALS AND METHODS 3.1. 1 Sample Preparation The scientific community has been actively pursuing the study of IBAD for specific applications such as tri-biological coatings, anti-corrosion coatings, optical coatings, superconducting buffer layers, and coatings for temperature sensitive substrates such as polymer. IBAD (Figure 3-1) combines evaporation with concurrent ion beam bombardment in a high vacuum environment. Energetic ions (with a depth penetration of typically less than 20 nm) were employed to produce engineered nano-crystals “stitched” to a substrate (utilizing billions and billions of directed and parallel ionic hammers). Ion bombardment is also the crucial factor for controlling other film properties such as surface morphology, density, stress level, crystallinity, grain size, grain orientation, and chemical composition (22).