Time-dependent morphology and adhesion of osteoblastic cells on titanium model surfaces featuring scale-resolved topography

Abstract

The role of micrometer and submicrometer surface roughness on the interaction of cells with titanium model surfaces of well-defined topography was investigated using human bone-derived cells (MG63 cells). The early phase of interactions was studied using a kinetic morphological analysis of adhesion, spreading and proliferation of the cells. By SEM and double immunofluorescent labeling of vinculin and actin, it was found that the cells responded to nanoscale roughness by a higher cell thickness and a delayed apparition of the focal contacts. A singular behavior was observed on nanoporous oxide surfaces, where the cells were more spread and displayed longer and more numerous filopods. On electrochemically microstructured surfaces with hemispherical cavities, arranged in a hexagonal pattern, the MG63 cells were able to go inside, adhere and proliferate in cavities of 30 or 100 μm in diameter, whereas they did not recognize the 10 μm diameter cavities. Cells adopted a 3D shape when attaching inside the 30 μm diameter cavities. Condensation of actin cytoskeleton correlated with vinculin-positive focal contacts on cavity edges were observed on all microstructured surfaces. Nanotopography on surfaces with 30 μm diameter cavities had little effect on cell morphology compared to flat surfaces with same nanostructure, but cell proliferation exhibited a marked synergistic effect of microscale and nanoscale topography.

Introduction

Events leading to integration of an implant into bone, and hence determining the long-term performance of the device, take place largely at the interface formed between the tissue and the implant. The development of this interface is complex and is influenced by numerous factors, including surface chemistry and surface topography of the foreign material [1], [2], [3], [4], [5]. To improve bone-tissue integration, various techniques have been used to increase the surface roughness of titanium implants, including machining/micromachining, particle blasting, titanium plasma-spraying, chemical/electrochemical etching, particle blasting and chemical etching, electrochemical anodization, or pulsed laser ablation [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18] . Topographic features on implants range from millimeters to nanometers and are all believed to be relevant to the biological response of the host [2], [13], [14], [15]. More specifically, micrometer and/or nanometer scale topographies affect different aspects of cell behavior such as cell adhesion, cell proliferation, cell differentiation, cell morphology, cell orientation, contact guidance, tissue organization, mechanical interlocking, production of local factors and microenvironments, and also cell selection [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]. In addition to producing the desired topographies, the surface roughening processes can also alter surface composition, surface charge and/or surface energy of the implant [13], [14], [35], [36], [37]. Many in vivo studies have compared the effects of the surface structure on the biomechanical stability of the device, on the morphology of the bone surrounding it and/or on the extent of the bone-implant interface [6], [8], [10], [11], [12], [16], [17], [18], [38]. Various results have been obtained, depending on the roughness amplitude but also on the method used to produce the surface topography [8], [9], [10], [11], [12], [16], [17], [18], [39].

Cell adhesion is one of the initial events essential to subsequent proliferation and differentiation of bone cells before bone tissue formation [40], [41]. Many in vitro evaluations of cell adhesion on substrates with various and usually complex topographies have been performed [19], [23], [29], [33], [40], [42], [43], [44], [45], [46], [47], [48]. However, up to now little is known concerning the main topographical properties influencing the cell response to implant surfaces. In order to obtain precise information about the effects of the surface topography on cell behavior, one should be able to study independently the micro topography (structures larger than 1 μm), the nanotopography (structures smaller than 1 μm) and the superposition of both, all this without changing the chemical characteristics of the surfaces. This requires the use of model surfaces. Recently, scale-resolved electrochemical surface structuring of bulk titanium has been reported [49]. Optimized through-mask electrochemical micromachining (EMM) of mechanically polished titanium led to well-defined microstructures comparable to cell size. Hexagonal arrays of closely spaced smooth hemispherical cavities of 10–100 μm diameter were made reproducibly using computer-controlled dissolution and precise charge control. Porous anodization and chemical etching, respectively, permitted to generate nanotopography on flat and on previously microstructured surfaces.

The aim of the present study is to use such well defined titanium model surfaces for the investigation how microscale and nanoscale roughness separately and in combination affect the early phase of cell-surface interactions that occur between some hours and some days. For this a kinetic morphological analysis of adhesion, spreading and proliferation of bone-derived MG63 cells was carried out. These cells exhibit the characteristics of immature human osteoblasts and have been widely used in the literature for the evaluation of implant surfaces.