Effect of roughness, wettability and morphology of engineered titanium surfaces on osteoblast-like cell adhesion

Abstract

Texturization of surfaces is usually advantageous in biomaterial engineering. However, the details of the textured surfaces can be more determining on cell adhesion and proliferation, rather than their roughness degree. Titanium is extensively used as a dental implant material in the human body. In this paper, the effect of four surface treatments on commercially pure titanium has been evaluated. These treatments were polishing (pTi); hydrofluoric acid (HF) etching (eTi); Al₂O₃ blasting (bTi); Al₂O₃ blasting + HF etching (beTi). Roughness and fractal dimensions were obtained from atomic force microscopy. Wettability was measured using water sessile drops. Morphology and surface chemical composition were analyzed with scanning electron microscopy and energy dispersive X-ray (EDX). MG-63 cell cultures were performed at different times (180 min, 24 h, 48 h, 72 h). Lowest roughness was found in pTi samples followed by eTi, bTi and beTi samples. Etching generated surfaces with the highest fractal dimension and negative skewness. Young contact angles were similar except for pTi and bTi surfaces. Silicon and aluminum traces were found in pTi and bTi samples, respectively. Cell adhesion (≤24 h) was greater on bTi and beTi surfaces. After 48 h, cell proliferation, mediated by specific morphologies, was improved in eTi samples followed by beTi surfaces. For the same surface chemistry, cell growth was driven by topography features.

Introduction

The success of a dental implant is based on the osseointegration that is defined as the direct contact between the bone tissue and the dental implant surface, without fibrous tissue growing at the interface [1], [2]. Surface characteristics of the implant play an important role for the evolution of bone tissue of the recipient site, after implantation [3]. Surface properties control the amount and quality of cells adhered on the implant and consequently, the tissue growth. Most determining surface properties for cell adhesion are surface topography and surface chemical reactivity. Accordingly, surface engineering of biomaterials is oriented to modify their surface texture and/or surface chemistry.

The topography of a surface substantially affects the macroscopic behaviour of a material [4]. Currently, the influence of surface topography on biological response is a matter of investigation. At cellular level, biological responses, such as the orientation and migration of cells and the cellular production of organized cytoskeletal arrangements, are directly influenced by the surface topography [5]. There are evidences that a suitable surface roughness, at nano- and microscopic scale, can lead to a successful osseointegration of titanium implants [6]. Osteoblast differentiation, proliferation and matrix production [7] as well as the production of local growth factors and cytokines are affected by surface roughness [8].

Biomolecule adsorption onto implant surfaces “in vivo” is indeed a dynamic process driven by the physico-chemical interactions between adsorbent surface and macromolecule [9]. This precursor process develops a “conditioning film” which will modulate the cellular host response. Surface energy, which is intimately related to wettability [10], is a useful quantity that has often correlated strongly with biological interaction. Hence, implant wettability can become determining for the protein adsorption and consequently, for the cell adhesion [11], [12], [13]. For instance, hydrophobic surfaces (i.e. surfaces with low water wettability) presumably decelerate primary interactions with the aqueous biosystem. Thus, it is usually reported that biomaterial surfaces with moderate hydrophilicity, improved cell growth and higher biocompatibility [14]. However, cell adhesion can decrease as the implant wettability is further decreased. This points out to the existence of a range of optimal surface energies [15]. Otherwise, interfacial reactions “in vivo” change relevant physical and chemical surface parameters, such as the surface energy, affecting the long-term stability of implants [16].

Modification of the physico- and physico-chemical surface properties of a biomaterial can improve interaction with cells. In particular, several surface treatments have been applied to optimize the surface topography of titanium implants in bone-contact applications. Most extending treatments are sandblasting and acid etching [17]. The main purpose of these texturization treatments is to achieve greater bone-to-implant contact [18], in order to reduce healing times and accelerate integration into the host tissue. Texturing of dental implants improves the mechanical adhesion to bone but, at the same time, the asperities and grooves may act as preferable sites for protein adsorption.

The evaluation of the topography of biomaterial surfaces is important because it usually improves the biological responses found during osteointegration and the long-term response of the bone–implant interface. However, one major difficulty arises in the choice of the topography parameters, which are actually relevant for the interactions between biomaterial surface and surrounding biological medium [19]. Generally, the description of a surface may be performed at three levels, according to the degree of surface information: height/spatial distribution, topology and morphology. The height/spatial distribution provides a statistical description of the surface roughness using amplitude or horizontal lengths and hybrid parameters, both in 2D and 3D analysis [20]. The topology description of a surface is understood as the set of intrinsic properties related to its structure such as connectedness, compactness. The topography of most engineering surfaces is topologically self-affine over a range of scales [21]. The fractal dimension (D_f) measures, in statistical sense, the structural complexity of a self-affine surface, i.e. the random roughness organization [22]. In other adhesion studies, the fractal dimension correlated better with adhesion than did conventional measurements of surface roughness [23], [24]. For this reason, fractal analysis might be more helpful than conventional roughness descriptions in order to elucidate the complex mechanisms occurring at the implant surface in contact with the surrounding biological tissues [19]. Otherwise, the morphological analysis, usually performed by SEM, provides the details and the figure of the surface topography at higher resolution; even although this raw information without post processing is merely qualitative.

In surface engineering of biomaterials, it is important to determine when cell spreading is modulated by surface energy, topography or both, at short and long-term times after implantation. In addition, rather than conventional approaches, fractal dimension might be used to quantify the role of the features of implant topography on the cell growth. Accordingly, the aim of this work is to evaluate the effect of four texturization treatments of titanium surfaces on the adhesion and growth of osteoblast-like cells, from the topography (roughness, fractal dimension and morphology) and the water wettability induced by each treatment.