Biomimetic modification of titanium dental implant model surfaces using the RGDSP-peptide sequence: A cell morphology study

Abstract

Surface topography and (bio)chemistry are key factors in determining cell response to an implant. We investigated cell adhesion and spreading patterns of epithelial cells, fibroblasts and osteoblasts on biomimetically modified, smooth and rough titanium surfaces. The RGD bioactive peptide sequence was immobilized via a non-fouling poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) molecular assembly system, which allowed exploitation of specific cell–peptide interactions even in the presence of serum. As control surfaces, bare titanium and bio-inactive surfaces (scrambled RDG and unfunctionalized PLL-g-PEG) were used. Our findings demonstrated that surface topography and chemistry directly influenced the attachment and morphology of all cell types tested. In general, an increase in cell number and more spread cells were observed on bioactive substrates (containing RGD) compared to bio-inactive surfaces. More fibroblasts were present on smooth than on rough topographies, whereas for osteoblasts the opposite tendency was observed. Epithelial cell attachment did not follow any regular pattern. Footprint areas for all cell types were significantly reduced on rough compared to smooth surfaces. Osteoblast attachment and footprint areas increased with increasing RGD-peptide surface density. However, no synergy (interaction) between RGD-peptide surface density and surface topography was observed for osteoblasts neither in terms of attachment nor footprint area.

Introduction

The success of an implant in dental applications depends on its strong anchorage to the surrounding bone [1] in order to withstand the continuous cyclic loading that occurs during mastication. Several factors, such as surgical technique, implant design, surface topography and surface (bio)chemistry are known to influence the bone ingrowth to an implant [2]. The optimization of the surface properties based on topographical and (bio)chemical surface modification [3], [4] has become a key issue in the development of improved devices.

Topographical modification of titanium surfaces using either geometrically defined features [5], [6] or random structures [7], [8], [9], [10], [11], [12], [13], [14], [15] has been shown to affect cell behavior in vitro [16] and in vivo [17], [18], [19], [20]. For example, osteoblasts exhibit a more mature osteoblastic phenotype on rougher titanium surfaces approximately one week after cell seeding [21], while fibroblasts [5], and in some cases epithelial cells [22], [23], [24] show a preference for smoother surfaces.

One approach for (bio)chemical (biomimetic) surface modification [25], [26] is the immobilization of small peptides found in extracellular matrix (ECM) proteins to promote cell adhesion. By using peptides present in the tissue of interest as a bridging unit between cell receptors and surface, different cellular pathways can in theory be activated [27]. Perhaps the most investigated peptide sequence is RGD (arginine–glycine–aspartic acid) [28], derived from fibronectin and recognized by almost all α/β integrins [29].

A number of systems using polymers have been developed to immobilize peptides onto a biomaterial surface [30]. However, many such surfaces lack protein-resistance so that proteins present in the medium adsorb to the surface and interfere with the direct study of peptide–cell interactions. An effective way to minimize protein adsorption onto a surface is the use of a poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) molecular assembly system (Fig. 1) [31], [32], [33]. These PEGylated (non-fouling [34]) surfaces reduce adsorbed protein mass to as low as <5 ng/cm² [35], [36]. Osmotic and entropic repulsion effects as well as screening of interfacial charges [37] appear to be involved in producing the anti-fouling effects. Furthermore, peptide sequences can be covalently attached via vinyl sulfone–cysteine coupling reaction to the poly(ethylene glycol) side chains [38]. The thickness of the adsorbed polymeric layer (1–1.5 nm in dry state and 5–10 nm in wet state [39]) is small in comparison to the dimensions of topographic features of rough surfaces such as the commercially used dental implant surface SLA from Institut Straumann that exhibits topographical features in the range of 0.5–50 μm [12]. Moreover, the topography of SLA surface is thought to protect the polymeric coating from damage caused by mechanical handling during surgical preparation and implantation.

The main goal of this study was to determine cell response as a function of the topographical and chemical surface modification. Cells were selected on the basis of tissues that contact dental implants: Epithelium (Porcine Epithelial Cells), connective tissue (Swiss Balb/c 3T3 Mouse Murine Mesenchymal Embryo Fibroblasts) and bone (Rat Calvarial Osteoblasts). Cell attachment, cell morphology and distribution of cytoskeletal elements (microtubules and vinculin) were investigated using fluorescent microscopy techniques.