Biomimetic modification of titanium dental implant model surfaces using the RGDSP-peptide sequence: A cell morphology study
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.
Section snippets
Preparation
Samples were produced using the epoxy replica technique as described by Wieland et al. [11]. In brief, replicas of a rough SLA CP Ti disc (Institut Straumann, Basel, Switzerland) and a smooth Tissue Culture Polystyrene (TCPS; Multidish 24 well, Nunc, Milian, Basel, Switzerland) surface were taken using vinyl polysiloxane as the impression material (PROVIL novo Light; Unor, Dietikon, Switzerland), followed by replicating these negative structures by casting into postive structures using
Results
In this section, only significant differences between important chemistries, namely between bioactive surfaces (TiO2 and RGD at high surface concentrations) and bio-inactive surfaces (PEG and RDG), are listed. For detailed (significant) information about other pair comparisons (especially in the case of osteoblasts at low RGD concentrations) see Table 3, Table 4.
Discussion
Our findings have demonstrated that surface topography and chemistry directly influenced the attachment and morphology of epithelial cells, fibroblasts and osteoblasts. In particular the use of the protein-resistant PLL-g-PEG system as a background allowed a more direct and powerful control of cell responses than an uncoated or physicochemically modified surface (e.g. hydrophobic/hydrophilic; see for example [50]). In general, a higher cell attachment and spreading rate was observed on the
Conclusion
Our study on cell attachment to surfaces with controlled surface topographies and (bio)chemistries demonstrated that surface roughness directly influenced cell attachment and cell footprint areas of fibroblasts and osteoblasts but not of epithelial cells under the cell culturing conditions used in this study. The presence or absence of cell–cell contacts seemed to have a more important influence on the behavior of epithelial cells. Surface chemistry was found to determine both the number of
Acknowledgement
The authors acknowledge Dr. Janos Vörös for scientific discussion and Karim H. Soto for statistical advice. This project was supported by the ITI Foundation for the Promotion of Oral Implantology, Basel, Switzerland and by the Swiss Federal Commission for Technology and Innovation CTI (CTI Project no. 7404.2).
References (60)
- et al.
Understanding and controlling the bone–implant interface
Biomaterials
(1999) - et al.
RGD modified polymers: biomaterials for stimulated cell adhesion and beyond
Biomaterials
(2003) - et al.
Peptide functionalized poly(l-lysine)-g-poly(ethylene glycol) on titanium: resistance to protein adsorption in full heparinized human blood plasma
Biomaterials
(2003) - et al.
Optical grating coupler biosensors
Biomaterials
(2002) - et al.
Culture and origin of epithelium-like and fibroblast-like cells from porcine periodontal-ligament explants and cell-suspensions
Arch Oral Biol
(1976) - et al.
The adhesiveness of normal and Sv40-transformed Balb/C 3t3-cells—effects of culture density and shear rate
Eur J Cancer Clin Oncol
(1982) - et al.
“Gap guidance” of fibroblasts and epithelial cells by discontinuous edged surfaces
Exp Cell Res
(2005) Effects of cell–cell contact on spreading of pigmented retina epithelial-cells in culture
Exp Cell Res
(1977)- et al.
Micropatterned surfaces modified with select peptides promote exclusive interactions with osteoblasts
Biomaterials
(2002) The development of the ITI (R) DENTAL IMPLANT SYSTEM—Part 1: a review of the literature
Clin Oral Implants Res
(2000)
The detachment strength and morphology of bone cells contacting materials modified with a peptide sequence found within bone sialoprotein
J Biomed Mater Res
Materials for enhancing cell-adhesion by immobilization of cell-adhesive peptide
J Biomed Mater Res
Production of microfabricated surfaces and their effects on cell behavior
Grooved titanium surfaces orient growth and migration of cells from human gingival explants
J Dent Res
Implant materials, designs, and surface topographies: their effect on osseointegration. A literature review
Int J Oral Max Implants
Design and surface characteristics of 13 commercially available oral implant systems
Int J Oral Max Implants
Measurement and evaluation of the chemical composition and topography of titanium implant surface
Use of Ti-coated replicas to investigate the effects on fibroblast shape of surfaces with varying roughness and constant chemical composition
J Biomed Mater Res
Wavelength-dependent roughness: a quantitative approach to characterizing the topography of rough titanium surfaces
Int J Oral Max Implants
Osseointegrated titanium implants—requirements for ensuring a long-lasting, direct bone-to-implant anchorage in man
Acta Orthop
Principles of cell behavior on titanium surfaces and their application to implanted devices
Titanium for dental applications (II)
The titanium–bone cell interface in vitro: the role of the surface in promoting osteointegration
The development of the ITI (R) DENTAL IMPLANT SYSTEM—Part 2. 1998–2000: steps into the next millennium
Clin Oral Implants Res
Early loading of nonsubmerged titanium implants with a sandblasted and acid-etched (SLA) surface: 3-year results of a prospective study in partially edentulous patients
Int J Oral Max Implants
The use of reduced healing times on ITI (R) implants with a sandblasted and acid-etched (SLA) surface: early results from clinical trials on ITI (R) SLA implants
Clin Oral Implants Res
Early loading of sandblasted and acid-etched (SLA) implants: a prospective split-mouth comparative study—one-year results
Clin Oral Implants Res
Mechanisms involved in osteoblast response to implant surface morphology
Annu Rev Mater Res
Substratum roughness alters the growth, area, and focal adhesions of epithelial cells, and their proximity to titanium surfaces
J Biomed Mater Res A
Cited by (202)
Apolipoprotein E facilitates titanium implant osseointegration by regulating osteogenesis-lipogenesis balance
2023, International Journal of Biological MacromoleculesCorrosion resistance characteristics of a Ti-6Al-4V ELI alloy fabricated by electron beam melting after the applied post-process treatment methods
2021, Biocybernetics and Biomedical EngineeringThe impact of antifouling layers in fabricating bioactive surfaces
2021, Acta BiomaterialiaPhenolic and epoxy-based copolymers and terpolymers
2019, Advanced Functional Polymers for Biomedical Applications