Elsevier

Biomaterials

Volume 32, Issue 13, May 2011, Pages 3395-3403
Biomaterials

The effects of combined micron-/submicron-scale surface roughness and nanoscale features on cell proliferation and differentiation

https://doi.org/10.1016/j.biomaterials.2011.01.029Get rights and content

Abstract

Titanium (Ti) osseointegration is critical for the success of dental and orthopedic implants. Previous studies have shown that surface roughness at the micro- and submicro-scales promotes osseointegration by enhancing osteoblast differentiation and local factor production. Only relatively recently have the effects of nanoscale roughness on cell response been considered. The aim of the present study was to develop a simple and scalable surface modification treatment that introduces nanoscale features to the surfaces of Ti substrates without greatly affecting other surface features, and to determine the effects of such superimposed nano-features on the differentiation and local factor production of osteoblasts. A simple oxidation treatment was developed for generating controlled nanoscale topographies on Ti surfaces, while retaining the starting micro-/submicro-scale roughness. Such nano-modified surfaces also possessed similar elemental compositions, and exhibited similar contact angles, as the original surfaces, but possessed a different surface crystal structure. MG63 cells were seeded on machined (PT), nano-modified PT (NMPT), sandblasted/acid-etched (SLA), and nano-modified SLA (NMSLA) Ti disks. The results suggested that the introduction of such nanoscale structures in combination with micro-/submicro-scale roughness improves osteoblast differentiation and local factor production, which, in turn, indicates the potential for improved implant osseointegration in vivo.

Introduction

Integration of titanium (Ti) implants with the surrounding bone is critical for successful bone regeneration and healing in dental and orthopedic applications. The desire to accelerate and improve osseointegration drives many implantology research and development efforts, particularly for patients whose bones have been compromised by disease or age. Previous work has shown that the surface characteristics of implants have a direct influence on tissue response by affecting protein adsorption and by modulating cell proliferation and differentiation [1], [2]. Surface characteristics such as roughness [3], [4], chemistry [5], [6], [7] and energy [8], [9] have been reported to significantly influence cell differentiation, local factor production and, consequently, bone growth and osseointegration [10], [11].

Surface modification strategies for metallic implants to improve osseointegration have attempted to mimic the characteristics of bone [12], [13], [14], [15]. During bone remodeling, previously-formed bone is resorbed by osteoclasts, in part to remove micro-cracks before new bone is formed in these primed regions [16], [17]. Resorption lacunae left by osteoclasts, created through acidification and proteinase activity [18], have a distinct hierarchical structural complexity [19], [20]. Resorption lacunae consist of microscale pits (up to 100 μm in diameter and 50 μm in depth [21], [22], [23]) with submicro-scale roughness formed by the irregular acid-etching at the ruffled border of the osteoclast [18], [19] and nanoscale features created by the collagen fibers left on the surface [20], [22].

Several studies have shown that increases in surface micro- and submicro-scale roughness, with feature sizes comparable to those of resorption pits and cell dimensions, lead to enhanced osteoblast differentiation and local factor production in vitro [24], [25], increased bone-to-implant contact in vivo [26], [27] and improved clinical rates of wound healing [28], [29]. Surface nanoscale roughness, which directly corresponds to the sizes of proteins and cell membrane receptors, could also play an important role in osteoblast differentiation and tissue regeneration (Fig. 1).

The effect of nanoscale surface roughness on osteoblast response has drawn the attention of several research groups over the last decade [30], [31], [32], [33]. The literature on this topic is dominated by studies on the initial interactions between osteoblasts and nano-modified polymeric substrates, and such work has indicated that nanoscale roughness can significantly affect cell adhesion [34], proliferation [35], and spreading [36]. Similar results have been found for ceramic [37] and metallic [38] substrates. However, other studies report either a decrease in osteoblast proliferation with an increase in nanoscale roughness [39], or no effect of nanoscale roughness on proliferation [40] in the absence of microscale surface roughness [12], [41].

Relatively few studies have examined the effects of nanostructured surfaces on osteoblast differentiation [12], [36], [37], [42], [43]. Some reports have indicated that increased osteoblast proliferation on nanostructured surfaces coincided with an increase in alkaline phosphatase (ALP) synthesis, increased Ca-containing mineral deposition [37], and higher immunostaining of osteocalcin (OCN) and osteopontin [36]. Gene expression studies have shown an increase in the expression of RUNX2, osterix (OSX), and bone sialoprotein (BSP) in osteoblasts grown on nano-roughened surfaces [42], [43]. Two studies [12], [41] examined the protein levels of different differentiation markers and local factors, and both of these studies reported an increase in differentiation, and an increase in factors PGE2 and active TGF-β1, when submicro- to nanoscale roughness was introduced to micro-rough substrates.

More recent studies have focused on the hierarchical combination of both micro- and nanoscale roughness to promote osseointegration on clinically-relevant surfaces [12], [13], [14], [44], [45]. Although some of these studies have reported promising results of increased osteoblast proliferation and differentiation, it has been challenging to create a tailored hierarchical surface without altering other underlying characteristics of the substrate (particularly the microscale roughness and surface chemistry) [13], [14], [45]. For this reason, it has been difficult to decouple the effects of nanoscale features from those of other surface features, such as surface micro-roughness, surface chemistry, and/or surface energy. Additionally, the simultaneous increase in osteoblast proliferation and differentiation caused by nanoscale roughness remains controversial due to some contradictory results [39], [40], [44], which may have been influenced by differences in the types of cells and in the types of nanoscale surface modifications used in these experiments.

The objectives of the present study were twofold. First, we aimed to develop a simple and scalable oxidation-induced surface modification process of clinical relevance in order to alter the nanoscale topography of Ti substrates without greatly affecting surface chemistry or the starting micro-/submicro-scale roughness. Second, we aimed to evaluate the influence of such modified nanoscale surface topography, with and without additional micro-/submicro-scale roughness, in vitro on the differentiation and local factor production of human osteoblast-like MG63 cells.

Section snippets

Titanium disks

Ti disks with a diameter of 15 mm were punched from 1 mm thick sheets of grade 2 unalloyed Ti (ASTM F67 unalloyed Ti for surgical implant applications) and supplied by Institut Straumann AG (Basel, Switzerland). After degreasing the disks in acetone, the disks were exposed at 55 °C for 30 s to an aqueous solution consisting of 2% ammonium fluoride, 2% hydrofluoric acid and 10% nitric acid to generate “pre-treated” (PT) Ti disks. The PT disks were further sandblasted with corundum grit

Results

Scanning electron microscopy (Fig. 2, Fig. 3, Fig. 4) confirmed that a modest temperature oxidation treatment could be used to introduce nanoscale structural features to the Ti surfaces. In this study, the oxidation temperature (i.e., 740 °C) and gaseous environment (i.e., synthetic air) were fixed while the duration of the process was varied. The surfaces of the starting PT samples were relatively smooth on the microscale (CLM Sa = 0.43 ± 0.02 μm), although surface pits, presumably resulting

Discussion

In the present study, a simple, readily-scalable (non-line-of-sight) oxidation-based surface modification process was developed that resulted in the superimposition of a high density of nanoscale structures on Ti substrates (as revealed by SEM and AFM analyses) in the absence or presence of appreciable microscale roughness. This nanoscale modification (NM) treatment did not affect surface chemistry (as revealed by XPS measurements) or wettability (as revealed by water contact angle

Conclusions

A simple and readily-scalable (non-line-of-sight) oxidation-based surface modification process has been developed that superimposes a high density of nanoscale structures on the surfaces of Ti samples without greatly affecting other surface properties (e.g., microscale roughness, hydrophobicity). The nanoscale structures are not unlike the nanoscale topography associated with collagen fibrils left by the osteoclasts after bone resorption. The results suggest that, while the nanostructures alone

Acknowledgments

This research was supported by USPHS AR052102, and the ITI Foundation. RAGI is partially supported by a fellowship from IFARHU-SENACYT. Support for the work of TM, YC, and KHS was provided by the Air Force Office of Scientific Research (Dr. Charles Lee, program manager). The PT and SLA disks were provided by Institut Straumann AG.

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