Differential responses of osteoblast lineage cells to nanotopographically-modified, microroughened titanium–aluminum–vanadium alloy surfaces
Introduction
Bone and joint injuries are among the most reported health problems in the United States [1]. Although orthopaedic implants provide a good option for joint replacements, with success rates continually improving, they still have undesirable failure rates in patients who are compromised by disease or age (i.e., patients who are often the ones most in need) [2], [3].
Surface topographical modifications at the micrometer and nanometer scales have driven improved success rates for dental implants by mimicking the hierarchical structure of bone associated with regular bone remodeling [4], [5]. In this process, damaged bone is resorbed by osteoclasts, which produce resorption lacunae containing high microroughness generated after mineral dissolution under the ruffled border [6], as well as superimposed nanoscale features created by the collagen fibers exposed at the surface [7]. New bone formation by osteoblasts is coupled with these primed surfaces, possibly after recognition of structural and chemical cues [8], [9]. Thus, surface topographical modifications have been exploited for implant design in order to achieve direct and intimate contact between the bone and the surface of the implant (osseointegration). Indeed, the beneficial effects of microroughness for bone formation have been well established in the literature [10], and the addition of nanostructures to the implant surface (to mimic more closely the natural structure of bone) has shown promising results in vitro [11], in vivo [12] and clinically [13], [14], validating the biological relevance of nanotopography for bone formation.
Titanium (Ti) and its alloys are widely-used metals for dental and orthopaedic implant applications due to their favorable weight-to-strength ratio and good biological performance in bone. Implant surface modifications at the microscale involve adding to, removing from, or deforming material on the bulk metallic substrate (e.g., acid etching, sandblasting) to generate features that are comparable in size or larger than cells [15], [16]. More recently, surface nanomodifications have been developed to directly restructure the oxide layer formed on the implant surface using different techniques, such as coatings [17], hydrothermal reactions [18], and surface oxidation [19], [20]. The generated oxide nanostructures can then interact with proteins and other small molecules that will eventually influence early cell behavior and long-term osseointegration [21].
The differentiation state required to respond to the surface topographical cues by the initial osteoblast lineage cells (to populate the surface of an implant) remains to be elucidated, with bone marrow mesenchymal stem cells (MSCs) or immature osteoblasts as possible candidates. Several recent studies using MSCs in vitro consider these cells as initial colonizers of the implant surface due to their higher mobility and ability to differentiate into osteoblasts and other cell types [22], [23]. Many of these studies culture the MSCs using exogenous factors, such as β-glycerophosphate, dexamethasone, and bone morphogenetic protein-2 (BMP-2) [24], [25], to force their differentiation into osteoblasts, which could be obscuring the real effects of the surface nanotopography [26]. We have recently demonstrated that human MSCs can differentiate into osteoblasts when cultured on Ti surfaces possessing microscale roughness, even in the absence of these media supplements [27]. However, it is not known if osteogenic differentiation of MSCs is a general response to microrough metal surfaces, including Ti alloys, or if it is specific to commercially pure Ti. How the addition of nanoscale features to a microrough surface will affect such differentiation is also unclear.
The goal of the present study has been to test the hypothesis that nanostructural features on implant surfaces can enhance the osteogenic differentiation of osteoblast-lineage cells in the absence of any exogenous soluble factors. To test this hypothesis, we have superimposed nanostructures on microrough Ti6Al4V surfaces and examined the responses of human MSCs and primary human osteoblasts without the addition of exogenous soluble factors.
Section snippets
Titanium alloy specimens and surface modification treatments
Titanium alloy rods (ASTM F136 wrought Ti6Al4V ELI alloy for surgical implant applications) 15 mm in diameter were cut into 1.5 mm thick disks and either machined to create a relatively smooth surface (control specimens referred to herein as “sTiAlV” specimens), or double-acid-etched with a proprietary process (Titan Spine LLC, Mequon, WI) to produce a microrough surface (specimens referred to herein as “rTiAlV” specimens). These disk specimens were provided by Titan Spine LLC. Some of the
Characterization of nanomodified surfaces
SE images of the original sTiAlV and rTiAlV surfaces revealed that both were relatively smooth at the nanoscale, with some sub-microscale features left from the machining or double-acid-etch treatment (Fig. 1A and B). However, the surfaces of the titanium alloy specimens that had received the 740 °C oxidation treatment for 45 min (specimens NMsTiAlV and NMrTiAlV) possessed high and homogeneous coverages of nanoscale protuberances (referred to herein simply as nanostructures) with diameters that
Discussion
Surface nanomodification of dental and orthopaedic implants is becoming a common approach to enhance osseointegration [5]. Although several scientific reasons have been postulated for beneficial effects of nanostructures on the surfaces of osseous implants [7], fundamental questions remain to be answered regarding the initial cellular responses to these nanostructural features in vitro and in vivo. In addition, variations in various parameters of published in vitro reports (e.g., the size and
Conclusions
The present paper demonstrates that the differentiation state of osteoblast-lineage cells can determine their response to oxidation-induced surface nanostructures on a titanium alloy in terms of the production of osteoblast differentiation markers, which has implications for clinical evaluation of new implant surface nanomodifications. The osteoblastic differentiation of primary human osteoblasts but not osteoblastic differentiation of MSCs was highly sensitive to nanostructures superimposed by
Acknowledgments
This research was supported by USPHS AR052102 and the ITI Foundation. RAG is partially supported by a fellowship from the Government of Panama (IFARHU-SENACYT). Support for the work of TM and YC was provided by the U.S. Air Force Office of Scientific Research (Award No. FA9550-09-1-0162). Support for the work of KHS was provided by the U.S. Department of Energy, Office of Basic Energy Sciences (Award No. DE-SC0002245). The sTiAlV and rTiAlV specimens as well as the Endoskeleton® TT implants
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