Titanium alloy surface oxide modulates the conformation of adsorbed fibronectin to enhance its binding to α5β1 integrins in osteoblasts

Although the outcomes of orthopaedic and dental implant procedures are usually successful, in many instances the long-term stability and functionality of the implant cannot be achieved. Implant loosening and failure is still a significant problem with a sizeable percentage of hip arthroplasties. In fact, 25% of hip-replacement procedures are revisions for previous implant failure (1). Despite the reported long-term predictability of dental implants (2,3), failures do occur in 10% of cases within a 5-year period (4). The quality and quantity of bone that forms at the implant–skeletal interface is generally believed to be one of the major determinants for implant success (5). Therefore, improving fixation by enhancing the attachment and function of osteoblastic cells at the implant surface is likely to decrease substantially the likelihood of implant failure. Commercially pure titanium (cpTi) and its alloy [titanium-6/aluminum-4/vanadium (Ti6Al4V)] are commonly used as implantable materials. Commercially pure titanium is used in dentistry and in maxillofacial reconstruction (6). The Ti6Al4V alloy exhibits superior strength and a lower modulus of elasticity that, coupled with its excellent biocompatibility, make it a favoured material for orthopaedic applications (7). The Ti6Al4V alloy has also been successfully utilized in the dental field (8). Both titanium and its alloys form an active oxide layer that readily interacts with cell-surface proteins and with extracellular matrix proteins produced by cells. It is this superficial oxide found on both metals, TiO2 being the most abundant, that provides a biocompatible interface with peri-implant tissues (7,9). Altering the surface of titanium-implant materials has been shown to affect protein adsorption, cell–substrate interactions, and tissue development (10). However, the mechanisms by which surface oxide properties modulate the bioactivity of bound osteogenic proteins to influence osseointegration are poorly understood. Proteins of the extracellular matrix, which function, in part, as sites of attachment for osteoblasts in skeletal tissues (11), have been tested as implant surface coatings to facilitate osteoblast attachment and to promote osseointegration. Our laboratory and others have studied extracellular matrix proteins such as fibronectin or human bone sialoprotein (hBSP), or hBSP peptides, following their non-covalent adsorption (12–16) or covalent grafting (17–20) to the implant surface oxide to increase surface coverage by osteoblasts. Cell adhesion to fibronectin and to other matrix proteins is primarily mediated by integrin receptors that recognize the tripeptide arginine-glycine-aspartic acid (RGD) as an important protein-binding site (20). However, when fibronectin is adsorbed to an implant surface, it is not known to what degree binding sites in the protein are available for interactions with integrin receptors or how the integrin-binding activity of fibronectin might be influenced by the properties of the surface oxide. Our laboratory has demonstrated that pretreating the Ti6Al4V surface oxide with heat (600 °C) or radiofrequency plasma glow discharge (RFGD) dramatically increases the number of osteoblastic cells that attach to adsorbed fibronectin (21,22). We have also shown that the pretreatment of the titanium alloy, Ti6Al4V, with heat or RFGD increases the surface oxide’s negative net charge (23). Other studies have found that fibronectin adsorbed to negatively charged surfaces exhibits a more extended or relaxed conformation (24–26) and a higher cell-attachment activity (26–28) compared with less-charged surfaces. These findings suggest that substrates with negatively charged functional groups can increase the functional presentation of the integrin-binding domain of fibronectin by inducing conformational changes in the protein upon its adsorption. Such structural changes in the adsorbed protein would hypothetically promote cell attachment by increasing the binding of fibronectin to integrin receptors. Our earlier studies (21,22) did not directly measure the effects of surface pretreatments of Ti6Al4V on fibronectin conformation or the binding of the adsorbed protein to specific integrin receptors. Therefore, in the current study we endeavoured to clarify the biological mechanism for the effects of pretreatment with heat or with RFGD on osteoblastic cell-attachment activity to fibronectin. To achieve this goal, we tested the hypothesis that these pretreatments increase the exposure of the fibronectin integrin-binding domain and its availability to form complexes with integrin receptors, putatively through protein-conformational changes.