The effect of surface chemistry modification of titanium alloy on signalling pathways in human osteoblasts
Introduction
Titanium alloy (Ti–6Al–4V) is well established as a primary metallic biomaterial for orthopaedic implants. It is spontaneously covered by a surface oxide layer of 1.5–10 nm, the property of which contribute, in part, to its excellent biocompatibility. These include low toxicity, great stability with low corrosion rates and favourable mechanical properties compared to other metals. However, the host response to Ti–6Al–4V is not always favourable, whereby a fibrous layer may form at the skeletal tissue–device interface, resulting in implant failure. Therefore, there is a need to develop novel micro-engineered surfaces to provide better biological outcomes. Various surface modifications have been applied to Ti–6Al–4V in an attempt to enhance bone differentiation and promote direct contact between bone and implant material. However, to date, none of these modifications have generated a stable interface strong enough to support functional loading for long periods. Hence, the specific need to develop implants with surface coatings designed to improve bone anchorage through enhanced osseointegration.
We propose that improved bond strength and integration rate of an implant in osseous tissue will be achieved specifically by altering the interfacial chemistry of biomaterial with bioactive molecules known to be important in bone formation. To achieve this, we utilized two methods; one using sol–gel technology and the other using ion beam modification. The latter during the last few decades has become an interesting topic in the field of bioengineering, due to its wide variety of applications, while calcium phosphate films, specifically nanoscale coatings, are of great interest for a variety of biomedical and engineering applications.
Bone mineral is composed of nanocrystalline platelets, originally described as hydroxyapatite (HAp). It is now agreed that bone apatite can be better described as carbonate hydroxyapatite (CHAP) and approximated by the formula (Ca,Mg,Na)10(PO4,CO3)6(OH)2 and the composition of commercial CHAP is similar to bone mineral apatite. The biological apatites (bone) are not pure HAp but contain trace elements including magnesium and zinc. Mg deficiency significantly and progressively diminishes bone formation, leading to osteoporosis [1], while Zn stimulates bone formation [2] and its deficiency has been linked to the development of osteoporosis [3].
Interactions of bone cells with implant surfaces in vitro [4], [5], [6] and in vivo [7] are mediated by adhesion receptors belonging to the integrin superfamily which recognize binding domains within proteins of the extracellular matrix [8]. Integrin-mediated adhesion to extracellular proteins activates multiple cytoskeletal-associated and intracellular signalling proteins, such as focal adhesion kinase (FAK) and Shc. FAK associates with Shc protein creating a Grb2-binding site. The Shc–Grb2-complex activates Ras, leading to stimulation of the mitogen-activated protein kinase (MAPK) signalling cascade [9]. The phosphorylation, extracellular regulated kinases 1/2 (Erk1/2) translocate to the nucleus, providing a link between cytoplasmic signalling molecules and nuclear proteins. The nuclear target proteins of MAPK include c-fos and c-jun (members of the activated protein-1). Activated protein-1 (AP-1) sites are located in the promoters of several genes expressed by osteoblasts such as osteocalcin and type I collagen, hence AP-1 plays an important role in bone development [10], [11].
The intracellular signalling cascade triggered as a result of the surface chemistry modification of biomaterials remains largely unknown. Therefore, in this study we chose to investigate important signalling pathways of bone development and growth, in order to address the question of how surface chemistry modifications modulate these pathways in osteoblasts. Our results suggest that surface-modified Ti–6Al–4V with CHAP or Mg enhanced the expression of key signalling proteins in human bone cells and that may in turn lead to enhanced gene expression of extracellular matrix proteins at the skeletal tissue–device interface.
Section snippets
Materials and methods
Polished disks of Ti–6Al–4V supplied by DePuy Orthopaedics, Inc., USA, 15 mm in diameter and 1 mm thick with a surface finish of <50 nm Ra, were sonicated using Milli-Q water and sterilised using ethylene oxide.
Sol–gel coatings
SEM images of anodized and CHAP-coated samples after micro-tensile (adhesion) testing showed higher fracture toughness (results not shown). The production of a homogeneous sol–gel solution was necessary to ensure good mechanical properties for the coating. The single coating layer was ∼70 nm. No cracking of the coatings was observed for any of the samples. Adhesive bonding to the substrate was also found to be good for all films, as evidenced by the lack of spalling in all samples. Another
Discussion
During the last decade considerable efforts have been made to alter surface characteristics of prostheses to improve the initial bonding of device and skeleton in the non-cemented joint prosthesis. This paper is an extension of a previous study in which we successfully showed that surface chemistry modification of biomaterials potentiates bone growth in vitro [4], [17], [21] and in vivo [22]. The present study introduced another concept of surface modification using sol–gel coating of CHAP onto
Conclusion
Modifying Ti–6Al–4V with CHAP or Mg modulated key signalling proteins such as Shc; a common point of integration between integrins and the Ras/Mapkinase pathway. Ras/Mapkinase pathway was also upregulated, suggesting its role in mediating osteoblastic cell interactions with biomaterials. The signalling pathway involving c-fos was also shown to be transiently upregulated in osteoblasts cultured on the Mg– and CHAP–modified Ti–6Al–4V. Results presented indicate that surface modification with CHAP
Acknowledgements
The authors gratefully acknowledge support from the National Health and Medical Research Council, Australia; Australian Research Council; DePuy, Orthopaedics Inc. (Warsaw, IN); Japan Society of Promotion of Science; the Australian Academy of Science; and the Australian Institute of Nuclear Science and Engineering (AINSE); Professor Khachigian, UNSW.
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