Biological responses of osteoblast-like cells to different Titanium surface by anodizing modification

Kim, Myung Joo; Kim, Chang Whe; Lim, Young Jun; Park, Hyun Joo

Abstract

STATEMENT OF PROBLEM: To improve a direct implant fixation to the bone, various strategies have been developed focusing on the surface of materials. The surface quality of the implant depends on the chemical, physical, mechanical and topographical properties of the surface. The different properties will interact with each other and a change in thickness of the oxide layer may also result in a change in surface energy, the surface topography and surface, chemical composition. However, there is limited the comprehensive study with regard to changed surface and biologic behavior of osteoblast by anodization. PURPOSE OF STUDY: The aim of this study was to analyze the characteristics of an oxide layer formed and to evaluate the cellular biologic behaviors on titanium by anodic oxidation (anodization) by cellular proliferation, differentiation, ECM formation and gene expression. And the phospholipase activity was measured on the anodized surface as preliminary study to understand how surface properties of Ti implant are transduced into downstream cellular events. METHODS AND MATERIALS: The surface of a commercially pure titanium(Grade 2) was modified by anodic oxidation. The group 1 samples had a machined surface and other three experimental specimens were anodized under a constant voltage of 270 V(Group 2), 350 V(Group 3), and 450 V(Group 4). The specimen characteristics were inspected using the following five categories; the surface morphology, the surface roughness, the thickness of oxide layer, the crystallinity, and the chemical composition of the oxide layer. Cell numbers were taken as a marker for cell proliferation. While the expression of alkaline phosphatase and Runx2 (Cbfa1) was used as early differentiation marker for osteoblast. The type I collagen production was determined, which constitutes the main structural protein of the extracellular matrix. Phospholipase A2 and D activity were detected. Results. (1) The anodized titanium had a porous oxide layer, and there was increase in both the size and number of pores with increasing anodizing voltage. (2) With increasing voltage, the surface roughness and thickness of the oxide film increased significantly (p<0.01), the TiO2 phase changed from anatase to rutile. During the anodic oxidization, Ca and P ions were more incorporated into the oxide layer. (3) The in vitro cell responses of the specimen were also dependant on the oxidation conditions. With increasing voltage, the ALP activity, type I collagen production, and Cbfa 1 gene expression increased significantly (p<0.01), while the cell proliferation decreased. (4) In preliminary study on the relation of surface property and phospholipase, PLD activity was increased but PLA2 activity did not changed according to applied voltage. CONCLUSION: The anodized titanium shows improved surface characteristics than the machined titanium. The surface properties acquired by anodization appear to give rise more mature osteoblast characteristics and might result in increased bone growth, and contribute to the achievement of a tight fixation. The precise mechanism of suface property signaling is not known, may be related to phospholipase D.