Overexpression of prolyl-4-hydroxylase gene generates harder mineralized tissue on titanium

Objectives: Harder and stiffer peri-implant bone would help establish stronger anchorage of titanium implants. We previously demonstrated the following: 1) mineralized tissue cultured on titanium is harder and stiffer than that cultured on a polystyrene surface; 2) collagen synthesis is accelerated in osteoblasts cultured on titanium compared with those on polystyrene; 3) the rate of collagen synthesis is correlated with the gene expression of prolyl-4-hydroxylase alpha-subunit (P4Ha), an essential enzyme for collagen triple-helix formation (J Biomed Mater Res, in press). We hypothesized that P4Ha has a key role in determining biomechanical nature of bone-titanium integration. The objective of this study is to examine the effect of P4Ha gene overexpression on biomechanical properities of mineralized tissue on titanium. Methods: Rat bone marrow-derived osteoblastic cells were seeded on titanium-coated polystyrene dish prepared by electron-beam physical vapor deposition technology. A non-viral DNA expression vector transcribing rat P4Ha was constructed, and then osteoblastic cells were transfected with the vector using cationic lipid-based gene transfer protocol. The expression of P4Ha was analyzed using RT-PCR at day 2 post-transfection. At day 28, Hardness and elastic modulus of mineralized cultures were measured by a nano-indention with a setting of 400 mN/min loading rate and 5000 nm maximum depth. Interfacial adhesion of the mineralized tissue was assessed by nano-scrach test as a critical load defined as the force to delaminate the tissue from the substrates. Results: Two days post-transfection, P4Ha mRNA was expressed 6 times more than the control culture. Nanoindentation revealed that the hardness, elastic modulus and critical load of P4Ha-transfected mineralized tissue were increased by 2.0-fold, 2.1-fold and 1.3-fold, respectively, compared to those of the control culture (p<0.05, t-test). Conclusions: Prolyl-4-hydroxylase gene overexpression enhances hardness, stiffness and interfacial strength of mineralized tissue on titanium, providing an opportunity to pursue a novel strategy for improved bone-titanium integration.