Supplementary Material for: Intrinsic Surface Effects of Tantalum and Titanium on Integrin α5β1/ ERK1/2 Pathway-Mediated Osteogenic Differentiation in Rat Bone Mesenchymal Stromal Cells

Background/Aims: Accumulating evidence demonstrates the superior osteoinductivity of tantalum (Ta) to that of titanium (Ti); however, the mechanisms underlying these differences are unclear. Thus, the objective of the present study was to examine the effects of Ta and Ti surfaces on osteogenesis using rat bone mesenchymal stromal cells (rBMSCs) as a model. Methods: Ta and Ti substrates were polished to a mirror finish to minimize the influences of structural factors, and the intrinsic surface effects of the two materials on the integrin α5β1/mitogen-activated protein kinases 3 and 1 (ERK1/2) cascade-mediated osteogenesis of rBMSCs were evaluated. Alkaline phosphatase (ALP) activity, Alizarin Red staining, real-time polymerase chain reaction, and western blot assays of critical osteogenic markers were conducted to evaluate the effects of the two substrates on cell osteogenesis. Moreover, the role of the integrin α5β1/ERK1/2 pathway on the osteoinductive performance of Ta and Ti was assessed by up- and down-regulation of integrin α5 and β1 with RNA interference, as well as through ERK1/2 inhibition with U0126. Results: Osteogenesis of rBMSCs seeded on the Ta surface was superior to that of cells seeded on the Ti surface in terms of ALP activity, extracellular matrix calcification, and the expression of integrin α5, integrin β1, ERK1/2, Runt-related transcription factor 2, osteocalcin, collagen type I, and ALP at both the mRNA and protein levels. Moreover, down-regulation of integrin α5 or integrin β1, or ERK1/2 inhibition severely impaired the osteoblastic differentiation on the Ta surface. By contrast, over-expression of integrin α5 or integrin β1 improved osteogenesis on the Ti substrates, while subsequent ERK1/2 inhibition abrogated this effect. Conclusion: The integrin α5β1/ERK1/2 pathway plays a crucial role in regulating rBMSCs osteogenic differentiation; thus, the greater ability of a Ta surface to trigger integrin α5β1/ERK1/2 signaling may explain its better osteoinductivity. The different effects of Ta and Ti surfaces on rBMSC osteogenesis are considered to be related to the conductive behaviors between integrin α5β1 and the oxides spontaneously formed on the two metals. These results should facilitate the development of engineering strategies with Ta and Ti surfaces for improved osteogenesis in endosteal implants.