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.

背景与目的：越来越多的研究证据表明，钽（tantalum, Ta）的骨诱导活性优于钛（titanium, Ti），但二者性能差异背后的分子机制尚不明确。因此，本研究以大鼠骨髓间充质基质细胞（rat bone mesenchymal stromal cells, rBMSCs）为模型，探究钽与钛表面对成骨过程的影响。方法：本研究将钽与钛基材抛光至镜面光洁度，以尽可能降低结构因素对实验的干扰，并评估两种材料的本征表面效应，通过整合素α5β1/丝裂原活化蛋白激酶3和1（ERK1/2）信号通路调控大鼠骨髓间充质基质细胞成骨过程的情况。实验通过碱性磷酸酶（alkaline phosphatase, ALP）活性检测、茜素红染色、实时荧光定量聚合酶链式反应及关键成骨标志物的蛋白质印迹实验，评估两种基材对细胞成骨过程的影响。此外，本研究通过RNA干扰技术上调或下调整合素α5与β1的表达，以及使用U0126抑制ERK1/2活性，探究整合素α5β1/ERK1/2通路在钽与钛骨诱导性能中的作用。结果：接种于钽表面的大鼠骨髓间充质基质细胞，其成骨活性在ALP活性、细胞外基质钙化程度，以及整合素α5、整合素β1、ERK1/2、Runt相关转录因子2、骨钙素、I型胶原与ALP的mRNA及蛋白表达水平上，均优于接种于钛表面的细胞。此外，下调整合素α5或整合素β1的表达，或抑制ERK1/2活性，会严重削弱钽表面细胞的成骨分化能力。与之相反，上调整合素α5或整合素β1的表达可增强钛基材表面的成骨过程，但后续抑制ERK1/2活性会抵消这一促进效果。结论：整合素α5β1/ERK1/2通路在调控大鼠骨髓间充质基质细胞成骨分化中发挥关键作用；因此，钽表面更高效地激活整合素α5β1/ERK1/2信号通路，或可解释其更优异的骨诱导活性。钽与钛表面对大鼠骨髓间充质基质细胞成骨过程的不同影响，可能与整合素α5β1与两种金属表面自然形成的氧化层之间的相互作用特性相关。本研究结果可为开发钽与钛表面改性工程策略提供理论依据，以提升骨内植入物的成骨效果。