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.

研究背景与目的：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），但二者差异背后的分子机制尚不明确。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：将钽与钛基底抛光至镜面光洁度，以最大限度降低结构因素的影响，进而评估两种材料的本征表面效应，即通过整合素α5β1（integrin α5β1）/丝裂原活化蛋白激酶3和1（ERK1/2）信号通路介导的大鼠骨髓间充质基质细胞成骨过程。本研究采用碱性磷酸酶（ALP）活性检测、茜素红染色、实时荧光定量聚合酶链式反应以及关键成骨标志物的蛋白质印迹实验，以评价两种基底对细胞成骨的影响。此外，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，以此探究整合素α5β1/ERK1/2通路在钽与钛的骨诱导性能中发挥的作用。结果：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及蛋白表达水平方面，接种于钽表面的大鼠骨髓间充质基质细胞的成骨活性均优于接种于钛表面的细胞。进一步实验发现，下调整合素α5或整合素β1的表达，或抑制ERK1/2活性，均会严重削弱钽表面的成骨细胞分化能力。与之相反，上调整合素α5或整合素β1的表达可增强钛基底上的成骨过程，而后续ERK1/2抑制则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过程中发挥关键作用；因此，钽表面更强的整合素α5β1/ERK1/2信号激活能力，或是其骨诱导活性更优的原因。钽与钛表面对大鼠骨髓间充质基质细胞成骨的差异效应，可能与整合素α5β1和两种金属表面自发形成的氧化物之间的相互作用有关。本研究结果可为开发基于钽与钛表面的工程化策略提供理论依据，以提升骨内种植体的成骨效果。