Department of Chemistry and Biochemistry, Bioscience Institute, São Paulo State University, UNESP, Campus Botucatu, Botucatu, São Paulo, Brazil.
Department of Chemistry and Biochemistry, Bioscience Institute, São Paulo State University, UNESP, Campus Botucatu, Botucatu, São Paulo, Brazil; Electron Microscopy Center, IBB, UNESP, Botucatu, SP, Brazil.
Colloids Surf B Biointerfaces. 2018 Mar 1;163:321-328. doi: 10.1016/j.colsurfb.2017.12.049. Epub 2017 Dec 28.
Over the last several years, we have focused on the importance of intracellular signaling pathways in dynamically governing the biointerface between pre-osteoblast and surface of biomaterial. Thus, this study investigates the molecular hallmarks involved in the pre-osteoblast relationship with different topography considering Machined (Mc), Dual Acid-Etching (DAE), and nano hydroxyapatite-blasted (nHA) groups. There was substantial differences in topography of titanium surface, considering Atomic Force Microscopy and water contact angle (Mc = 81.41 ± 0.01; DAE = 97.18 ± 0.01; nHA = 40.95 ± 0.02). Later, to investigate their topography differences on biological responses, pre-osteoblast was seeded on the different surfaces and biological samples were collected after 24 h (to consider adhesion signaling) and 10 days (to consider differentiation signaling). Preliminary results evidenced significant differences in morphological changes of pre-osteoblasts mainly resulting from the interaction with the DAE and nHA, distinguishing cellular adaptation. These results pushed us to analyze activation of specific genes by exploring qPCR technology. In sequence, we showed that Src performs crucial roles during cell adhesion and later differentiation of the pre-osteoblast in relationship with titanium-based biomaterials, as our results confirmed strong feedback of the Src activity on the integrin-based pathway, because integrin-ß1 (∼5-fold changes), FAK (∼12-fold changes), and Src (∼3.5-fold changes) were significantly up-expressed when Src was chemically inhibited by PP1 (5 μM). Moreover, ECM-related genes were rigorously reprogrammed in response to the different surfaces, resulting on Matrix Metalloproteinase (MMP) activities concomitant to a significant decrease of MMP inhibitors. In parallel, we showed PP1-based Src inhibition promotes a significant increase of MMP activity. Taking all our results into account, we showed for the first time nano hydroxyapatite-blasted titanium surface creates a biointerface able to govern Src-dependent osteoblast metabolism as pre-requisite to ECM remodeling.
在过去的几年中，我们一直专注于细胞内信号通路在动态调控前成骨细胞与生物材料表面之间生物界面的重要性。因此，本研究调查了涉及不同形貌的前成骨细胞与表面关系的分子特征，考虑了机械加工（Mc）、双酸蚀刻（DAE）和纳米羟基磷灰石喷砂（nHA）组。原子力显微镜和水接触角表明钛表面的形貌有很大差异（Mc=81.41±0.01；DAE=97.18±0.01；nHA=40.95±0.02）。之后，为了研究它们在生物学反应中的形貌差异，将前成骨细胞接种在不同的表面上，并在 24 小时（考虑粘附信号）和 10 天（考虑分化信号）后收集生物样本。初步结果表明，前成骨细胞的形态变化有显著差异，主要是由于与 DAE 和 nHA 的相互作用，从而区分细胞的适应性。这些结果促使我们通过探索 qPCR 技术来分析特定基因的激活。随后，我们表明 Src 在细胞粘附和随后的前成骨细胞分化过程中发挥关键作用，与基于钛的生物材料有关，因为我们的结果证实了 Src 活性对整合素途径的强烈反馈，因为整合素-β1（变化约 5 倍）、FAK（变化约 12 倍）和 Src（变化约 3.5 倍）在前成骨细胞中被化学抑制 Src 时显著上调（5μM 的 PP1）。此外，不同表面会严格重新编程细胞外基质相关基因，导致基质金属蛋白酶（MMP）活性增加，同时 MMP 抑制剂的含量显著降低。平行地，我们表明基于 PP1 的 Src 抑制会促进 MMP 活性的显著增加。综合所有结果，我们首次表明纳米羟基磷灰石喷砂钛表面创建了一个生物界面，能够控制 Src 依赖性成骨细胞代谢，作为细胞外基质重塑的先决条件。
