Key Laboratory of Biorheological Science and Technology, Ministry of Education College of Bioengineering, Chongqing University, Chongqing 400044, China; First Affiliated Hospital, College of Medicine of Zhejiang University, & Key Laboratory of Clinical In Vitro Diagnostic Techniques of Zhejiang Province, Hangzhou, Zhejiang 310003, China.
Biomedical Engineering Center, Medical School of Ningbo University, Ningbo, Zhejiang 315211, China.
Acta Biomater. 2018 Jul 1;74:489-504. doi: 10.1016/j.actbio.2018.04.043. Epub 2018 Apr 25.
We used surface-modified titanium (Ti) substrates with a multilayered structure composed of chitosan-catechol (Chi-C), gelatin (Gel) and hydroxyapatite (HA) nanofibers, which were previously shown to improve osteogenesis, as a platform to investigate the interaction of osteogenesis and angiogenesis during bone healing. Combined techniques of Transwell co-culture, wound healing assay, enzyme linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and immunohistochemical staining were used to evaluate adhesion, morphology and migration of adipose-derived mesenchymal stem cells (Ad-MSCs) and human umbilical vein endothelial cells (HUVECs) grown on different Ti substrates. We investigated the effect of substrates on the osteogenic differentiation of Ad-MSCs and reciprocal paracrine effects of Ad-MSCs on HUVECs or vice versa. The multilayered Ti substrates directly regulated the cellular functions of Ad-MSCs and angiogenic HUVECs and mediated communication between them by enhancing paracrine effects via cell-matrix interactions in vitro. The in vivo results showed that the change of microenvironment induced by surface-modified Ti implants promoted the adhesion, recruitment and proliferation of MSCs and facilitated coupled osteogenesis and angiogenesis in bone healing. The study proved that multilayer-film-coated Ti substrates positively mediated cellular biological function in vitro and improved bone healing in vivo.
Recent studies have revealed that osteogenesis and angiogenesis are coupled, and that communication between osteoblasts and endothelial cells is essential for bone healing and remodeling processes; however, these conclusions only result from in vitro studies or in vivo studies using transgenic murine models. Relatively little is known about the communication between osteoblasts and endothelial cells in peri-implants during bone healing processes. Our results revealed the cellular/molecular mechanism of how multilayered Ti substrates mediate reciprocal paracrine effects between adipose-derived mesenchymal stem cells and human umbilical vein endothelial cells; moreover, the interactions between the cell-matrix and peri-implant was proven in vivo with enhanced bone healing. This study contributes to our understanding of the fundamental mechanisms of angiogenesis and osteogenesis that affect peri-implantation, and thus, provides new insights into the design of future high-quality orthopedic implants.
我们使用表面改性的钛（Ti）基底，其具有由壳聚糖-儿茶酚（Chi-C）、明胶（Gel）和羟基磷灰石（HA）纳米纤维组成的多层结构，先前的研究表明这可以改善成骨作用，作为研究骨愈合过程中成骨作用和血管生成相互作用的平台。我们采用 Transwell 共培养、划痕愈合实验、酶联免疫吸附测定（ELISA）、实时定量聚合酶链反应（qRT-PCR）、Western 印迹和免疫组织化学染色等联合技术，评估在不同 Ti 基底上生长的脂肪间充质干细胞（Ad-MSCs）和人脐静脉内皮细胞（HUVEC）的黏附、形态和迁移。我们研究了基底对 Ad-MSCs 成骨分化的影响，以及 Ad-MSCs 对 HUVEC 或反之的旁分泌效应。多层 Ti 基底通过细胞-基质相互作用增强旁分泌作用，直接调节 Ad-MSCs 和血管生成 HUVEC 的细胞功能，并介导它们之间的交流。体内结果表明，表面改性 Ti 植入物引起的微环境变化促进了 MSC 的黏附、募集和增殖，并促进了骨愈合过程中的成骨和血管生成的偶联。该研究证明，多层膜涂层 Ti 基底可在体外积极介导细胞生物学功能，并改善体内骨愈合。
最近的研究表明，成骨作用和血管生成作用是偶联的，成骨细胞和内皮细胞之间的交流对于骨愈合和重塑过程至关重要；然而，这些结论仅来自于体外研究或使用转基因鼠模型的体内研究。关于骨愈合过程中在植入物周围的成骨细胞和内皮细胞之间的交流知之甚少。我们的研究结果揭示了多层 Ti 基底如何介导脂肪间充质干细胞和人脐静脉内皮细胞之间的相互旁分泌作用的细胞/分子机制；此外，体内实验证明了细胞-基质与植入物周围的相互作用，并增强了骨愈合。该研究有助于我们理解影响植入物周围的血管生成和成骨作用的基本机制，从而为设计未来高质量的骨科植入物提供了新的见解。
