Biophysical signals, biomaterial surface characteristics and hMSC differentiation

描述（由申请人提供）：随着人口老龄化，对骨移植修复骨缺损的需求正在迅速增加。使用自体移植物和同种异体移植物已成功治疗骨缺损。然而，这些都不是理想的方法，因为自体移植物的可用性有限，并且可能导致供体部位发病，而同种异体移植物可能会被免疫排斥并有可能传播疾病。使用假定的人间充质干细胞（hMSC）与生物相容性支架相结合，在治疗骨缺损方面具有巨大潜力，否则只能通过自体移植或同种异体移植来治疗骨缺损。不幸的是，hMSC 的体外扩增虽然可以增加 hMSC 的治疗潜力，但却降低了它们的成骨潜力。在体外扩增 hMSC，同时保持甚至增强其成骨潜力的能力，可以大大增强其治疗潜力。因此，该项目的目标是确定特定的生物材料表面特征和生物物理信号，它们在优化 hMSC 向成骨细胞谱系的分化过程中协同相互作用。我们的总体假设是生物材料的表面特征，特别是纳米级的形貌，使细胞对流体流动敏感，从而增加流体流动对 hMSC 扩张的影响，同时也增强 hMSC 的成骨潜力。利用独特的流体流动方案、新颖的生物材料表面特征、原子力显微镜、基因工程和转基因动物模型，我们将确定一个优化 hMSC 向成骨细胞谱系分化的环境以及参与该机制的信号转导途径。然后我们将检查这些预处理的 hMSC 是否比未预处理的 hMSC 在体内更具成骨性。我们将通过在 5 年时间内完成 4 个目标来实现这一目标： 目标 1，在存在或不存在特定信号通路抑制剂的情况下，检查表面形貌对 hMSC 粘附、增殖和分化的影响；目标 2，确定 hMSC 在具有不同纳米级形貌的表面上的刚度和机械敏感性；目标 3，检查在存在和不存在 PLC/钙调神经磷酸酶和 PLC/ERK 信号通路抑制剂的情况下流体流动对 hMSC 增殖和分化的影响；目标 4，检查接种到 HA/TCP 支架上的 hMSC 的体内成骨作用。通过完成这些目标，我们不仅将开发骨组织工程的新策略，还将提供 hMSC 增殖和分化调节的机制见解。公共卫生相关性：随着老年人口的增加，对肌肉骨骼病理学新治疗方法的需求也将增加。利用成体干细胞的组织工程就是这样的方法之一。该项目将开发结合纳米技术、成体干细胞和生物物理力的新型肌肉骨骼组织工程方案，从而制定替代疾病、损伤和衰老导致的骨质流失的策略。

DESCRIPTION (provided by applicant): The need for bone grafts to repair bone defects is rapidly accelerating as our population ages. Bone defects have been successfully treated using autografts and allografts. However, these are less than ideal approaches since autograft availability is limited and can result in donor-site morbidity while allografts can be immunologically rejected and have the potential to transmit disease. The use of putative human mesenchymal stem cells (hMSC) combined with biocompatible scaffolds has great potential for treating bone defects, which would otherwise be treated with autografts or allografts. Unfortunately, the expansion of hMSC in vitro, which could increase the therapeutic potential of hMSC, reduces their osteogenic potential. The ability to expand hMSC in vitro while maintaining, or even enhancing, their osteogenic potential, could greatly enhance their therapeutic potential. Therefore, the goal of this project is to identify specific biomaterial surface characteristics and biophysical signals that interact synergistically in optimizing hMSC differentiation toward the osteoblastic lineage. Our overall hypothesis is that biomaterial surface characteristics, specifically nanoscale topography, sensitize cells to fluid flow thus increasing the effect of fluid flow on expansion of hMSC while also enhancing the osteogenic potential of hMSC. Using unique fluid flow protocols, novel biomaterial surface characteristics, atomic force microscopy, genetic engineering and transgenic animal models we will identify an environment that optimizes differentiation of hMSC towards the osteoblastic lineage and the signal transduction pathways involved in this mechanism. We will then examine whether these pre-treated hMSC are more osteogenic in vivo than non pre-treated hMSC. We will accomplish this through the completion, over a 5 year period, of 4 aims: Aim 1, Examine the effect of surface topography on adhesion, proliferation and differentiation of hMSC, in the presence and absence of inhibitors of specific signaling pathways; Aim 2, Determine stiffness and mechanosensitivity of hMSC on surfaces with varying nanoscale topographies; Aim 3, Examine the effect of fluid flow, in the presence and absence of inhibitors of the PLC/calcineurin and PLC/ERK signaling pathways, on hMSC proliferation and differentiation; and Aim 4, Examine in vivo osteogenesis of hMSC seeded onto HA/TCP scaffolds. By completing these aims we will not only develop novel strategies for bone tissue engineering but also provide mechanistic insight into the regulation of hMSC proliferation and differentiation. PUBLIC HEALTH RELEVANCE: As the aged population increases the need for novel therapeutic approaches to musculoskeletal pathology will also increase. Tissue engineering exploiting adult stem cells is one such approach. This project will develop novel musculoskeletal tissue engineering protocols combining nanotechnology, adult stem cells and biophysical forces that will lead to strategies to replace bone loss to disease, injury and aging.