A novel metformin-nanomineral scaffold as enhancer of craniofacial bone regeneration and angiogenesis via dental pulp stem cells

项目总结 这项建议的长期目标是开发广泛适用、成本效益高的骨组织工程平台， 将二甲双胍与干细胞相结合，可再生大型、临界大小的口腔和颅面部骨骼缺损。虽然组织 使用干细胞、支架和生长因子的工程学为自体骨提供了一种有吸引力的、侵入性较小的替代方案 移植物的成功很大程度上取决于细胞对局部缺氧微环境的适当适应和重建。 有功能的微血管系统。血管内皮生长因子(VEGF)是一种重要的血管内皮细胞生长因子。 骨再生中的成骨/血管生成偶联。因此，开发新的和负担得起的基于干细胞的组织工程 增强血管内皮生长因子介导的血管生成的策略可能显著促进骨骼再生。我们的团队和其他人 最近有报道称，全球数百万糖尿病患者使用的低成本药物二甲双胍可以诱导成骨细胞 来自不同组织来源的干细胞的分化。这表明二甲双胍可以在局部改变用途 提供配方，以加强干细胞为基础的骨再生。我们提出了这一概念，通过配制一种钙 含有二甲双胍的磷酸盐骨水泥(CPC)在培养中释放时上调成骨细胞的表达 牙髓干细胞(DPSCS)的标志物和矿化细胞外沉积增加，这是一种易于获取和 取之不尽、用之不竭的后天干细胞。耐人寻味的是，我们发现二甲双胍也能显著增加 在低氧条件下，血管内皮生长因子的分泌在DPSCS中进一步放大。二甲双胍的成骨作用有 与AMP激活的蛋白激酶(AMPK)信号通路的激活有关，这是一种主要的感觉 细胞生物能量学的机制。而在肝细胞中，二甲双胍通过激活 AMPK通过上游的肝细胞激酶B1(LKB1)，这是一个机制上的，翻译相关的问题，仍然存在 难以捉摸的是DPSCs是否主要依赖LKB1来促进骨形成和新生血管对局部的反应 二甲双胍缓释片。我们将检验基于DPSC的颅面骨再生和骨再生的中心假设 AMPK通过一种功能性的， 具有催化活性的LKB1。体外和体内研究将通过两个特定的目标来扩展我们的结果。目标1将 在DPSCS中，从CPC支架中释放的二甲双胍是否诱导成骨和促血管生成反应 LKB1/AMPK依赖方式。目标2将检验以下假设：在DPSCS中，具有功能的LKB1/AMPK细胞 局部注射对促进颅面骨再生和新生血管的反应是必要的 二甲双胍从CPC支架中释放。我们预计我们的结果将产生新的、有价值的基本和翻译 将导致具有成本效益的组织工程平台的信息，其中二甲双胍负载支架组合 干细胞将促进颅面和骨科的骨再生。

PROJECT SUMMARY The long-term goal of this proposal is to develop widely applicable, cost-effective bone tissue engineering platforms that combine metformin with stem cells to regenerate large, critical-sized oral and craniofacial skeletal defects. Although tissue engineering using stem cells, scaffolds and growth factors offers an attractive, less invasive alternative to autologous bone grafts, its success highly depends on the proper adaptation of cells to a local hypoxic microenvironment, and reestablishment of a functional microvasculature. It is well established that vascular endothelial growth factor (VEGF) is a key mediator of osteogenic/angiogenic coupling in bone regeneration. Thus, developing novel and affordable stem cell-based tissue engineering strategies that potentiate VEGF-mediated angiogenesis may significantly enhance skeletal regeneration. Our group and others recently reported that metformin, a low-cost drug used by millions of diabetics worldwide induces the osteoblastic differentiation of stem cells derived from various tissue sources. This suggests that metformin could be repurposed in a local delivery formulation to potentiate stem cell-based bone regeneration. We have advanced this concept by formulating a calcium phosphate cement (CPC) containing metformin that when released in culture upregulated the expression of osteogenic markers and increased mineralized extracellular deposits in dental pulp stem cells (DPSCs), an easily accessible and inexhaustible source of postnatal stem cells. Intriguingly, we have found that metformin also induces a significant increase in VEGF secretion that is further amplified in DPSCs exposed to hypoxic conditions. The osteogenic action of metformin has been associated with the activation of the AMP-activated protein kinase (AMPK) signaling pathway, a master sensing mechanism of cellular bioenergetics. While in hepatocytes, metformin reduces high blood glucose production by activating AMPK via the upstream kinase liver kinase B1 (LKB1), a mechanistic, translationally relevant question that still remains elusive is whether DPSCs rely mainly on LKB1 to enhance bone formation and neovascularization in response to locally delivered metformin. We will test the central hypothesis that DPSC-based craniofacial bone regeneration and neovascularization in response to locally delivered metformin is enhanced by AMPK activation through a functional, catalytically active LKB1. In vitro and in vivo studies will expand our results through two specific aims. Aim 1 will determine whether in DPSCs, metformin released from CPC scaffolds induces osteogenic and pro-angiogenic responses in an LKB1/AMPK-dependent manner. Aim 2 will test the hypothesis that in DPSCs, a functional LKB1/AMPK cellular response is necessary to enhance craniofacial bone regeneration and neovascularization in response to locally delivered metformin released from CPC scaffolds. We anticipate our results will yield new, valuable basic and translational information that will lead to cost-effective tissue engineering platforms where metformin-loaded scaffolds in combination with stem cells will enhance craniofacial and orthopedic bone regeneration.