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 ...