Project Summary Stem-cell based strategy has been widely considered as a promising approach to replace damaged pulp and dentin structures and restore their biological functions in regenerative endodontics. However, regeneration of functional tubular dentin has been a challenge owing to the lack of understanding dental pulp stem cell (DPSC) polarization and differentiation as well as the underlying mechanism that are prerequisite for tubular dentin regeneration. A unique biomimetic 3D platform was recently developed to study DPSC polarization and differentiation in the PI’s group. This platform can precisely manipulate single cells on each microisland of the 3D platform and is an excellent tool to decipher the biophysical and biochemical signals that initiate and regulate DPSC polarization at the single cell level. A variety of biophysical factors were screened using this 3D platform, and the nanofibrous architecture and tubular structure of the matrix were identified to be the two critical biophysical factors that initiate and regulate DPSC polarization. The combination of RNA-seq with laser microdissection techniques and a conditional knockout model further identified that TGF-1 is a crucial biochemical factor for odontogenesis. Consequently, the hypothesis of this work is that DPSC polarization is controlled by a dynamic signaling network composed of a set of critical biophysical and biochemical factors, and integration of these factors in scaffolding design will regenerate tubular dentin from DPSCs. Therefore, the overall objective of this proposal is to explore and optimize the parameters that initiate/modulate DPSC polarization, understand the underlying mechanism of DPSC polarization, and regenerate tubular dentin in vivo. To accomplish the overall objective for this project, three specific aims are proposed. Aim 1 will explore how the biomimetic tubular matrix modulates DPSC polarization and differentiation. Aim 2 will examine how TGF-1 regulates DPSC polarization and differentiation. Aim 3 will integrate TGF-1 into the biomimetic tubular matrix to regenerate tubular dentin in vivo. Successfully completing this work will fundamentally advance the understanding of DPSC polarization and differentiation, and greatly promote the ability to develop new bio- inspired matrices to regenerate functional tubular dentin for regenerative dental therapies.