PROJECT SUMMARY / ABSTRACT Reparative dentinogenesis occurs after intense injury that leads to odontoblast death (i.e., deep caries or pulp exposure). During this process, signaling factors sequestered in the dentin matrix and pulp-supportive tissue are released and activate the recruitment, migration of resident Mesenchymal Stem cells (MSCs) in dental pulp to the site of injury. At the site of injury, these MSCs differentiate and give rise to cells secreting reparative dentin, also called osteodentin. Reparative dentin is a thin band of poorly organized mineralized tissue containing a mixture of non-columnar odontoblast-like and osteoblast-like cells. Although the reparative dentin/osteo-dentin provides an impermeable hard-tissue barrier to the dental pulp, its histological and molecular characteristic is very different from physiological dentin and cannot be considered a true regeneration of tubular dentin devoid of osteoblasts. The overall goal of proposed studies is to test the hypothesis that signaling pathways induced by trauma/inflammation to the pulp during pulp exposures results in abnormal and sustained activation of Runx2 in dental pulp. The sustained activation of Runx2 leads to impaired odontoblasts differentiation and accumulation of osteoblasts at the site of injury and together contribute to the formation of atubular and poorly organized mineralized reparative dentin. To address this sequence, we will use examine the effects of conditional modulation of levels of Runx2 (loss of function and gain of functions) on the late stages of odontoblast and osteoblasts differentiation. Three specific aims have been proposed. Experiments in Aim #1 and Aim 2 will examine the effects of conditional modulation of levels of Runx2 (loss of function and gain of functions) in odontoblasts and osteoblasts on the late stages of odontoblast (Aim 1) and osteoblast (Aim 2) differentiation during reparative dentinogenesis in vivo and during mineralization of primary pulp cultures. Experiments in Aim #3 will examine the effects of impaired odontoblast differentiation and accumulation of osteoblasts during reparative dentinogenesis the organization of collagen fibers and tubular vs. atubular dentin. Ultimately, the results of this study will provide a better understanding of mechanisms leading to reparative dentinogenesis and will be critical for the development of improved treatments for vital pulp therapy and improved dentin regeneration.