Project Summary/Abstract The ability to regenerate tissue in mammals has remained elusive. While the use of stem cell populations in the context of bio-scaffolds has shown promise as a potential means of replacing lost, damaged, or diseased tissue, significant challenges remain. An alternative approach is to attempt to evoke a classical in situ regenerative response emulating that seen in lower species such as newts. While this trait was thought to be lost in evolution, our observation (Heber-Katz) that the MRL mouse and related strains have a significant spontaneous regenerative capability demonstrates that the trait is retained in mammals. Studies over the past almost 25 years have culminated in the identification of the HIF-1 (hypoxia inducible factor) pathway as the central actor regulating regeneration in mice. The up-regulation of HIF-1 in non-regenerating mice have allowed these mice to regenerate ear holes with cartilage and hair follicles; and jaw bone and associated soft tissues after induced periodontal disease (PD). In both cases, a classical regenerative de-differentiation response was seen. This was achieved using the PHD inhibitor 1,4-DPCA in novel injectable biomaterial constructs (Messersmith) leading to the stabilization of high levels of HIF-1 in vivo. The mechanisms that were identified in the PD model involved inflammatory cytokines, bone remodeling osteoblasts, Tregs and CXCR4 expression. Using a CXCR antagonist, AMD3100, the regenerative response could be blocked and supports the need for Tregs and certain stem and myeloid cells. In this current proposal, we provide preliminary results showing that two new upstream target molecules DEL-1 (Hajishengallis) and PAR1 (Heber-Katz) with known agonists have been identified which affect HIF-1 expression. We will explore the role of HIF-1 and these two molecules in the regenerative response in young and aged mice using both the ear hole model and the PD model. We will use advanced molecular design to produce a biomaterial capable of achieving single dose and local delivery vs the current three dose delivery system. In addition to yielding a novel soft and bone tissue regeneration therapy, we believe that this system provides an impressive landscape of phenomena that will yield important mechanistic information about in-situ regenerative responses in oral tissues. In Aim 1, we will create new biomaterials to yield local drug release using mucoadhesive polymers; in Aim 2, we will examine the role of the anti-inflammatory molecular DEL-1 in aged mice with PD, a molecule having direct affects on HIF-1 levels; and in Aim 3, we will examine the role of PAR1, another molecule which affects HIF-1 levels in the ear hole and periodontal injury models, in both young and aged mice.