Pre-vascularization of an Integrated 3D Human Osteo-Mucosal Model - Summary: This proposal builds upon our recent completion of an NIH grant titled “Development of an Integrated 3D Human Osteo-Mucosal Model,” for which our team stands as the sole entity to have successfully developed this model. During that project, we engineered an osteo-mucosal construct, overcoming various challenges, particularly in establishing robust adhesion between the soft and hard tissue sections. Now, with this proposal, we aim to bridge the gap between our previous grant and future directions. More specifically, given that the osteo-mucosal construct is inherently critical-sized, the challenge of vascularization hinders the advancement of this research for pre-clinical examinations. It is evident that non-vascularized constructs are prone to failure when implanted into critical-sized defects. Therefore, this proposal is dedicated to addressing this challenge by focusing on the pre-vascularization of the previously developed osteo-mucosal construct and evaluating its effectiveness in vivo. To achieve this goal, we will implement key angiogenesis strategies that synergistically activate pre- vascularization. More specifically, we will harness the synergistic effects of intracellular and extracellular mechanisms to further promote pre-vascularization. Specifically, we will utilize FG-4592 (Roxadustat), a hypoxia- inducible factor prolyl hydroxylase inhibitor (HIF-PHI), known for its intracellular mechanism. FG-4592 stabilizes HIF-1α by inhibiting HIF-prolyl hydroxylases, thereby preventing its protease degradation within the cells. This process facilitates the expression of relevant genes and promotes angiogenesis. Notably, FG-4592 is currently undergoing global Phase III clinical trials for the treatment of anemia. In addition to FG-4592, our model incorporates an aptamer targeting an extracellular mechanism. This aptamer binds effectively to both VEGF receptors 1 and 2 (VEGFR-1 and -2), with a notably stronger affinity for the latter, thereby facilitating the promotion of angiogenesis. The osteo-mucosal construct will be created using 3D-printed scaffolds loaded with these pro-angiogenic factors and seeded with HUVECs and supporting cells. The research involves comprehensive in vitro testing to promote blood vessel formation, followed by in vivo studies using a rat alveolar bone model with overlaying gingiva to evaluate the construct's integration with host tissue, and formation of well-defined and interconnected vascular networks. The success of this approach will be measured by the density and functionality of the blood vessels with capillary density of 50-100 vessels/mm² in bone tissue and 30-60 vessels/mm² in mucosal tissue. The osteo-mucosal complex presents one of the most challenging multi-tissue defects. It represents a key example of critical-sized defects, and its successful regeneration can establish a robust approach for treating other critical-sized defects involving both hard and soft tissues. Furthermore, a laboratory-grown osteo-mucosal construct derived from a patient's own cells proves immensely beneficial for oral surgeries. Moreover, it offers an alternative to animal models for studying biomaterial-oral tissue interactions and for screening oral diseases.
