PROJECT SUMMARY This K01 award describes a 3-year training program to support Dr. Franca's transition to scientific independence. Dr. Franca is a dentist-scientist, and her long-term goal is to lead a research program to (i) develop immunomodulatory biomaterials for dental and craniofacial regeneration and (ii) to create tissue chips to understand and treat oral diseases. The scientific goal of this K01 is to develop an immunomodulatory scaffold for dental pulp regeneration. Despite undeniable progress to develop regenerative strategies for the dental pulp, necrotic teeth present challenging conditions for regeneration. Even after careful disinfection, a previously infected root canal's microenvironment has remnant lipopolysaccharide (LPS) that activates immune cells into a pro-inflammatory state incompatible with tissue regeneration. Recently, early events of the immune response were shown to highly influence tissue engineering strategies' success. Especially, macrophage responses to biomaterials have been identified as essential mediators of scaffold remodeling and promising targets for immune-mediated regenerative strategies. These cells can respond to external stimuli by changing their phenotype from pro-inflammatory to pro-regenerative, with many intermediate states, in a process known as M1/M2 polarization. These two states are highly plastic, function synergistically, and can be modulated by the scaffold's properties. When a scaffold for dental pulp regeneration is placed in contact with LPS-contaminated dentin, it can be expected that M1 activation will persist without providing the pro-regenerative environment necessary for tissue formation. Small antibodies or nanobodies have been used to neutralize LPS in Gram-negative infections and target pathogens in scarcely accessible tissue microenvironments. Unfortunately, this therapeutic tool has not been used for dental regeneration purposes. Our overarching hypothesis is that an immunomodulatory scaffold that neutralizes LPS and increases the M2/M1 ratio in the scaffold-tissue interface will provide a sustainable pro-regenerative microenvironment more favorable to dental pulp tissue formation. Hence, our goal is to engineer a novel nanobody-laden photocrosslinkable hydrogel with fine-tuned stiffness and the ability to neutralize dentin LPS. This scaffold will enable controllable guidance of M1 macrophages activation, minimizing their presence within the root canal so that incoming monocytes can polarize into M2 cells, infiltrate the hydrogel, and secret an anti-inflammatory cytokine milieu to foster dental pulp tissue formation. We propose the following aims: (Aim 1) to determine the effect of scaffold's stiffness on M1/M2 macrophage polarization, (Aim 2) to neutralize dentin LPS and reduce M1 polarization, and (Aim 3) to evaluate the nanobody-laden scaffold's immunomodulatory capacity in vivo. Successful completion of these aims will generate data on the interplay between scaffold's propert...