Anti-Inflammatory Mesenchymal Stem Cell Therapy for Dental Applications Dr. Vining is a general dentist and Ph.D. student, training with Professor David Mooney at Harvard School of Engineering and Applied Sciences. Dr. Vining's long-term research goal is to address how biophysical parameters of the extracellular microenvironment can be used to develop novel anti-inflammatory therapies in oral and craniofacial health. Specifically, the proposed research will examine whether mechanical stiffness of the microenvironment can be used to control the anti-inflammatory function of MSCs for therapeutic use in oral and craniofacial health. Dr. Vining is committed to become an independent clinician-scientist, so he seeks this mentored clinical scientist award to further his growth and development as a clinician-scientist in oral health sciences and engineering. The proposed research plan will investigate the hypothesis that hydrogel stiffness can be used to control the anti-inflammatory paracrine function of encapsulated bone marrow-derived MSCs in vitro and to enhance their in vivo function in a rodent dental pulp injury model. In Specific Aim 1, Dr. Vining will examine how the level of mechanical stiffness affects the immunomodulatory secretory and transcriptional behavior of adult human bone marrow-derived MSCs in a collagen-alginate interpenetrating network hydrogel. The MSCs' behavior will be characterized in varying matrix stiffness by measuring expression and secretion of pro- and anti-inflammatory factors. Based on the optimal matrix stiffness in bulk hydrogels from Aim 1, in Specific Aim 2 Dr. Vining will investigate the in vitro functional effects of human bone marrow-derived MSCs on human macrophages M1 versus M2 surface markers. In Specific Aim 3, Dr. Vining will investigate the in vivo anti-inflammatory function of MSCs, which are tuned by the stiffness of their microenvironment to increase secretion of anti-inflammatory factors as shown in Aims 1 - 2. He will test the mechanically-tuned human MSCs in vivo by locally injecting MSCs encapsulated in the hydrogel from Aims 1 - 2 into a rodent dental pulp injury model. MSCs' functional effects on the host inflammatory response will be measured by a myeloperoxidase probe, histology and immunohistochemistry, and gene expression. Despite being widely investigated in medicine, MSC immunotherapies have largely been overlooked in dentistry. Dr. Vining proposes to apply these findings to develop a novel MSC-immunotherapy for oral and craniofacial applications. These aims will have an impact on basic science knowledge by exploring how biophysical cues modulate MSCs' immunomodulatory paracrine signaling. As part of Dr. Vining's career development plan, he will take courses in engineering and applied sciences, as well as cell biology and immunology, to help him achieve his research objectives. Dr. Vining will develop his professional skills through participating in research seminars and study groups and presenting at annual major scientific conferences. Dr. Vining will be mentored by his Ph.D. advisor, Professor Mooney, throughout his Ph.D. thesis via formal, bi-weekly individual meetings. Dr. Kai Wucherpfennig will provide co-mentoring for the immunology aspects of the project. A key component of Dr. Vining's training will involve informal training in the lab via interaction with various members of the Mooney laboratory, and collaborating laboratories at Harvard University. Dr. Vining will learn how to be a good teacher and mentor through teaching activities through Harvard Faculty of Arts and Sciences and School of Engineering and Applied Sciences. This plan lays a framework for Dr. Vining's successful progression from mentored Ph.D. student, to independent post- doctoral researcher, and finally independent principal investigator in a tenure-track academic position.