Craniofacial bone loss in children is common due to traumatic and congenital causes. Costs attributed to just the repair of craniofacial bone loss in the US currently is estimated to be up to $549 million/year, and there is no regenerative option for pediatric cases. Autologous bone graft harvest is the only surgical approach to replace craniofacial bone and revision surgeries are necessary due to the limited supply. Bone graft harvest requires prolonged operative time, overnight stay in the hospital, hip pain and risk of hip fracture. Current bone regenerative strategies exist for adult spine surgery patients (BMP2); however none exist for children due to potential carcinogenicity of BMP2 in children. Our group identified Jagged1 as a potential bone regenerative therapy based on in vitro and in vivo studies following deletion of Jagged1 from the cranial neural crest (CNC) (JAG1CKO). Human mutations in JAGGED1 cause Alagille syndrome which has cardiac, biliary and craniofacial bone loss phenotypes and our model is a phenocopy of Alagille craniofacial bone loss. Our team recently described that Jagged1 delivery in vitro and in vivo induces mineralization and ossification through non-canonical Notch signaling suggesting that Jagged1 therapy is a potential bone regenerative strategy. Our long-term goal is to develop bone regenerative approaches to treat craniofacial bone loss in children. The overall objective in this application is to engineer, characterize and test a Jagged1-hydrogel as pre-clinical data to develop human bone regenerative therapies. The central hypothesis is that Jagged1-hydrogel delivery can induce human pediatric bone progenitors (HBO) to terminally differentiate to form bone through a Notch non- canonical pathway using in vitro and in vivo pre-clinical models. The rationale for the proposed research is that the development of bone regenerative Jagged1 osteoinductive hydrogels is the first step towards a novel bone tissue engineering solution for children. Guided by strong preliminary data, including a novel non-canonical pathway, the hypothesis will be tested by pursuing two specific aims: 1) Engineer Jagged1-presenting hydrogel to induce pediatric HBO to form bone through Notch non-canonical signaling in vitro; 2) Test the in vivo ability of Jagged1 hydrogels to regenerate bone in a pre-clinical animal model of craniofacial bone loss. In Aim 1 we will engineer a synthetic hydrogel to present Jagged1, and then block canonical Notch signaling to test and characterize the optimal cell-instructive cues for HBOs to mineralize through the Jagged1 non-canonical pathway. In Aim 2 we will test the ability of optimized Jagged1-hydrogels in vivo to induce bone formation using HBO cells in a murine craniofacial traumatic bone loss model and induce mesenchymal stem cells to induce bone in an in vivo pre-clinical animal model. The proposed research is innovative in that we will develop a novel Jagged1-hydrogel therapy targeting the Notch no...