PROJECT SUMMARY/ABSTRACT Facial disfigurement can have devastating effects on one’s quality of life. Approximately 0.26 million Americans per annum have reconstructive procedures, such as the use of tissue flaps, to correct congenital and acquired craniofacial defects. Management of large volumetric craniofacial muscle tissue defects remains a challenge. Up to one quarter of patients have repeat procedures due to flap failure, tissue rejection and limited availability of tissue; and there remains a subset of patients for whom treatment completely fails resulting in continued facial disfigurement, financial burden and challenges with societal integration. The ability to generate craniofacial muscle tissue containing the patient’s own cells would provide a more predictable, life-changing treatment for a serious problem. Previous work has demonstrated the importance of craniofacial muscle- derived cells and scaffolds for engineering the craniofacial skeletal muscles. The project proposes to engineer craniofacial muscle tissue by seeding 3-D printed biomimetic scaffolds with porcine craniofacial muscle-derived cells. The goals are to (1) generate new knowledge on the use of craniofacial muscle-derived cell populations for the formation of muscle tissue; (2) produce 3-D biomimetic scaffolds to support craniofacial muscle development; (3) bioengineer craniofacial muscle tissue for implantation. The vision is that permanent restoration of craniofacial soft tissue defects can be achieved by implantation of precision-engineered autologous craniofacial skeletal muscle tissue. The hypothesis is that successful craniofacial muscle tissue engineering applications will incorporate autologous craniofacial skeletal muscle-derived cells, 3-D printed biomimetic scaffolds, application of mechanical load and insulin-like growth factor 1 (IGF-1), a growth factor important for muscle cell proliferation and differentiation. The approach is to enrich porcine craniofacial muscle-derived cell populations with muscle stem cells (satellite cells) responsible for regeneration. The cells will be seeded into 3-D printed biomimetic scaffolds and subjected to load and IGF- 1 to improve regeneration and promote muscle fiber hypertrophy leading to a tissue suitable for implantation into the craniofacial region. Aim 1 is to produce 3-D printed biomimetic scaffolds and assess response of an enriched porcine craniofacial muscle-derived cell population within the scaffolds. Aim 2 is to determine response of engineered porcine craniofacial skeletal muscle tissue to mechanical stimulation and IGF-1 delivered within customized bioreactors. Aim 3 is to determine the regenerative capability of engineered porcine craniofacial skeletal muscle tissue in vitro and in vivo. This project ultimately aims to produce a functional craniofacial tissue for restoration of craniofacial soft tissue defects through the combination of three innovative elements: (1) craniofacial muscle-derived cell populati...