A majority of all fractures in current US armed conflicts are open fractures, in which a soft tissue injury is sustained along with the bone fracture. Even with gold standard treatment, in which muscle flaps are used to cover bony defects, patients often do not regain normal function of their extremity, highlighting the necessity for tissue engineering strategies for this complex clinical problem. Due to a substantial amount of tissue damage and debridement treatment in composite injuries, a large volume of cells and extracellular matrix (ECM) proteins that are necessary for tissue healing are removed from the body. In the replacement of large volumes of tissue, nutrient transfer necessitates a vascular supply to maintain the viability of delivered cells. The objective of this project was to examine the regenerative potential of engineered matrix constructs and stem cells on composite bone & muscle defects. We hypothesized that stem cells delivered on engineered matrix constructs into the muscle defect will aid in muscle regeneration and promote bone healing, ultimately resulting in superior functional limb recovery. These studies established multiple preclinical platforms for testing tissue engineering strategies as well as models that can be used to gain insights on the healing of VML and composite VML/bone defects. From some of the insights gained on the vascularization of the defect sites, a vascular treatment strategy was tested within these platforms and shown to have varying results in the treatment of complex multi-tissue injuries.

Severe extremity trauma often involves significant damage to multiple tissue types, including bones, skeletal muscles, peripheral nerves, and blood vessels. Such injuries present unique challenges for reconstruction, and improving structural and functional outcomes of intervention remains a pressing, unmet clinical need. While tissue engineering/regenerative medicine (TE/RM) therapeutics offer promising potential to overcome the status quo limitations of surgical reconstruction, very few products have transitioned to clinical practice. Improving treatment options will likely require advancing our understanding of the biological interactions occurring in the repair of damaged tissues. Bone tissue is known to be innervated and highly vascularized, and both tissue types are involved in normal bone physiology. However, the degree to which these tissue relationships influence the repair of large, multi-tissue defects remains unknown. Accordingly, the goal of this thesis was to investigate tissue regeneration in two novel composite injury models. First, we characterized interactions in a composite bone and nerve injury model where a segmental bone defect was combined with a peripheral nerve gap. Our results indicated that although tissue regeneration was not impaired, the composite injury group experienced a marked functional deficit in the operated limb compared to single-tissue injury. Second, we developed a model of composite bone and vascular extremity trauma by combining a critically-sized segmental bone defect with surgically-induced hind limb ischemia to evaluate the effects on BMP-2-mediated bone repair. Interestingly, our results demonstrated a stimulatory effect of the recovery response to ischemia on bone regeneration. Finally, we investigated early vascular growth and gene expression as potential mechanisms coupling the response to ischemia with bone defect repair. Although the response to ischemia promoted robust vascular growth in the thigh, it did not directly augment vascularization at the site of bone regeneration. In addition, the stimulatory effects of ischemia on bone regeneration could not be explained by gene expression alone based on the genes and time points investigated. Taken together, this thesis presents pioneering work on a new thrust of TE/RM research – tissue regeneration in models of composite injury. This work has provided new insights on the complexity of composite tissue repair, specifically in regard to the relationship between vascular tissue growth and bone healing. Going forward, successful leverage of models of composite tissue injuries will provide valuable test beds for screening new technologies, advance the understanding of tissue repair biology, and ultimately, may produce new therapeutic interventions for limb salvage and reconstruction that improve outcomes for extremity trauma patients.