Regenerative Engineering of Complex Extremity Trauma

Introduction:: Lower extremity trauma is a multifaceted injury consisting of an open bone fracture and soft tissue damage that requires staged surgical management. These kinds of complex injuries are 4-5 times more likely to result in delayed or failed bone union and patients often have poor long-term limb function, chronic pain, and frequent delayed amputation. Current therapies do not effectively restore the pairing of functional muscle and stable bone to restore limb function. Therefore, there is an unmet clinical need for a regenerative approach that can facilitate structural and functional healing of bone, muscle, and the complete limb.

In prior work, we have demonstrated that spatial patterning cues from nanoscale extracellular matrices modulate angiogenesis and skeletal muscle myogenesis. We have shown that when these patterned materials are transplanted into a severe muscle injury in mice, that large volumetric muscle defects can be regenerated and re-innervated comparable to native tissue. With growing evidence of a regenerative inter-dependency of bone and muscle healing outcomes, we hypothesized that a muscle-focused regenerative strategy would support improved tissue and limb healing. Herein we describe the extension of topography mediated myogenesis from a purely muscle injury to a composite muscle/bone mouse injury model.


Materials and Methods:: Engineered muscle scaffolds were fabricated from dialyzed Rat Tail Collagen I and using a shear-based extrusion method. Scaffolds were seeded with green fluorescent protein (GFP+) primary mouse muscle cells that were pre-differentiated into myotubes followed by transplantation into the muscle defect of the complex injury model.

To evaluate the therapeutic efficacy of the engineered muscle, we developed a novel, complex injury mouse model which entails a segmental bone defect and a critically sized muscle injury. Briefly, fractured tibias were stabilized using a custom 3D printed biocompatible resin plate fastened to the tibia with stainless steel screws. This was followed by surgically creating a transverse 2 mm bone defect in the mid-diaphysis of the tibial shaft. The muscle defect was then created by surgically excising 20% of the TA muscle. The muscle and skin were closed with suture and the leg was immobilized with a splint for 7 days to discourage weight bearing. Bone healing and callus formation were monitored by microCT on Day 7, 28 and 56. On Day 56, functional muscle response was measured by in situ muscle physiology. The tibia and TA were harvested for histological analysis of osteogenesis and myogenesis.


Results, Conclusions, and Discussions:: Engineered muscle scaffolds enhanced muscle function and bone formation. The TA muscles receiving engineered muscle scaffolds demonstrated increased TA muscle cross-sectional area (p < 0.01) and a max contractile force (p < 0.05) compared to muscles that were left untreated (. The muscle that did not receive treatment resulted in smaller tissue size, suggestive of muscle atrophy. Decreased bone volume (p < 0.05) and bone mineral density (p < 0.05) of the bone defect adjacent to the muscle treatment was observed compared to the injury without muscle treatment. These readouts indicate improved bone healing in the muscle-treated group due to progressed osteogenesis and more mature chondrification of a fully formed lumen and absence of soft callus tissue. Furthermore, treatment with a muscle scaffold supported greater survival rate (68% improved to 83%) as well as reduced limb morbidity. Studies are currently underway to assess dynamic weight bearing, pain sensitivity, and circulating inflammatory markers/cytokines throughout the injury and healing process. Taken together, treatment of a complex tissue injury with an engineered muscle scaffold, supported the structural and functional regeneration of both injured muscle and bone, which more closely recapitulate native myofibers and ossified bone morphology. Our findings demonstrate a regenerative relationship between muscles healing on bone outcomes and highlights the restorative potential of our approach for the treatment of complex lower extremity injuries.


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