Local muscle loss associated with open fractures remains an obstacle to functional recovery and bone healing. Muscle cells secrete bioactive myokines that elicit autocrine and paracrine effects and initiate signaling pathways for regenerating damaged muscle and bone. Mesenchymal stem/stromal cells (MSCs) are under investigation for the regeneration of both muscle and bone through their potent secretome. Compared to monodisperse cells, MSC spheroids exhibit a more complex secretome with heightened therapeutic potential. We hypothesized that the osteogenic potential of myokines would be enhanced when myoblasts were exposed to the MSC spheroid secretome. Conditioned media from MSC spheroids increased osteogenic response of MC3T3 pre-osteoblasts compared to myokines from L6 myoblasts alone. This effect was synergistically enhanced when conditioned media of MSC spheroids was serially delivered to myoblasts and then osteoprogenitor cells in vitro. We then delivered myoblast-stimulated conditioned media in the presence or absence of syngeneic rat bone marrow stromal cells (rBMSCs) from alginate hydrogels to a rat critical-sized segmental defect. We observed increased bone formation in defects treated with conditioned media compared to rBMSCs alone, while bone formation was greatest in defects treated with both conditioned media and rBMSCs over 12 weeks. This foundational study demonstrates a novel approach for capitalizing on the paracrine signaling of muscle cells to promote bone repair and provides additional evidence of the synergistic interaction between muscle and bone.

BACKGROUND: Osteosarcoma (OS) survival rates and outcome have not improved in 50 years since the advent of modern chemotherapeutics. Thus, there is a critical need for an improved understanding of the tumor microenvironment to identify better therapies. Extracellular matrix (ECM) deposition and hypoxia are known to abrogate the efficacy of various chemical and cell-based therapeutics. Here, we aim to mechanistically investigate the combinatorial effects of hypoxia and matrix deposition with the use of OS spheroids. METHODS: We use two murine OS cell lines with differential metastatic potential to form spheroids. We form spheroids of two sizes, use ascorbate-2-phosphate supplementation to enhance ECM deposition, and study cell response under standard (21% O2) and physiologic (5% O2) oxygen tensions. Finally, we examine chemotherapeutic responses to doxorubicin treatment. RESULTS: ECM production and oxygen tension are key determinants of spheroid size through cell organization based on nutrient and oxygen distribution. Interestingly, highly metastatic OS is more susceptible to chemotherapeutics compared to less metastatic OS when matrix production increases. Together, these data suggest that dynamic interactions between ECM production and oxygen diffusion may result in distinct chemotherapeutic responses despite inherent tumor aggressiveness. CONCLUSION: This work establishes OS spheroids as a valuable tool for early OS tumor formation investigation and holds potential for novel therapeutic target and prognostic indicator discovery.