Extracellular Matrix Vesicles and Intercellular Communication of Osteoblast-lineage Cells

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PROJECT SUMMARY Autologous bone grafting, the current gold standard for treating critical sized bone defects and chronic nonunions, has limited success, leaving a need for adjunct treatments to contribute to overall bone healing. Extracellular vesicles, of which there are many types, are a promising new cell-free, membrane-bound therapeutic in the field of regenerative medicine. Specific to the field of bone regeneration and repair are matrix vesicles; small extracellular vesicles released by mineralization-competent cells that become anchored in the extracellular matrix and are a necessary component of mineralized bone formation via endochondral ossification. They are involved in cellular signaling via small, non-coding microRNA in the growth plate, suggesting that they may play a similar role in bone. Previous research indicates that chondrocyte-derived matrix vesicles are enriched with microRNA and other cell signaling molecules that contribute to their ability to influence proliferation and differentiation of target cells, however the role and mechanism of action of osteoblast-derived matrix vesicles, particularly in bone as opposed to the growth plate, remains to be elucidated. With this information, the biological mediation of bone development and regeneration that matrix vesicles provide may translate into novel therapeutic potential for orthopedic pathologies, including critical size bone defects and fracture nonunions. Therefore, we hypothesize that matrix vesicles produced by osteoblast-lineage cells, as a specific subset of extracellular vesicle, use their microRNA cargo to enhance osteogenic differentiation and proliferation, drive osteogenesis, and aid in bone defect healing. First, we aim to determine the relationship between osteoblast- derived matrix vesicles and another class of extracellular vesicle, the exosome. Next, we aim to determine the pathways targeted by matrix vesicle microRNA cargo. Finally, we aim to determine the extent to which osteoblast-derived matrix vesicles modulate osteogenesis and bone defect healing. To test these aims, we will use osteoblast-lineage cells to derive matrix vesicles and characterize protein expression, size, and morphology. We will also use in vitro experimental models to assess pathway involvement and evaluate co-culture response with osteoblast-like cells. To test the therapeutic potential, we will use a translatable in vivo model of a mouse long bone defect and a biorthogonal injectable hydrogel to deliver matrix vesicles. We expect to find that osteoblast-derived matrix vesicles are a specialized subclass of extracellular vesicle with microRNA cargo that targets the canonical Wnt pathway to activate cellular signaling and leads to increased osteoblastic differentiation of target cells when co-cultured in vitro. Our in vivo model is expected to demonstrate improved healing upon treatment with hydrogel-delivered matrix vesicles. All of which together demonstrates the role matrix vesicles play in the coordinated effort to form new bone and their viability as a therapeutic option to improve bone healing.