Dual Delivery of Engineered EVs and Growth Factor for Bone Regeneration

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Project Summary/Abstract The inflammatory conditions and the complex physiological changes associated with chronic immunological responses to wound healing in type 2 diabetes (T2DM) challenge the outcomes of the treatments for bone regeneration. The related morbidity and costs associated with bone healing failures are considerable and increasing. Recent discoveries have demonstrated that the immune system is tightly linked to bone physiology and that MSC derived extracellular vesicles (EVs) play a prominent role in immunomodulation of bone repair. While recombinant human bone morphogenetic protein 2 (rhBMP2) mediated bone regeneration is well established, the challenges of controlling inflammation arising from rhBMP2 usage is exacerbated in the presence of immunomodulating systemic diseases such as T2DM present a significant knowledge gap that limit clinical outcomes. Based on preliminary studies of inflammation informed MSC EV microRNA (miRNA) composition and effects on immunomodulation and bone regeneration, we hypothesize that a dual delivery of engineered MSC EVs with anti-inflammatory properties and rhBMP2 using a combinatorial delivery system with self-assembling leucine zipper (LZ) peptide-based hydrogels and alginate beads can enhance bone healing in T2DM. We propose three specific aims to test this hypothesis. In aim 1, we will test our preliminary data driven hypothesis that a subset of altered miRNAs in preconditioned MSC EVs contributes to the enhanced immunomodulatory function of the inflammatory preconditioned MSC derived EVs. We will also generate engineered MSC EVs that overexpress candidate miRNA with anti-inflammatory properties to further study the mechanistic aspects by which individual EV related miRNAs control the immunomodulation functionality. In aim 2, we will create a novel dual delivery system with LZ-hydrogels and alginate beads that contain engineered EVs and rhBMP2 respectively. We will test our hypothesis that 1) EVs with anti-inflammatory properties (generated from aim 1) can be released during the inflammatory phase of healing (up to 7 days) from a self-assembling LZ- hydrogel and 2) rhBMP2 that is encapsulated in alginate beads with an extended-release profile (4-6 weeks) can provide a long term osteoinductive signal to promote/enhance bone regeneration. In aim 3, using a diabetic mouse calvarial defect model, we will test the hypothesis that engineered MSC EVs containing anti-inflammatory miRNA will suppress inflammation upon wounding while sustained released rhBMP2 promote bone healing. Overall, the results of these first studies of the combinatorial usage of engineered and MSC EVs rhBMP2 will provide a foundation for continued definition of MSC EV-mediated immunomodulation, and the regenerative properties and applicability of the dual delivery system to bone healing in T2DM. Further, these studies will also provide a framework for the use of growth factors and engineered EVs in combination for tissue engineering applications.