Project Summary/Abstract Both soft and hard tissue wound healing are impaired in type 2 diabetes (T2DM). Diabetes negatively impacts fracture healing, bone regeneration and osseointegration of endosseous implants. The complex physiological changes associated with diabetes are often manifest in immunological responses to wounding and repair where macrophages play a prominent role in determining outcomes. Recent discoveries have demonstrated that the immune system is tightly linked to bone physiology and immunomodulation of bone repair is affected by key interaction involving macrophages and mesenchymal stem cells (MSCs). Yet, a fundamental knowledge gap exists with respect to the nature of and the mechanisms that govern this interaction in the presence of T2DM. My recent published study has revealed that the conditioned medium from diabetic mouse macrophages impairs osteogenic differentiation of MSCs. My studies also show that macrophages secrete phenotype-dependent extracellular vesicles (EVs) that affect the level of bone repair. Here, I hypothesize that diabetic macrophage EVs mediate specific paracrine control of osteogenesis. To test this hypothesis, I propose two independent but thematically related aims. In Aim 1, I will characterize and compare wild type mouse macrophage EVs (wtEVs) and diabetic mouse macrophage EVs (dbEVs) at the structural and functional level and define the effects of these EVs on MSC osteogenic differentiation quantitatively. I will demonstrate the role of macrophage miRNAs cargo on osteoinduction by interfering miRNA function in Argonaute 2 (involved in RISC complex formation and miRNA function) knockdown MSCs. In addition, I will validate that dbEVs contain miRNAs that negatively influence the process of osteogenesis by generating EVs from DICER (required for mature miRNA formation) knockout macrophages. In Aim 2, I will identify significantly distinct miRNAs in dbEVs, their target genes and signaling pathways involved in osteogenesis by bioinformatic approach. I will evaluate the functionality of these miRNAs on osteoinduction by utilizing mimics/antagomirs that increase or eliminate the effects of identified miRNAs. I will study selected target genes at the level of miRNA interaction to affirm the direct effects on gene regulation and downstream effects of these key miRNAs on osteoinductive pathways. Translationally, I will generate functionally engineered EVs (FEEs) by engineer the candidate miRNAs (promote osteogenesis) that rescue dbEV effects into MSC EVs. The FEEs will be characterized structurally and functionally in vitro. Further I will utilize calvarial bone defect model to evaluate the function of selected miRNAs within FEEs on bone healing in diabetic mice. Overall, these mechanistic studies will explore the significance of the macrophage EV-mediated immunomodulation that occurs between macrophages and MSCs in the context of bone healing in the presence of T2DM. These studies will refine knowledge of diabetic pathology and provide potential targets for therapeutic intervention.

Project Narrative Bone healing is impaired in type 2 diabetes (T2DM). This project will study the role of diabetic macrophage derived-extracellular vesicles and their unique/novel miRNA act in concert with mesenchymal stem cells to control bone repair and regeneration. Successful completion of this work will refine knowledge of diabetic pathology and provide potential targets for therapeutic intervention as well as further innovative ways to control inflammation and regeneration that influences bone repair in T2DM.