Biomimetic Apoptotic Particles for Macrophage-driven Oral Bone Regeneration

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ABSTRACT Adequate bone quality, quantity, and wound repair in the oral cavity are crucial for treatment eligibility, as well as short and long-term success of dental implants. Successful restoration of a functional dentition requires an understanding of the endogenous bone repair process. Often overlooked, one of the first steps in osseous wound repair after trauma, such as in a dental extraction, is cell death and subsequent apoptotic cell (AC) clearance (efferocytosis) by macrophages. As a result of efferocytosis, macrophages secrete a variety of factors that facilitate regeneration, and swiftly alter their behavior in response to a multitude of physical and biological microenvironmental cues. CC-motif chemokine ligand 2 (CCL2), which is secreted by macrophages, mediates mesenchymal stem/progenitor cell (MSPC) recruitment to the wound site. Although previous work has largely focused on biochemical signals that drive CCL2 production, preliminary data in the current proposal suggests that the physical nature of AC engulfment drives cytoskeletal events and subsequent mechanotransductive signaling. Engulfment of apoptotic cells induces changes in macrophage shape, actin organization, and nuclear architecture that likely initiates CCL2 production. Understanding efferocytosis-induced intracellular forces and resulting signaling will inform the design of apoptotic cell mimics (ACM) as a regenerative therapy for patients whose age or co-morbidities impair wound healing capacity. The goals of this project are to determine the role of efferocytosis-induced macrophage mechanotransduction in bone repair and to promote reparative macrophage behavior using ACM, hence catalyzing the endogenous osseous wound healing response. The overall hypothesis is that macrophages promote osteogenic repair through efferocytosis-driven biophysical signaling, which can be recapitulated using apoptotic cell mimicry for a regenerative advantage. The two aims proposed are: 1) to connect CCL2 expression and macrophage phenotype with AC and ACM engulfment-induced changes to the cyto- and nucleoskeleton, and 2) to optimize and deliver ACM to promote macrophage-driven osteogenesis and improve bone repair in a clinically relevant oral osseous wound healing model. To accomplish these aims, first an in vitro co-culture system that allows for macrophage engulfment of AC and ACM will be used. This will be a valuable tool to identify and validate genes and proteins associated with mechanotransduction and osteogenic-repair that are similarly altered in both experimental groups. ACM with tunable size, stiffness, and degradability will be optimized to maximize CCL2 secretion given its role in MSPC recruitment. Next, we will use CCL/R2 genetic knockout mouse models to confirm the role of CCL2 in bone regeneration. We anticipate ACM treatment will induce cytoskeletal changes that result in CCL2 production and promote bone repair. The outcomes of this project will identify underexplored effects of post- efferocytosis macrophage mechanotransduction and reparative activity, providing mechanistic insights into bone regeneration, which can be used to establish new bone regeneration therapies for patients.