Cell-Based Vascular Therapeutics for Bone Regeneration

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Ruehle, Marissa Ashley
Guldberg, Robert E.
Willett, Nick J.
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Bone is a highly vascularized tissue, and adequate vascularity is an essential requirement for proper bone healing. Revascularization is a challenge in critical-sized defects, especially those with concomitant muscle damage typical of traumatic injury. Patients with these injuries heal slowly and exhibit higher rates of infection and non-union, underscoring the critical importance of vasculature to bone healing. Additionally, the bone defect environment is a complex niche, involving mechanical cues in addition to a host of biochemical signals. It is well known that mechanical loading affects bone growth and remodeling, and while flow-mediated mechanics influence the vasculature, remarkably little is known about the effects of bulk matrix deformation on neovascularization. The overall objective of this thesis was to leverage mechanical cues to enhance vascular network formation and to use enhanced vascularization to improve bone regeneration. First, we evaluated the effect of multicellular microvascular fragments (MVF) co- delivered with BMP-2 to a model of composite bone-muscle trauma using collagen sponge, the clinically available BMP-2 delivery vehicle. MVF did not improve bone healing as hypothesized; however, we also investigated the effect of a modestly increased BMP-2 dose, which did significantly improve functional healing. While MVF maintained viability within the collagen sponge in vitro, they first dissociated to single cells, which we speculated may have prevented their inosculation with the host vasculature. Next, we developed and characterized decorin-supplemented collagen gels for use as both an in vivo co-delivery vehicle for MVF and BMP-2 and as a dimensionally stable biomaterial scaffold to investigate the effects of compressive loading on MVF growth in vitro. Despite in vitro results demonstrating synergistic effects of BMP-2 and MVF, there was no effect of MVF on bone healing, and MVF significantly decreased early revascularization following injury. However, the addition of decorin increased the compressive properties and dimensional stability of collagen while still supporting robust in vitro MVF growth. We then evaluated the effects of dynamic compressive loading on MVF growth. While the vasculature has long been recognized as mechanosensitive, the effects of abluminal forces experienced by healing tissues on angiogenesis are poorly understood. We demonstrated that delayed compressive loading led to longer, more extensively branched microvascular networks than early loading at all strain magnitudes tested. Across strain magnitudes, delayed loading increased vascular network length and branching compared to non-loaded controls; however, early high strain loading inhibited network formation. Gene expression analysis revealed differential mechanoregulation of gene expression profiles by early vs. delayed loading. Genes associated with angiogenic sprout tip cells were downregulated by early loading and upregulated by delayed loading. Delayed loading also led to the upregulation of genes involved in cell adhesion and migration. Using a pharmacological inhibitor, we established that the YAP mechanotrasduction pathway is involved in the pro-angiogenic response to delayed loading. Overall, this thesis has tested MVF as a therapeutic for bone healing, developed and characterized a novel biomaterial for in vitro and in vivo applications, and increased fundamental knowledge about the effects of bulk loading on neovascularization. These findings can be leveraged to more effectively treat composite bone-muscle defects, both through future tissue engineering work and with physical rehabilitation regimens informed by knowledge of loading effects on nascent vasculature.
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