Effects of crosslinking and pro-inflammatory signals on vascularization of biomaterials in vivo

Alicia Clark

doi:10.17918/hwwp-5c38

Current biomaterials used to support the repair and regeneration of damaged or diseased tissues often utilize crosslinked and non-crosslinked collagen, the most prominent protein in the human body. Crosslinked materials can be advantageous due to their increased mechanical strength, slower degradability, and structure that more closely mimics that in human tissues. One major challenge in collagen-based biomaterials is a lack of healthy vascularization after implantation and its integration in the body. Macrophages are one of the primary cells involved in angiogenesis and in response to implanted biomaterials, which suggests that biomaterials can modulate macrophage behavior and may have potential to facilitate enhanced angiogenesis. Previous research has suggested that pro inflammatory M1 macrophages in crosslinked scaffolds initiate angiogenesis, however, the effect of crosslinking and macrophage behavior on vascularization is still poorly understood in vivo. Therefore, the goal of this study was to determine the effects of chemical crosslinking and the release of pro inflammatory, M1-stimulating, interferon gamma (IFNg) on macrophage behavior and blood vessel infiltration into the scaffolds and within the scaffold cross-section edges in vivo. Following pilot studies to optimize methods for blood vessel measurements, the work for this thesis come from 2 studies, one of which is a repeat of the other (n=6 mice total, 3 mice for each study). One scaffold from each group (non-crosslinked collagen control scaffold, non-crosslinked collagen scaffold with adsorbed IFNg, crosslinked collagen scaffold, and crosslinked collagen scaffold with IFNg) was implanted subcutaneously into the dorsal region of 8-week-old, C57BL/6J male mice. After 14 days, the mice were perfused with anti-mCD31 via a tail-vein injection to visualize blood vessels, and then sacrificed. Scaffolds were then explanted from the mice and processed for cryo-sectioning and staining. Blood vessels within the scaffolds and within the scaffold cross-section edges from each group were manually counted from z-stack images and averaged per mouse. Measurements of the furthest distances that the blood vessels traveled into each scaffold cross-section were also acquired. One cross section from the Disc Center range and one cross-section from each of the Disc Edge ranges were randomly selected per group per mouse to analyze for both studies. Results from both studies showed that crosslinking changed the effects of IFNg compared to non-crosslinked scaffolds. IFNg decreased vascularization within non-crosslinked scaffolds and increased vascularization within crosslinked scaffolds. In scaffolds without IFNg, vascularization was increased in non-crosslinked scaffolds, but decreased in crosslinked scaffolds compared to scaffolds with IFNg. These results suggest that complex interactions between scaffold properties and immunomodulatory drugs affect macrophage behavior. These results also suggest that the immune response can be controlled to affect desired vascularization.

Effects of crosslinking and pro-inflammatory signals on vascularization of biomaterials in vivo

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Effects of crosslinking and pro-inflammatory signals on vascularization of biomaterials in vivo

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