Date on Master's Thesis/Doctoral Dissertation

12-2022

Document Type

Master's Thesis

Degree Name

M. Eng.

Cooperating University

University of Louisville

Department

Bioengineering

Degree Program

JB Speed School of Engineering

Committee Chair

Soucy, Patricia S.

Committee Co-Chair (if applicable)

LeBlanc, Amanda Jo

Committee Member

O'Toole, Martin G.

Author's Keywords

Angiogenesis; Adipose-Derived; non-cellular; stromal; vascular; fraction

Notes

Gelatin methacrylate hydrogels were employed to encapsulate either adipose-derived stromal vascular fraction or its non-cellular components in order to be characterized and evaluated in-vitro and in-vivo for their ability to promote angiogenesis.

Abstract

Microvascular disease is hallmarked by pathophysiological conditions such as endothelial senescence, intimal thickening which impairs vasodilation, and regression of the capillary beds causing tissue ischemia in the myocardium or in peripheral vascular networks. Adipose-derived stromal vascular fraction (SVF) has previously demonstrated the ability to revascularize tissue. Increasing evidence shows that regenerative cells elicit their therapeutic benefit by paracrine mechanisms, leaving open extracellular vesicles (EVs) as a potential crux of the cell therapy paradigm. To test this idea, three types of gelatin methacrylate hydrogels were employed: SVF gels, EV gels derived from SVF, and blank control gels, which were used in-vitro and in-vivo to evaluate the effect of cell versus cell-free therapies for revascularizing tissues. Fischer 344 Green Fluorescent Protein positive (GFP+) male and female 6-month-old rats were used as donors for SVF, which was harvested by enzymatic digestion, centrifugation, and filtration steps. Cells were placed directly into gelatin methacrylate (GelMA) hydrogels. EVs were harvested from SVF tissue culture supernatants prior to being loaded in gel constructs. EVs were isolated by ultracentrifugation at 100,000g from cell culture supernatants and detected by spectral flow cytometry (Cytek Aurora) utilizing violet laser side scatter height and characterized by expression of CD9 and CD63 markers. This detection method was also used to estimate the number of particles from a known number of SVF cells. Degradation studies of unloaded GelMA hydrogels were performed using six or eight percent (w/v) concentration over 60 days in PBS. Hydrogels were further characterized by viability assays of the SVF gels to ensure the gel synthesis process, specifically UV exposure, was not cytotoxic to SVF. EV hydrogels were also characterized by their release over 7 days. A plug-in-field assay was used to determine the vasculogenic efficacy of the groups in vitro by staining the constructs with Griffonia Simplicifolia Lectin I (GS1+) and DAPI after seven and fourteen days in culture; plugs were explanted, and images were analyzed for cellular presence and expression of GS1 and αSMA markers. Finally, experimental and control gels were implanted bilaterally into the dorsal subcutaneous space of Rag1 mice for 14 days. Prior to explant, dextran tetramethylrhodamine was infused, gels were explanted and stained for cell markers, and imaged via confocal microscopy. Analysis of images acquired from in vivo experiments were compared for angiogenic metrics such as vascular density, indicated by the dextran infusion.

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