Date of Award

Winter 3-2-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering

First Advisor

Mary Caldorera-Moore

Abstract

Over 150,000 patients undergo lower extremity amputation every year in the United States, most commonly caused by complications due to diabetes mellitus, peripheral vascular disease, and trauma. Diseased or damaged tissues that are unable to naturally repair themselves must either be fully removed or replaced, otherwise the injuries can lead to further complications such as infection or death. Tissue resection and amputation, as forms of removing damaged tissue, are not favorable to patients as they can cause pain, reduce mobility, and negatively impact quality of life. However, replacing lost or damaged tissues with donor tissues carries the risks of tissue rejection, the need for lifelong immunosuppressive medications post-operation, and critical shortages of donor tissues. Therefore, there is a need for alternative treatment solutions to repair or replace lost or damaged tissues. One major research area is the combination of biomaterial scaffolds and stem cells to develop and restore new, functional tissues. This research aims to produce an injectable, in-situ crosslinking hydrogel capable of encapsulating stem cells for use in tissue regeneration or wound repair applications by utilizing the beneficial antibacterial and biocompatible properties of chitosan and the effective, non-toxic crosslinker genipin to produce an effective, proactive wound dressing. It was hypothesized that combining the absorptive, antibacterial, pH regulating effect, and 3-dimensional (3D) structure of the chitosan-genipin hydrogels with stem cells would more effectively promote tissue regeneration than either component individually. Herein, a thermally-driven, injectable chitosan-genipin hydrogel capable of in-situ crosslinking at 37 °C and cell encapsulation was developed and characterized. Material characterization studies presented that the in-situ crosslinking hydrogels had a gelation time of 150 minutes, an average elasticity of 449 Pa, and an average fluid uptake of 793% over 14 days. In vitro characterization studies presented that the in-situ crosslinking hydrogels were biocompatible with keratinocytes over 7 days averaging between 80 – 157% viability compared to control cells, and that stem cells remained viable when encapsulated within the in-situ-crosslinking hydrogels over 14 days, averaging >80% cell viability, and their migration rate out of the hydrogel was significantly reduced compared to non-encapsulated cells. Two in vivo studies were performed to evaluate the wound healing efficacy of the in-situ crosslinking chitosan-genipin hydrogels and cell-encapsulated chitosan-genipin hydrogels. The in-situ-crosslinking chitosan-genipin hydrogel was able to provide a stable matrix for the attachment and growth of new bone tissue and retain stem cells at the defect site, while encapsulated stem cells promoted chondrogenesis. In a full-thickness wound model, the in-situ-crosslinking chitosan-genipin hydrogels accelerated wound closure compared to control treatments, and both in-situ-crosslinking hydrogels and slow-frozen chitosan-genipin hydrogel sheets reduced inflammation and improved re-epithelialization of the injury site. These promising results confirm that the chitosan-genipin in-situ-crosslinking hydrogels are an excellent platform for promoting tissue regeneration and provide a viable matrix for stem cell encapsulation and delivery to an injury site.

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