Date of Award

2019

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy in Biomedical Engineering (PhD)

Administrative Home Department

Department of Biomedical Engineering

Advisor 1

Dr. Megan C. Frost

Committee Member 1

Dr. Feng Zhao

Committee Member 2

Dr. Jeremy Goldman

Committee Member 3

Dr. Caryn Heldt

Abstract

Diabetic foot ulcers (DFU) are one of the most challenging complications associated with diabetes mellitus (DM). Every 1 in 4 patients with DM are expected to develop an ulcer in their life time. The time course of healing is on average 90 to 120 days which increases the risks of infection, amputations and death. Nitric oxide (NO) is a signaling molecule produced in the body that has been established in the literature to serve many roles in the body, including as a vasodilator, neurotransmitter immune modulator and antimicrobial agent. Emerging evidence has shown that NO is indispensable in every stage of wound healing and its dysregulation is a major hinderance in the healing of DFU. The problem associated with harnessing the biological effects of NO is that it is very reactive and consequently it is extremely difficult to measure. As a result, majority of the studies in the literature measure its metabolic products (nitrate and nitrite) to correlate NO function. The challenge with relying on NO metabolites is that they can only be analyzed at static time points as an accumulation measure and they do not give any information about the dynamic NO changes taking place in the wound environment. Moreover, the NO dose produced by specific cells during the course of cell migration and proliferation varies depending on the stage of wound healing. The function of NO is very complex with its effects ranging from protective to deleterious depending on the dose and duration of the treatment and even the surrounding host environment. It is therefore of paramount importance to understand the real-time NO profile produced and exposed to the cells that are actively involved in the wound healing process and to analyze how the profile changes in physiological and pathological conditions. This dissertation presents the real-time NO levels produced from normal and chronic wound fibroblast cells cultured in normal and high glucose conditions when stimulated by inflammatory mediators that are commonly present in the wound environment. The results showed that the real-time NO levels produced by fibroblasts are higher in normal glucose compared to high glucose cell culture conditions (8.42±1.16 vs 3.26±0.79 nmols/105 cells). The real-time NO levels were significantly increased in normal glucose with stimulation compared to normal glucose without stimulation. High glucose conditions in the absence and presence of stimulation showed no statistical difference between the two groups. The NO levels produced by fibroblasts obtained from biopsy samples from patients treated at a chronic wound care clinic was suppressed. Overall there was no statistical difference within the experimental groups. The NO levels produced by the cells did not correlate with the nitrite and nitrate detected in the fluid obtained from the wound site (nanomolar vs micromolar concentration respectively). The results were compared with the well characterized macrophage cell line RAW 264.7 which also showed higher NO levels (161.80± 29.05 vs 46.30±12.9 nmols/106 cells) in normal glucose compared to high glucose conditions and no statistical difference in the nitrite levels in normal and high glucose conditions. The results clearly indicate the need for real-time NO monitoring to determine the level and duration of NO production by cells in the wound bed and that the use of nitrite and nitrate does not provide accurate information regarding NO dysregulation in chronic non-healing wounds. The work presented herein will facilitate the development of treatments that address not only the actual dose of NO treatment needed in the wound but also potentially indicate factors that may interfere with the NO function.

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