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theses

Chronic wounds are wounds that recur or fail to heal by six weeks and cost the United States $150 million through prolonged and frequent hospital visits. Chronic wounds can be caused by poor blood circulation, compromised immune systems, and limited mobility. Current treatments include daily dressing changes that maintain moist wound environments, surgical debridement, skin grafts, and vacuum assisted wound closure. There is a need for an accelerated wound therapy that is inexpensive, nonsurgical, and reduces the patient’s discomfort.

Insulin treatments have shown promise in wound healing because it is the main initiator of the mitogen-activated protein kinase – extracellular signal-regulated kinases (MAPK-ERK) pathways and the phosphoinositide 3-kinase – protein kinase B (PI3K-Akt) pathway. This leads to the stimulation of haptotactic migration, collagen production, cell proliferation and a reduction in apoptosis. Pilot studies in the lab showed accelerated healing in a chronic wound model by using RIN-m, which are insulin secreting cells (ISCs) derived from pancreatic rat tumor cells. The results showed that wounds that were treated with ISCs closed at day 28 of the study, while the wounds treated by the controls did not close during the 35-day study. Since cancer cells also promote growth and resist apoptosis it is necessary to establish that these results stem from the insulin treatment and not the use of cancer cells. The goal of this present study is to evaluate the effects of cell lines derived from RIN-m, namely RIN-5F and RIN-14B, which secrete only insulin and somatostatin respectively.

These cells were combined with prepolymer solutions to create polyethylene glycol diacrylate (PEGDA) hydrogels. Conditioned media were used in an insulin ELISA to determine insulin concentrations. The conditioned media was also used to perform scratch assays using HaCaT keratinocytes to model an in vitro wound and monitor the rate of scratch closure.

The ELISA showed that the cell bioactivity and insulin release were not impeded by encapsulation, with the insulin concentrations at expected levels. RIN-5F had high levels of insulin secretion, and RIN-14B had low levels of insulin secretes. Since RIN-14B secretes somatostatin, which suppresses insulin, these results are expected. The experimental groups treated with RIN-5F conditioned media had the smallest gap area at the end of the 48 hour study than the experimental groups treated with RIN-14B conditioned media in the scratch assay. Since the RIN-5F experimental groups stimulated keratinocytes to close gaps faster than the RIN-14B experimental groups, it strongly suggests that the results of the wound healing, both in these experiments and in previous lab work, stem from the insulin treatment and not simply the use of cancer cells.

ETD_10891

doi:10.7282/t3-a22b-j697

M.S.

Includes bibliographical references

ETD graduate

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