Abstract
Diabetic foot ulcers (DFU) are chronic, non-healing, wounds, and are the leading cause of non-traumatic amputations in the United States. Patients with DFUs represent a multi-billion dollar burden on health care, and the costs become more urgent with higher percentages of the U.S. population being classified as pre-diabetic each year. Current standard treatments are poor at curing DFUs, and there is no therapy to prevent their recurrence. Mesenchymal stem cells (MSC), which are capable of secreting cytokines that are helpful to the wound healing process, have great potential to improve treatments. However, therapies utilizing MSCs suffer from poor engraftment to target tissues due to unknown reasons. Engraftment and healing properties of MSCs can be improved through scaffold delivery and preconditioning treatments. Scaffolds provide cells 3-D extracellular matrix support that mimics in vivo conditions, and allows for delivery of large cell counts to wounded patients. Pre-conditioning treatments come in a variety of modalities, including environmental acclimation, pharmacological agents, and naturally occurring cytokines. Specifically hypoxic pre-conditioning of MSCs has been used to enhance their paracrine activity, and to acclimate the cells to the surroundings they will be delivered into. Timolol, a pharmacological agent that acts as a ~2-adrenergic receptor (~2AR) antagonist, has been shown to improve the wound healing function of cells involved in the wound healing process. Platelet derived growth factor (PDGF) is a naturally produced cytokine important in the normal wound healing process. Although these pre-conditioning treatments have a positive impact on wound healing functions individually, there has been no characterization of these treatments in combination. The overarching goal of this project is to define pre-conditioning treatments for MSCs to maximize their healing potential for DFUs. The first goal was to determine the survival and distribution of pre-conditioned MSCs that were delivered in a scaffold to a wounded diabetic mouse. The second goal was to determine if PDGF had potential as a pre-conditioning treatment for DFUs. To accomplish the first goal, we first needed to decide on a scaffold to use for delivery. We found that Jntegra™ skin substitute would be the ideal candidate, since it had the highest MSC loading efficiencies. However, we needed to assess the survival of the MSCs that were loaded. After finding that MSCs were proliferating within the matrix after one week in vitro, we moved forward with in vivo experiments using Integra™ as our scaffold. These in vivo exerpiments were performed by inserting Integra™ seeded with pre-conditioned MSCs into circular wounds on a diabetic (db-/-) mouse. After 9 days of healing, the wound tissue was excised, formalin fixed, embedded in paraffin, and sectioned for further analysis. We determined the survival and distribution of MSCs within this mouse wound tissue using fluorescence in situ hybridization to discriminate between human MSCs and murine cells. We found that preconditioning treatments improved survival of MSCs and altered their migratory capabilities such that there were more MSCs in the wound bed as well. For the second goal, the first aim was to identify MSCs from donors with high expression of the PDGF receptor, PDGFr-~, and optimize the culture conditions for expression. We observed that MSCs from increasing passage and freeze/thaw cycles had remarkably lower expression of PDGFr-~. Thus, we performed all experiments using fresh MSCs from passage 3, which were deemed optimal for experiments. Next, we determined the optimal dosage of PDGF for MSC migration using single cell migration assays. We found that MSCs responded to PDGF in a dose dependent manner, and the optimal dose was 25ng/mL. After finding the optimal dose of PDGF for MSC migration, we sought to determine the global gene expression response to PDGF treatment at 25ng/mL. Isolated RNA has been sent to a commercial facility for RNA-sequencing. In summary, we found that combined pre-conditioning treatment of MS Cs improved their survival and distribution within wounded diabetic mice. We also found that PDGF has potential to be added as a pre-conditioning treatment given its positive effects on MSC migration.