The safety and efficacy of timolol treated mesenchymal stem cells in a scaffold on an in vivo diabetic wound model

Diabetic foot ulcers (DFU) are chronic, non-healing wounds on the feet of diabetic individuals and are the leading cause of non-traumatic amputations in the United States. Current treatments have success rates generally below 50%, however novel experiments using hypoxia (1% O2) preconditioned mesenchymal stem cells (MSCs) show the potential to improve healing rates through the secretion of cytokines that recruit cells vital to the healing process to the injury site. In addition, stress-induced epinephrine generated within the wound site has been shown to inhibit wound healing by disrupting the actions of these key cells through binding and activation of the β2-adrenergic receptor (β2-AR). These effects could be reversed by the application of a β2-AR inhibitor. Thus, the purpose of this study is to investigate the effects of treating MSCs with the general β2-AR antagonist Timolol at varying concentrations to determine if the compound improves their therapeutic potential. We analyzed three aspects of MSCs to determine if preconditioning or exposure to Timolol improved their therapeutic abilities. We first looked at MSC persistence and distribution throughout mouse wound tissue when delivered using two different collagen scaffolds, Integra™ and Oasis®. Control samples of MSCs attached to scaffolds and wound tissue treated with MSCs delivered in scaffolds were formalin fixed and paraffin embedded. After sectioning and removing the paraffin, samples were subjected to either fluorescence in situ hybridization (FISH) using a human specific centromeric probe or immunohistochemistry (IHC) using a HLA-E human specific antibody to visualize human cells within matrices. After repeated attempts, we were unable to distinguish MSCs from 3t3 fibroblast using FISH. However we were able to identify MSCs within a murine wound sample using IHC, but the conditions will need to be optimized to be able to accurately quantify the amount of MSCs persisting in wound tissue. A second goal was to examine the differentiation capacity of preconditioned MSCs when exposed to low dose (2μM) and high dose (7.9 mM) concentrations of Timolol. The studies showed that MSCs treated with 2μM Timolol retained their tri-lineage differentiation capacity in both normoxic (20-22% O2) and hypoxia (1% O2) conditioned cell cultures, however we could not determine if there were significant differences in the extent of differentiation between conditioned cultures. Lastly, we analyzed the effects of low and high dose concentrations of Timolol on the migration rates of MSCs cultured under hypoxia or normoxia conditions. We found the migratory rate of cells treated with 2μM Timolol moved significantly slower when preconditioned with hypoxia versus those cultured in normoxia. Additionally, 2μM Timolol treated cells moved significantly slower when hypoxia preconditioned versus normoxia preconditioning. This finding is important as it suggests that it is possible that the binding affinity of the β2-AR of MSCs may be affected by hypoxic conditioning and Timolol may elicit similar negative effects on the cell as epinephrine in it's absence. Overall, this study presented some insight into the healing potential and behavior of human MSCs when exposed to differing amounts of Timolol and suggests possible directions for investigators to optimize the therapeutic effects of MSCs delivered into tissue for healing chronic wounds.