Introduction

The pathogenesis of diabetic wound healing is multifactorial, with major complications resulting from impaired vascular architecture and function. Recent studies have indicated that progenitor cells released from the bone marrow play an integral role in the formation and repair of blood vessels throughout the body. Specifically, endothelial progenitor cells (EPCs) are mobilized from the bone marrow in response to injury, and these progenitor cells migrate through the circulation, home to the site of injury, and incorporate into the wound, where they contribute to vasculogenesis and tissue repair. This physiologic vasculogenic process is impaired in diabetics. For example, our laboratory has confirmed that diabetic animals have fewer circulating EPCs than wild-type animals at baseline and following peripheral ischemia despite having a normal amount of progenitor cells available in their bone marrow. Thus, we hypothesized that restoring EPC mobilization from the bone marrow with the CXCR4 antagonist, AMD3100, would improve diabetic wound healing.

Methods
In order to test this hypothesis, two 6-mm wounds were made on the dorsum of diabetic mice using a sterile skin punch biopsy.  The wounds were stented with a 12-mm O-ring silicone stent to prevent wound contraction and recapitulate healing by secondary intention.  Starting on post-operative day 3 and continuing until wound closure, AMD3100 or sterile saline (0.01mg/g) was injected intraperitoneally into the treatment group (n=20) and control group (n=20), respectively.  Photogramography was used to compare the percent wound closure between the two groups at days 7, 14, and 21.  Circulating endothelial progenitor cell number (Lin-, Sca-1+, ckit+) was compared between the two groups at numerous time points throughout the study period using flow cytometry.

Results

Diabetic mice treated intraperitoneally with AMD3100 showed a significantly improved wound closure rate compared to diabetic mice treated with sterile saline (p < 0.05). At 7, 14, and 21 days post-operatively, mice treated with AMD3100 had wound closures on average of 32.7%, 72.9%, and 90%, respectively; whereas the control mice treated had closures on average of 11.9%, 36.5%, and 66.7%.  FACS analysis of peripheral blood revealed that animals treated with AMD3100 also had increased circulating EPCs (Lin-, Sca-1+, ckit+ cells) compared to untreated controls: 3.7±1.0-fold increase at 1 hour, 11.9±1.7-fold increase at day 7, 19.6±1.9-fold increase at day 14. Interestingly, there was a 23.6-fold decrease in circulating EPCs in treated animals at day 21 compared to the controls.

Conclusion

AMD3100-induced EPC mobilization leads to an improved wound healing in diabetic mice by increasing the total number of circulating endothelial progenitor cells.  Furthermore, the rate of wound healing is directly proportional to the number of circulating EPCs.