Chemical Delay of Flaps through Endogenous Stem Cell Therapy

Background: Blood supply is a limiting factor in flap surgery. Endothelial progenitor cells (EPCs) migrate from the bone marrow into sites of ischemic injury to contribute to neovascularization. By disrupting the CXCR4/SDF-1 axis, AMD3100, a clinically approved stem cell mobilizer, increases circulating (c)EPCs.[1][2] In previous studies, we have shown that AMD3100 accelerates reossification in a distraction osteogenesis model and improves diabetic wound healing. Furthermore, others have shown that AMD3100 accelerates revascularization in ischemic hind limbs.[3, 4] We hypothesize that pretreating a flap with a topical chemoattractant (e.g. PDGF) will promote trafficking of AMD3100 mobilized bone marrow stem cells to improve flap survival.

Methods: Using a previously described reproducible murine ischemic dorsal skin flap model, wild-type (WT) FVB mice were randomized into 4 groups (n=10/group): untreated WT, AMD3100-treated WT (A+), PDGF-treated WT (P+), and AMD3100/PDGF-treated WT (A+P+). Prior to surgery, P+ and A+P+ mice were treated for 3 days with PDGF-BB (2μg). A+ and A+P+ received AMD3100 (5mg/kg, SC) 1 hour prior to surgery, and daily postoperatively. Flap survival was assessed photometrically. Histology, immunohistochemistry, ELISA, quantitative RT-PCR and in vitro migration assays toward SDF-1 and PDGF where performed at sequential timepoints. Flap vascularity was assessed by tissue oximetry measurements (SatO2%), color laser Doppler analysis (flux ratio) and CD31 immunofluorescence (vessels/hpf). cEPC number was determined by FACS.

Results: AMD3100 treatment increased cEPC levels (3.7±1.0-fold at 1 hour, p<0.05; 5.5±1.1-fold at day 7, p<0.02; and 13.2±0.5- fold at day 14, p<0.02). A+P+ had the greatest improvements in flap survival and flap vascularity (431.8±19.3 vessels/hpf vs. 155.3±16.1 vessels/hpf, p<0.001) compared to untreated WT mice. In the presence of AMD3100, EPC migration to SDF-1 was decreased 25.1±2.8% (p<0.05), while EPC migration towards PDGF-BB was unaffected (8.4±3.4% fewer, p>0.05).

Conclusion: These are the first preclinical chemical delay data to support a novel endogenous stem cell therapy to significantly improve flap survival.

Reference: 1.De Clercq, E., The AMD3100 story: the path to the discovery of a stem cell mobilizer (Mozobil). Biochem Pharmacol, 2009. 77(11): p. 1655-64. 2.Devine, S.M., et al., Rapid mobilization of functional donor hematopoietic cells without G-CSF using AMD3100, an antagonist of the CXCR4/SDF-1 interaction. Blood, 2008. 112(4): p. 990-8. 3.Tan, Y., et al., A novel CXCR4 antagonist derived from human SDF-1beta enhances angiogenesis in ischaemic mice. Cardiovascular Research, 2009. 82(3): p. 513-21. 4.Jiao, C., S. Fricker, and G.C. Schatteman, The chemokine (C-X-C motif) receptor 4 inhibitor AMD3100 accelerates blood flow restoration in diabetic mice. Diabetologia, 2006. 49(11): p. 2786-9.