A freeze-injured skin graft model for the quantitative study of basic fibroblast growth factor and other promoters of angiogenesis in wound healing.

A new in vivo model has been developed for the quantitative study of promoters and potential promoters of angiogenesis. Full thickness rat skin autografts received a reproducible and uniform freeze injury, before being applied to full thickness wounds, in order to delay revascularisation. Blood flow in the grafts was measured during the healing period using noninvasive (laser Doppler flowmetry) and invasive (133Xe clearance) techniques. The increase in blood flow over a period of 10-14 days was taken as an index of angiogenesis. These measurements were corroborated by histological assessment of the graft vasculature, using a laminin stain to highlight vascular basement membrane. Freeze injury delayed but did not ultimately prevent full graft revascularisation (p < 0.01 for laser Doppler flowmetry and 133Xe clearance). Application of the angiogenic agent basic fibroblast growth factor (bFGF), in slow release pellet form, stimulated angiogenesis in cryoinjured grafts in a dose-related fashion. Doses of 500 and 5000 ng bFGF produced significant stimulation (500 ng bFGF, p < 0.001, and 5000 ng bFGF, p < 0.01, for both laser Doppler flowmetry and 133Xe clearance; increased vessel profile counts, p < 0.05, at each time point tested for both doses) while 50 ng bFGF had no significant effect. By contrast, 500 ng bFGF had no measurable effect on uninjured grafts. In addition, bFGF-stimulated angiogenesis in cryoinjured grafts was antagonised by a neutralising antibody to bFGF, demonstrating the specificity of action of bFGF in this model.