Three-dimensional fibroblast culture implant for the treatment of diabetic foot ulcers: metabolic activity and therapeutic range.

Dermagraft is three-dimensional, allogeneic, human neonatal dermal fibroblast culture grown on a degradable scaffold and cryopreserved. Clinical trials for treatment of diabetic foot ulcers showed optimal healing within a therapeutic range of metabolic activity, determined by 3[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) reduction. Actions of Dermagraft in wound repair include colonization by cells and provision of growth factors and cytokines, both activities dependent on living cells. Cells in the cryopreserved culture showed 60% viability by dye exclusion and, when isolated, were able to proliferate in monolayer culture. Protein synthesis by Dermagraft was inhibited 70-98% by cryopreservation, but, if within the therapeutic range, recovered to 45-85% of the prefreeze value over 48 h. Subtherapeutic Dermagraft showed variable, low recovery. Expression of mRNA for vascular endothelial growth factor (VEGF), platelet-derived growth factor A chain, and insulin-like growth factor-1 was reduced >83% in subtherapeutic compared with therapeutic Dermagraft. Granulocyte colony-stimulating factor and VEGF protein secretion, determined by enzyme-linked immunosorbent assay (ELISA), and angiogenic activity also depended on therapeutic range. VEGF secretion dropped sharply with MTT reductase in subtherapeutic tissue. The data demonstrate the critical dependence of the therapeutic properties of this living dermal implant on recovery of protein synthesis, growth factor expression, and angiogenesis, determined by metabolic activity.