Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts

Abstract

The purpose of this study was to confirm the therapeutic effects of mixed sheets consisting of peripheral blood mononuclear cells (PBMNCs) and fibroblasts on cutaneous skin ulcers. Vascular endothelial growth factor (VEGF) secretion in mixed cell sheets was much higher than in PBMNCs and fibroblasts. Concerning the mechanism, transforming growth factor beta 1 and platelet-derived growth factor BB secreted from PBMNCs enhanced VEGF production in fibroblasts. In wounds created on the backs of diabetic mice, the therapeutic effect of mixed cell sheets was similar to that of daily treatment with trafermin, a recombinant human basic fibroblast growth factor. Although abnormal granulation tissue and inflammatory cell infiltration were observed in trafermin-treated wounds, the transplantation of mixed cell sheets resulted in the natural anatomy of subcutaneous tissues. The expression patterns of identical wound-healing factors in wounds were different between mixed sheet-transfected and trafermin-treated animals. Because mixed cell sheets transplanted into full-thickness skin defects were eliminated in hosts by day 21 in syngeneic transplantation models, allogeneic transplantation was performed using mice with different genetic backgrounds. The wound-healing rates were similar between the mixed cell sheet and trafermin groups. Our data indicated that mixed cell sheets represent a promising therapeutic material for cutaneous ulcers.

Treatment of Cutaneous Ulcers with Multilayered Mixed Sheets of Autologous Fibroblasts and Peripheral Blood Mononuclear Cells

“…We previously reported that mixed-cell sheets consist of autologous fibroblasts (1.25 × 10 5 cells) and PBMNCs (2.0 × 10 6 cells) on UpCell® 24-well plates [26]. To determine the maximum possible number of fibroblasts to configure mixed-cell sheets on these plates, we examined the number of fibroblasts present when the number of PBMNCs remained at 2.0 × 10 6 cells/well.…”

“…Therefore, we attempted to make multilayered sheets on a typical culture dish and detach them using a small amount of dispase. With this method, the multilayered sheets incorporated a subset of PBMMCs as in our previous study [26], and the population of PBMNCs was considered to contribute to angiogenesis [38]. …”

“…In our previous study in rabbits, hypoxic preconditioned PBMNCs enhanced cell adhesive ability and angiogenesis, and transplantation of the hypoxic preconditioned PBMNCs into muscle improved blood flow at the ischemic hindlimb [13]. Hypoxic preconditioning has also been reported to increase secretion of VEGF in single-layered sheets [26, 27]. Therefore, hypoxic preconditioning may be effective for promoting secretion of growth factors in multilayered sheets.…”

“…It has been reported that PBMNCs provide interleukins and growth factors such as TGF-β1 and PDGF-BB that are required for the inflammatory phase of the normal healing process [22-25]. TGF-β1 and PDGF-BB increase the expression levels of VEGF and collagen mRNAs in fibroblasts [26]. Accordingly, we developed a mixed-cell sheet consisting of autologous fibroblasts and PBMNCs that secrete necessary growth factors from the inflammatory phase to the tissue repair phase and confirmed its efficacy for ulcer treatment in mice and rabbits [26, 27].…”

“…TGF-β1 and PDGF-BB increase the expression levels of VEGF and collagen mRNAs in fibroblasts [26]. Accordingly, we developed a mixed-cell sheet consisting of autologous fibroblasts and PBMNCs that secrete necessary growth factors from the inflammatory phase to the tissue repair phase and confirmed its efficacy for ulcer treatment in mice and rabbits [26, 27]. The mixed-cell sheet had high angiogenic potency, promoted wound closure, and led to complete natural healing.…”