Living skin substitutes: Survival and function of fibroblasts seeded in a dermal substitute in experimental wounds

The healing of full-thickness skin defects requires extensive synthesis and remodeling of dermal and epidermal components. Fibroblasts play an importa nt role in this process and are being incorporated in the latest generation of artificial dermal substitutes. We studied the fate of fibroblasts seede d in our artificial elastin/collagen dermal substitute and the influence of the seeded fibroblasts on cell migration and dermal substitute degradation after transplantation to experimental full-thickness wounds in pigs, Wound s were treated with either dermal substitutes seeded with autologous fibrob lasts or acellular substitutes. Seeded fibroblasts, labeled with a PKH-26 f luorescent cell marker, were detected in the wounds with fluorescence micro scopy and quantitated with flow cytofluorometric analysis of single-cell su spensions of wound tissue. The cellular infiltrate was characterized for th e presence of mesenchymal cells (vimentin), monocytes/macrophages, and vasc ular cells. Dermal substitute degradation was quantitated by image analysis of wound sections stained with Herovici's staining. In the wounds treated with the seeded dermal substitute, fluorescent PKH-26-labeled cells were de tectable up to 6 d and were positive for vimentin but not for the macrophag e antibody. After 5 d, flow cytofluorometry showed the presence of 3.1 (+/- 0.9) x 10(6) (mean +/- SD, n = 7) PKH-26-positive cells in these wounds, w hereas initially only 1 x 10(6) fluorescent fibroblasts had been seeded. In total, the percentage of: mesenchymal cells minus the macrophages was simi lar after 5 d between wounds treated with the seeded and the acellular subs titutes. In the wounds treated with the seeded substitute, however, 19.5% o f the mesenchymal cells were of seeded origin. Furthermore, the rate of sub stitute degradation in the seeded wounds was significantly lower at 2-4 wk after wounding than in wounds treated with the acellular substitute, Vascul ar in-growth and the number of infiltrated macrophages were not different, In conclusion, cultured dermal fibroblasts seeded in an artificial dermal s ubstitute and transplanted onto full-thickness wounds in pigs survived and proliferated. The observed effects of seeded fibroblasts on dermal regenera tion appeared to be mediated by reducing subcutaneous fibroblastic cell mig ration and/or proliferation into the wounds without impairing migration of monocytes/macrophages and endothelial cells, Moreover, the degradation of t he implanted dermal substitute was retarded, indicating a protective activi ty of the seeded fibroblasts.