1. ^*Wound Healing Research Group, Department of Dermatology, Beverwijk, The Netherlands
2. ^†Wound Healing Research Group, Department of Cell Biology and Histology, Beverwijk, The Netherlands
3. ^‡Wound Healing Research Group, Department of Electron Microscopy, University of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands
4. ^§Burns Center, Red Cross Hospital and Burns Foundation, Beverwijk, The Netherlands

Correspondence: Dr E.N. Lamme, Academisch Medisch Centrum, Department of Dermatology, Neurozintuigen Lab., K2N-210, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands

Received 25 November 1997; Revised 14 August 1997; Accepted 2 September 1998.

Abstract

The healing of full-thickness skin defects requires extensive synthesis and remodeling of dermal and epidermal components. Fibroblasts play an important role in this process and are being incorporated in the latest generation of artificial dermal substitutes. We studied the fate of fibroblasts seeded 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. Wounds were treated with either dermal substitutes seeded with autologous fibroblasts or acellular substitutes. Seeded fibroblasts, labeled with a PKH-26 fluorescent cell marker, were detected in the wounds with fluorescence microscopy and quantitated with flow cytofluorometric analysis of single-cell suspensions of wound tissue. The cellular infiltrate was characterized for the presence of mesenchymal cells (vimentin), monocytes/macrophages, and vascular 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 detectable up to 6 d and were positive for vimentin but not for the macrophage antibody. After 5 d, flow cytofluorometry showed the presence of 3.1 (0.9) 10⁶ (mean SD, n = 7) PKH-26-positive cells in these wounds, whereas initially only 1 10⁶ fluorescent fibroblasts had been seeded. In total, the percentage of mesenchymal cells minus the macrophages was similar after 5 d between wounds treated with the seeded and the acellular substitutes. In the wounds treated with the seeded substitute, however, 19.5% of the mesenchymal cells were of seeded origin. Furthermore, the rate of substitute degradation in the seeded wounds was significantly lower at 2–4 wk after wounding than in wounds treated with the acellular substitute. Vascular in-growth and the number of infiltrated macrophages were not different. In conclusion, cultured dermal fibroblasts seeded in an artificial dermal substitute and transplanted onto full-thickness wounds in pigs survived and proliferated. The observed effects of seeded fibroblasts on dermal regeneration appeared to be mediated by reducing subcutaneous fibroblastic cell migration and/or proliferation into the wounds without impairing migration of monocytes/macrophages and endothelial cells. Moreover, the degradation of the implanted dermal substitute was retarded, indicating a protective activity of the seeded fibroblasts.