Wound healing/Plastic surgeryViability and Function of Autologous and Allogeneic Fibroblasts Seeded in Dermal Substitutes after Implantation
Introduction
We developed a bilayered acellular artificial skin (artificial dermis) composed of an upper silicone sheet and a lower collagen sponge [1, 2, 3, 4, 5] by modifying the material described by Yannas and Burke [6, 7]. When the artificial dermis is implanted into a full-thickness skin defect, fibroblasts infiltrate the artificial dermis, and the formation of new capillaries and the deposition of extracellular matrix are observed in the collagen sponge. The collagen sponge is biodegraded and dermis-like tissue is formed in 2 or 3 weeks. To shorten this period, we seeded fibroblasts into the sponge and cultured them in vitro to produce cultured artificial dermis before implantation. In our previous study, when fibroblasts were cultured in a collagen sponge, the pores became filled with secreted dermal extracellular matrix in 4 weeks [8].
Various types of cultured dermal substitute have been reported [9, 10, 11, 12, 13, 14]. Collagen gel, collagen/GAG matrix, spongy collagen matrix, and synthetic biodegradable matrix are used as scaffolds and seeded with autologous or allogeneic fibroblasts. The autologous and allogeneic cultured dermal substitutes have been used for clinical application and are reported to improve wound healing and reduce wound contraction [10, 11, 12].
Nevertheless, the viability and distribution of autologous and allogeneic fibroblasts seeded on dermal substitutes after implantation have not been reported and the usefulness of the seeded fibroblasts is still unclear. This is probably because it is difficult to distinguish seeded fibroblasts from infiltrated cells in vivo. In this study, we created full-thickness wounds on the backs of guinea pigs and applied an acellular collagen sponge and collagen sponges seeded with autologous or allogeneic fibroblasts labeled with fluorescent membrane dye. Then we evaluated the tissue regeneration and wounds based on the viability and distribution of the seeded fibroblasts.
Section snippets
Materials and methods
Animal experiments were carried out according to a protocol approved by the Animal Experimental Committee of Kyoto University Graduate School of Medicine.
In vitro culture
After 3 weeks of culturing in vitro, fibroblasts attached to the collagen fibers were PKH26-positive (Fig. 1A, B). Fibroblasts attached to the culture dish were also PKH-positive (Fig. 1C, D). PKH-labeled fibroblasts were thus detectable and viable after 3 weeks.
Evaluation of epithelized area
The appearance of the skin defects is shown in Fig. 2. One week after implantation, granulation was formed in the wound where the autologous dermal substitutes had been implanted. Two weeks after implantation, the wound covered with
Discussion
Cultured dermal substitutes with allogeneic fibroblasts have been reported to improve wound healing and are used clinically [10, 11, 12, 13, 14, 20, 21]. To produce autologous cultured dermal substitutes, we must obtain a skin sample from the patient. It takes several weeks to isolate and culture fibroblasts, and it is impossible to use autologous cells for emergency use. Therefore, allogeneic cultured substitutes are used for the treatment of skin defects or skin ulcers and some of them have
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