A major requirement for the microsurgical repair of contour defects of the skin, for example, following removal of a skin cancer on the face, is a mass of vascularised subcutaneous tissue. Such tissue can be generated in vivo using basic tissue engineering principles. In previous studies in our laboratory, we have used a model comprising an arteriovenous (AV) shunt loop sandwiched in artificial dermis, placed in a cylindrical plastic growth chamber, and inserted subcutaneously to grow new connective tissue progressively up to 4 weeks. To learn more about the basic growth characteristics with this model, the same AV shunt loop within a chamber without added extracellular matrix was inserted subcutaneously into the groins of rats for 2, 4, or 12 weeks (n = 5 per group). There was a progressive increase in the mass and volume of tissue such that the chamber was two-thirds full after 12 weeks. Histological examination showed that at 2 weeks there was evidence of fibroblast and vascular outgrowth from the AV shunt, with the formation of granulation tissue, surrounded by a mass of coagulated exudate. At 4 weeks the connective tissue deposition was more extensive, with a mass of more mature granulation tissue containing considerable collagen. By 12 weeks there was an extensive, well vascularized mass of mature fibrous tissue. The blood vessels and residual adventitia of the AV shunt were the likely source of growth factors and of the cells which populated the chamber with new maturing connective tissue. A patent AV shunt in an isolated chamber appears to be the minimal requirement for the generation of new vascularized tissue that is potentially suitable for microsurgical transplantation.
Isogenous fibroblasts derived from the skin of inbred Sprague-Dawley rats were cultured in vitro, labeled with bisbenzamide (BB) or carboxyfluorescein diacetate (CFDA), and seeded into polycarbonate growth chambers. After 24 h incubation in vitro, the chambers, either empty or containing an arteriovenous (AV) shunt, were implanted subcutaneously into the inguinal region of Sprague-Dawley rats and examined by fluorescence microscopy 2 or 7 days later. The AV shunt remained patent in all experiments. The density of labeled cells on the chamber surface in all chambers decreased in the first 2 days after insertion. At 7 days, the cell density in the empty chambers had not altered from the 2-day level, but the density in the AV shunt containing chambers had increased to almost three times the day 2 level (p = 0.013). It appears that an AV shunt can induce a significant proliferation of fibroblasts implanted adjacent to it. For at least 7 days after labeling, BB and CFDA provide a simple and effective method of quantitative detection of implanted fibroblasts. It is concluded that nutrients from the AV shunt implanted in a growth chamber result in a significant increase in the number of viable, matrix-synthesizing cells, compared with AV shunt-free controls.
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