Induction of vascular endothelial growth factor by fibrin as a dermal substrate for cultured skin substitute.
In the initial phase of wound healing, endogenous fibrin clots are known to form a provisional matrix and to promote angiogenesis. Growth factors such as vascular endothelial growth factor (VEGF) increase in wounds to stimulate angiogenesis. However, it remains unknown whether VEGF is induced when fibrin is used as a dermal substrate for cultured skin substitutes. The authors investigated the effect of fibrin gel as a dermal substrate for a cultured skin substitute, using human keratinocytes and dermal fibroblasts. A collagen-cultured skin substitute was also examined for comparison. VEGF in the culture supernatant in both types was measured by enzyme-linked immunosorbent assay, and VEGF mRNA was determined semiquantitatively by reverse-transcriptase polymerase chain reaction after 2 days of incubation. Experiments were performed using 12 cultured skin substitutes: four for histologic examination before transplantation, four for VEGF assay in vitro, and four for the transplantation to athymic mice. Three independent experiments were performed for each step. VEGF concentration in the fibrin-cultured supernatant was 84.3 +/- 11.8 pg/ml, whereas it was 27.8 +/- 4.68 pg/ml in the case of the collagen substrate. The relative levels of VEGF mRNA were 1.088 +/- 0.100 and 0.698 +/- 0.226, respectively. In in vivo transplantation, the fibrin-type cultured skin substitute showed an excellent take on the wound bed, and a normally proliferating keratinocyte layer with emergence of vascular endothelial cells in the transplanted floor was seen 3 days after transplantation. Vascular endothelial cells, which were identified using alkaline phosphatase stain, were significantly increased in the fibrin-type cultured skin substitute. The use of fibrin as a dermal substrate for cultured skin substitute increases the secretion of VEGF, improves regeneration of mature epidermal structure after in vivo transplantation, and promotes the migration of vascular endothelial cells.
Hojo
Motoharu
M
Department of Plastic Surgery, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
Inokuchi
Sadaki
S
Kidokoro
Masako
M
Fukuyama
Naoto
N
Tanaka
Etsuro
E
Tsuji
Chizuko
C
Miyasaka
Muneo
M
Tanino
Ryuzaburo
R
Nakazawa
Hiroe
H
D000818:Animals; D002455:Cell Division / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D020405:Dermis / Q000502:physiology*; D016228:Endothelial Growth Factors / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005337:Fibrin / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D015603:Keratinocytes / Q000166:cytology*; D008222:Lymphokines / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016038:Skin Transplantation / Q000473:pathology; D023822:Tissue Engineering / Q000379:methods*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D005337:Fibrin