The objective of this study was to establish a new method for reconstruction of a tissue-engineered skin containing melanocytes by employing tissue engineering. The keratinocytes, melanocytes and dermal fibroblasts were isolated and purified from human foreskin biopsies. Then the cells were used to construct a tissue-engineered skin containing melanocytes. The localization of melanocytes in the tissue-engineered skin was detected by DOPA staining, S-100 immunohistochemical staining and transmission electron microscope (TEM). The results showed that the melanocytes could be detected in the basal layer of the constructed skin and the melanocytes showed dendritic morphology. Moreover the constructed skins were used to repair the athymic mice skin defects. Animal experiment results indicated that the skin equivalents could successfully repair full thickness skin defects in athymic mice and generated black skins by 6weeks after grafting. Melanocytes located in the basal layer of the athymic mice skin could also be detected by using the S-100 immunohistochemical staining. Our established method is useful to repair the full-thickness skin defects.

Tongue morphology varies according to animal food habit and feeding environments. In particular, differences in food habit appear to be reflected in the morphology of the lingual papillae on the dorsal tongue surface [1][2][3] . Four types of lingual papillae are found on the tongues of human: filiform, fungiform, vallate and foliate. While, the pattern of papillae development differs in animals with more selective food habits 2,[4][5][6][7][8][9][10][11][12][13][14] . Recently, ferrets have been used as laboratory animals. The ferret is a carnivorous species belonging to the weasel family, but there have been no investigations to date describing the morphology of the lingual papillae in the ferret. In the present study, the surface morphology of ferret lingual papillae was observed by scanning electron (SEM) and light microscopes (LM), and then we focused on the food habits and discussed the morphology of the lingual papillae from the viewpoint of comparative anatomy. Materials and MethodsFour ferrets (Mustela putorius furo) were selected for this study. The animals were euthanized with intraperitoneal injection of an overdose of sodium pentobarbital (Nembutal, Abbott park, IL, USA). Following intravascular fixation with 2% glutaraldehyde which was adjusted to pH 7.4 with 0.1M phosphate-buffer (PB), and the tongues were removed. Three tongues were treated with tannic acid (1%, 4 h) and post-fixed with osmic acid (2%, 4 h). The tissues were then dehydrated with the ascending ethanol system, substituted t-butyl alcohol, and freeze-dried (JFD-310, JEOL), and observed by SEM (JSM-5500, JEOL). One tongue was cut trimmed a tissue block including the vallate papillae (width 5 mm), to confirm the presence of taste buds in the lingual papillae, because this region is the most possible location for the presence of foliate papillae in other animals, and this block was fixed in 10% formalin solution. Serial frontal sections (30 μm thick) were prepared using standard procedures (MICROM HM 500 M, Zeiss), and stained with hematoxylin-eosin, and examined under LM (MI-CROPHOT-FXA, Nikon). Parameters including diameter and height were measured according to the scale bars of the SEM images and the LM images. This animal study was approved by the Osaka Dental University Animal Research Committee (approval number 04-02005), and No foliate papillae were observed on the dorsal tongue surface of the ferret. The ferret belongs to the carnivore family but has a highly developed vallate papillae which are taste bud papillae and many taste glands. Thus we conclude that the ferrets need a large amount of saliva to swallow food because it demonstrates a large number of taste glands.

Many types of skin substitutes have been constructed using exogenous materials. Angiogenesis is an important factor for tissue-engineered skin constructs. In this study, we constructed a scaffold-free bilayered tissue-engineered skin containing a capillary network. First, we cocultured dermal fibroblasts with dermal microvascular endothelial cells at a ratio of 2 : 1. A fibrous sheet was formed by the interactions between the fibroblasts and the endothelial cells, and capillary-like structures were observed after 20 days of coculture. Epithelial cells were then seeded on the fibrous sheet to assemble the bilayered tissue. HE staining showed that tissue-engineered skin exhibited a stratified epidermis after 7 days. Immunostaining showed that the epithelium promoted the formation of capillary-like structures. Transmission electron microscopy (TEM) analysis showed that the capillary-like structures were typical microblood vessels. ELISA demonstrated that vascularization was promoted by significant upregulation of vascularization associated growth factors due to interactions among the 3 types of cells in the bilayer, as compared to cocultures of fibroblast and endothelial cells and monocultures.

Subjects with persistent left superior vena cava were classified on the basis of the presence and thickness of both superior venae cavae, the anastomotic ramus between the superior venae cavae (anastomotic ramus), and the presence of both azygos veins. Among subjects with persistent left superior vena cava, the percentage of those with weak development of the anastomotic ramus (41.5 %) or absence of an anastomotic ramus (35.8 %) was 77.3 %. In addition, 54.7 % of subjects had a left azygos vein. However, 88.7 % of subjects had a right azygos vein. In this classification, the most frequently observed types included the presence of both superior venae cavae, an anastomotic ramus, and both azygos veins (20.8 %). During student dissection practice sessions performed on 337 cadavers that were carried out from 2002 through 2010, a subject having a left superior vena cava (in 2002) and a subject having both superior venae cavae (in 2003) were detected. The former case was reported previously. The latter case is reported in this paper. The incidence of persistent left superior vena cava was 0.59 % (2/337 cadavers).

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