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Summary:. We developed a surgical technique to correct cryptotia using a subcutaneous pedicled flap raised from the retroauricular region. A skin paddle over the caudal part of the auricular sulcus is designed and transferred to the skin defect of the upper posterior surface of the auricle. This procedure has been performed on 17 ears in 14 patients with cryptotia since 1992. There were no postoperative complications, such as necrosis of the flaps or deformities, and cryptotia did not recur in any patient. A satisfactory auricular contour with a sufficiently deep auriculotemporal sulcus was preserved in all cases. The intraoperative procedure to correct cryptotia and outcomes obtained are presented herein and compared with other procedures.

Background: Recently, human adipose-derived stem cells (hASCs) were discovered in the human subcutaneous adipose tissue. PLTMax Human Platelet Lysate (PLTMax), a supplement refined from human platelets, has been reported to have proliferative effects on bone marrow mesenchymal stem cells. The proliferative effects of PLTMax on ASCs were investigated in this study. Methods: The ASCs in DMEM (serum-free), DMEM+PLTMax (1%, 2%, 5%, and 10%), and DMEM+FBS (10%) were cultivated for two, five, and seven days. The cell growth rate was examined, BrdU incorporation, and the cell cycle and Ki-67 immunostaining were performed. The cell growth rate was investigated when each inhibitor (PD98059, SP600125, SB203580, and LY294002) was added and phosphorylation of ERK1/2, JNK, p38, and Akt were examined by western blotting. The cell surface marker of hASCs was also analyzed. Results: The cells in the PLTMax (5%) group showed significantly more proliferation compared to the cells in control (serum-free) and FBS (10%) groups, and a significant increase in the number of cells in the S phase and G2/M phase. The number of Ki-67 positive cells increased significantly in the DMEM+ PLTMax (5%) and the FBS (10%) groups. The addition of inhibitors PD98059, SP600125, SB203580, and LY294002 decreased the proliferative effects of PLTMax on ASCs. Phosphorylation of ERK1/2, JNK, p38, and Akt was observed in both the PLTMax (5%) and the FBS (10%) groups. Conclusions: For human adipose stem cells, 5% PLTMax was the optimum concentration, which showed a significantly higher proliferative effect than 10% FBS. PLTMax is a useful medium additive, which can substitute FBS. The proliferative effects of PLTMax are suggested to function via multiple signaling pathways, similar to FBS.

Adipose tissue-derived stem cells (ASCs) have been recently isolated from human subcutaneous adipose tissue. ASCs may be useful in regenerative medicine as an alternative to bone marrow-derived stem cells. Changes in the oxygen concentration influence physiological activities, such as stem cell proliferation. However, the effects of the oxygen concentration on ASCs remain unclear. In the present study, the effects of hypoxia on ASC proliferation were examined.Normal human adipose tissue was collected from the lower abdomen, and ASCs were prepared with collagenase treatment. The ASCs were cultured in hypoxic (1%) or normoxic (20%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible factor (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these growth factors on ASC proliferation were investigated. Chromatin immunoprecipitation (ChIP) of the HIF-1α-binding hypoxia responsive element in FGF-2 was performed. HIF-1α was knocked down by siRNA, and FGF-2 expression was investigated.ASC proliferation was significantly enhanced in the hypoxic culture and was inhibited by ERK and Akt inhibitors. Hypoxia for 5-15 minutes stimulated the phosphorylation of ERK1/2 among MAP kinases and induced HIF-1α expression. The levels of VEGF and FGF-2 mRNA and protein in the ASCs were significantly enhanced in hypoxia, and FGF-2 increased ASC proliferation. The ChIP assay revealed an 8-fold increase in the binding of HIF-1α to FGF-2 in hypoxia. HIF-1α knockdown by siRNA partially inhibited the FGF-2 expression of ASCs induced by hypoxia.ASC proliferation was enhanced by hypoxia. HIF-1α activation, FGF-2 production, and the ERK1/2 and Akt pathway were involved in this regulatory mechanism.

We have reported that high-hydrostatic-pressure (HHP) technology is safe and useful for producing various kinds of decellularized tissue. However, the preparation of decellularized or inactivated skin using HHP has not been reported. The objective of this study was thus to prepare inactivated skin from human skin using HHP, and to explore the appropriate conditions of pressurization to inactivate skin that can be used for skin reconstruction. Human skin samples of 8 mm in diameter were packed in bags filled with normal saline solution (NSS) or distilled water (DW), and then pressurized at 0, 100, 150, 200 and 1000 MPa for 10 minutes. The viability of skin after HHP was evaluated using WST-8 assay. Outgrowth cells from pressurized skin and the viability of pressurized skin after cultivation for 14 days were also evaluated. The pressurized skin was subjected to histological evaluation using hematoxylin and eosin staining, scanning electron microscopy (SEM), immunohistochemical staining of type IV collagen for the basement membrane of epidermis and capillaries, and immunohistochemical staining of von Willebrand factor (vWF) for capillaries. Then, human cultured epidermis (CE) was applied on the pressurized skin and implanted into the subcutis of nude mice; specimens were subsequently obtained 14 days after implantation. Skin samples pressurized at more than 200 MPa were inactivated in both NSS and DW. The basement membrane and capillaries remained intact in all groups according to histological and immunohistological evaluations, and collagen fibers showed no apparent damage by SEM. CE took on skin pressurized at 150 and 200 MPa after implantation, whereas it did not take on skin pressurized at 1000 MPa. These results indicate that human skin could be inactivated after pressurization at more than 200 MPa, but skin pressurized at 1000 MPa had some damage to the dermis that prevented the taking of CE. Therefore, pressurization at 200 MPa is optimal for preparing inactivated skin that can be used for skin reconstruction.