Enhancements of the production of bilirubin and the expression of ß-globin by carbon monoxide during erythroid differentiation.

Heme is degraded by heme oxygenase to form iron, carbon monoxide (CO), and biliverdin. However, information about the catabolism of heme in erythroid cells is limited. In this study, we showed the production and export of bilirubin in murine erythroleukemia (MEL) cells. The production of bilirubin by MEL cells was enhanced when heme synthesis was induced. When mouse bone marrow cells were induced with erythropoietin to differentiate into erythroid cells, the synthesis of bilirubin increased. The expression of ß-globin was enhanced by CO at the transcriptional level. These results indicate that constant production of CO from heme regulates erythropoiesis.

Preparation of Inactivated Human Skin Using High Hydrostatic Pressurization for Full-Thickness Skin Reconstruction.

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.

Inactivation of Human Nevus Tissue Using High Hydrostatic Pressure for Autologous Skin Reconstruction: A Novel Treatment for Giant Congenital Melanocytic Nevi.

Giant congenital melanocytic nevi are intractable lesions associated with a risk of melanoma. High hydrostatic pressure (HHP) technology is a safe physical method for producing decellularized tissues without chemicals. We have reported that HHP can inactivate cells present in various tissues without damaging the native extracellular matrix (ECM). The objectives of this study were to inactivate human nevus tissue using HHP and to explore the possibility of reconstructing skin using inactivated nevus in combination with cultured epidermis (CE). Human nevus specimens 8 mm in diameter were pressurized by HHP at 100, 200, 500, and 1000 MPa for 10 min. The viability of specimens just after HHP, outgrowth of cells, and viability after cultivation were evaluated to confirm the inactivation by HHP. Histological evaluation using hematoxylin-eosin staining and immunohistochemical staining for type IV collagen was performed to detect damage to the ECM of the nevus. The pressurized nevus was implanted into the subcutis of nude mice for 6 months to evaluate the retention of human cells. Then, human CE was applied on the pressurized nevus and implanted into the subcutis of nude mice. The viability of pressurized nevus was not detected just after HHP and after cultivation, and outgrowth of fibroblasts was not observed in the 200, 500, and 1000 MPa groups. Human cells were not observed after 6 months of implantation in these groups. No apparent damage to the ECM was detected in all groups; however, CE took on nevus in the 200 and 500 MPa groups, but not in the 1000 MPa group. These results indicate that human nevus tissue was inactivated by HHP at more than 200 MPa; however, HHP at 1000 MPa might cause damage that prevents the take of CE. In conclusion, all cells in nevus specimens were inactivated after HHP at more than 200 MPa and this inactivated nevus could be used as autologous dermis for covering full-thickness skin defects after nevus removal. HHP between 200 and 500 MPa will be optimal to reconstruct skin in combination with cultured epidermal autograft without damage to the ECM.

A simple and highly sensitive method of measuring heme oxygenase activity.

Heme oxygenase (HO) is a rate-limiting step of heme degradation, which catalyzes the conversion of heme into biliverdin, iron, and CO. HO has been characterized in microorganisms, insects, plants, and mammals. Previously used assays of HO activity were complicated and had low sensitivity. We found that the use of an eel bilirubin-bound fluorescent protein, UnaG, can achieve a highly sensitive and simple assay of HO activity. Using several enzyme sources including human culture cells, homogenates of plant tissues, and recombinant yeast HO, data were successfully obtained. The present method can facilitate the examination of HO in various organisms.

Gelatin hydrogel impregnated with platelet-rich plasma releasate promotes angiogenesis and wound healing in murine model.

Platelet-rich plasma (PRP) contains numerous growth factors to promote wound healing and angiogenesis. The objective of this study was to explore the efficacy of biodegradable gelatin hydrogel impregnated with PRP releasate (PRPr) in the wound healing process compared with the single application of PRPr prepared from mouse PRP centrifuged by a double-spin method. Gelatin hydrogel disks with an isoelectric point of 5.0 were used in this study. A total of 180 mice (n = 45/group) were randomly assigned to the following 4 experimental groups: control group, biodegradable gelatin hydrogel group, PRPr group and gelatin hydrogel impregnated with PRPr (PRPrG) group. Wound area and epithelialization were compared on days 1, 5, 7, 14 and 21 post-wounding. After complete epithelialization, wound contraction was also evaluated. Neovascularization using immunohistochemical staining of von Willebrand factor was analyzed on day 14. The wound area of PRPrG on days 5, 7 and 14 was smaller than that in the other groups (p < 0.01). The epithelialization lengths of PRPrG on days 7 and 14 were significantly longer than the others (p < 0.01). The capillary formation of PRPrG was also superior to those in all other groups on day 14. On day 21, all wounds were completely epithelialized and PRPrG prevented wound contraction the most. It is concluded that the sustained-release system of gelatin impregnated with PRPr can stimulate angiogenesis and accelerate wound healing compared with the single application of PRP.

Adipogenesis using human adipose tissue-derived stromal cells combined with a collagen/gelatin sponge sustaining release of basic fibroblast growth factor.

We have developed a collagen/gelatin sponge (CGS) that can provide a sustained release of basic fibroblast growth factor (bFGF). In our previous study, it was shown that CGS impregnated with the appropriate dosage of bFGF accelerates dermis-like tissue formation two or three times earlier than an existing collagen sponge. In this study, adipogenesis was evaluated using CGSs disseminated with adipose tissue-derived stem cells (ASCs). Human ASCs were primarily isolated from human adipose tissue that was obtained during breast cancer surgery with informed consent at Kyoto University Hospital. ASCs were isolated from collagenase digests of adipose tissue. ASCs were labelled with PKH26. CGSs (8 mm diameter × 3 mm thickness) were impregnated with bFGF (0.1, 1, 7, 14 µg/cm(2) ) or normal saline solution. Then the labelled cells were disseminated (passage 3) on CGSs at a seeding density of 1 × 10(5) cells/cm(2) and implanted into the back subcutis of nude mice. Six weeks after implantation, adipogenesis at the administered site was evaluated. Immunohistological staining with von Willebrand factor (vWf) was performed to evaluate newly formed capillaries. Newly formed adipose tissue was observed macroscopically and histologically in all groups. The weight and area of regenerated adipose tissue were largest in the 1 µg/cm(2) bFGF group. Under a fluorescent microscope, newly formed adipose tissue in the bFGF-administered group was PKH-positive. These findings show that ASCs differentiated and formed adipose tissue. In this study, we showed that our CGSs impregnated with bFGF could be used as scaffolds with ASCs for adipogenesis.

Autologous skin reconstruction by combining epidermis and acellular dermal matrix tissue derived from the skin of giant congenital melanocytic nevi.

Giant congenital melanocytic nevi (GCMN) are defined as nevi greater than 20 cm in diameter. It is difficult to completely remove GCMN because of the lack of available skin grafts for covering the resultant defects. This study examined whether it is possible to produce reconstructed skin by combining epidermal and acellular dermal matrix (ADM) tissue derived from excised GCMN. GCMN skin samples were obtained with the informed consent of volunteer patients. The abilities of hypertonic saline (1 N NaCl), 0.05% trypsin, 0.1% SDS (sodium dodecyl sulfate), and phosphate buffered saline (PBS) to decellularize GCMN tissue were compared. The specimens were incubated in one of the test solutions at 37 °C for 48 h, before being washed with PBS at 4 °C for 14 days. Residual nuclei, residual DNA, nevus tissue viability, and the structural integrity of the basement membrane and capillaries were evaluated before treatment, and after 48 h' treatment with or without 7 or 14 days' washing. We tried to produce reconstructed skin by combining the resultant ADM with enzymatically separated GCMN epidermal tissue. The histological structure of the reconstructed skin was examined after it had been cultured for 5 days. In the SDS group, most cells had been removed after 48 h, and the DNA content of the ADM was significantly lower than in the other groups. As for viability, no significant difference was detected among the groups. The basement membrane and capillaries remained intact in all groups. After 5 days' culturing, the epidermis had become attached to the ADM in all groups, except the SDS group. SDS displayed a superior decellularization ability compared with the other methods; however, it cannot be used to produce reconstructed skin because of its toxicity. In conclusion, we produced reconstructed skin that was devoid of nevus cells by combining GCMN epidermal tissue with GCMN-derived ADM produced with NaCl or trypsin. This is a promising treatment strategy for giant nevus.

Treating a collagen scaffold with a low concentration of nicotine promoted angiogenesis and wound healing.

BACKGROUND: Nicotine, one of the major pharmacologically active agents of cigarette smoke, has various effects on cell proliferation, and it has recently been reported to have angiogenic effects. In our previous study, we showed that the topical administration of nicotine at a low concentration accelerated wound healing. This study aimed to evaluate the efficacy of nicotine and synergistic effects of combination treatment with nicotine and basic fibroblast growth factor (bFGF) in a murine excisional wound model treated with artificial dermis. METHODS: Full-thickness defects (8 mm in diameter) were created on the backs of mice, and artificial dermis was sutured to the defects. Phosphate-buffered saline (10 µL), nicotine (10(-3), 10(-4), or 10(-5) M), bFGF (0.5 µg), and both bFGF and 10(-4) M nicotine were topically administered to the artificial dermal tissue for 7 d. The mice were killed on day 14, and the wound area, neoepithelium length, and area of newly formed capillaries in the artificial dermis were evaluated. RESULTS: The wound areas treated with 10(-4) M nicotine, bFGF, or bFGF plus 10(-4) M nicotine were significantly smaller than those in the control group. In these three groups, the neoepithelium in the bFGF plus 10(-4) M nicotine group was significantly longer than that in the other groups. There was no significant difference between the neoepithelium lengths of the control and 10(-5) M nicotine groups. The 10(-3) M nicotine group displayed the least re-epithelization among the groups. CONCLUSIONS: In this study, 10(-4) M nicotine induced angiogenesis in, and accelerated the healing of, wounds treated with artificial dermis. bFGF and nicotine had synergistic effects, and the combined use of nicotine and bFGF is an effective wound healing method.