Michiharu Sakamoto

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.

A Comparison of Conventional Collagen Sponge and Collagen-Gelatin Sponge in Wound Healing

The objective of this study was to compare the effectiveness of the collagen-gelatin sponge (CGS) with that of the collagen sponge (CS) in dermis-like tissue regeneration. CGS, which achieves the sustained release of basic fibroblast growth factor (bFGF), is a promising material in wound healing. In the present study, we evaluated and compared CGSs and conventional CSs. We prepared 8 mm full-thickness skin defects on the backs of rats. Either CGSs or CSs were impregnated with normal saline solution (NSS) or 7 μg/cm2 of bFGF solution and implanted into the defects. At 1 and 2 weeks after implantation, tissue specimens were obtained from the rats of each group (n = 3, total n = 24). The wound area, neoepithelial length, dermis-like tissue area, and the number and area of capillaries were evaluated at 1 and 2 weeks after implantation. There were no significant differences in the CGS without bFGF and CS groups. Significant improvements were observed in the neoepithelial length, the dermis-like tissue area, and the number of newly formed capillaries in the group of rats that received CGSs impregnated with bFGF. The effects on epithelialization, granulation, and vascularization of wound healing demonstrated that, as a scaffold, CGSs are equal or superior to conventional CSs.

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.

Efficacy of gelatin gel sheets in sustaining the release of basic fibroblast growth factor for murine skin defects

BACKGROUND Gelatin has been used as a material sustaining the release of basic fibroblast growth factor (bFGF), which promotes fibroblast proliferation and capillary formation and accelerates wound healing. In the application of these materials, bFGF is impregnated immediately before application, and it is difficult to conform the shape to the wound. In this study, we prepared a pliable and plastic gelatin gel sheet (GGS) that sustains bFGF and conforms to the shape of the wound as a result of cross-linking just before application. In addition, we examined the sustained release profile of bFGF from GGS and its effect on wound healing in murine skin defects. MATERIALS AND METHODS A 13-wt% gelatin solution was mixed with bFGF before cross-linking with 1% glutaraldehyde solution. GGSs impregnated with 7 μg/cm(2) of bFGF were incubated in phosphate-buffered saline and collagenase solution, and GGS degradation and bFGF release were evaluated. In the murine experiments, GGSs treated without bFGF and GGSs impregnated with 1, 3.5, 7, or 14 μg/cm(2) of bFGF were applied to full-thickness skin defects created on the backs of C57BL/6JJcl mice, and the wound closure, epithelial length, extent of granulation tissue and capillary formation were compared. RESULTS bFGF was released according to the degradation of GGS in phosphate-buffered saline, and the remaining bFGF was released in collagenase solution. In the animal studies, epithelialization was accelerated in the GGSs treated with 1 and 3.5 μg/cm(2) of bFGF, and granulation tissue formation and angiogenesis were promoted based on the amount of bFGF impregnated into the GGS. CONCLUSIONS GGS impregnated with bFGF is capable of sustaining the release of bFGF, with consequent accelerated epithelialization, granulation tissue formation, and angiogenesis in vivo. GGS is a novel and promising wound dressing that sustains bFGF and can be adapted to the shape of various wounds in the treatment of both acute and chronic wounds.

An Exploratory Clinical Trial of a Novel Treatment for Giant Congenital Melanocytic Nevi Combining Inactivated Autologous Nevus Tissue by High Hydrostatic Pressure and a Cultured Epidermal Autograft: Study Protocol

Background Giant congenital melanocytic nevi (GCMNs) are large brown to black skin lesions that appear at birth and are associated with a risk of malignant transformation. It is often difficult to reconstruct large full-thickness skin defects after the removal of GCMNs. Objective To overcome this difficulty we developed a novel treatment to inactivate nevus tissue and reconstruct the skin defect using the nevus tissue itself. For this research, we designed an exploratory clinical study to investigate the safety and efficacy of a novel treatment combining the engraftment of autologous nevus tissue inactivated by high hydrostatic pressurization with a cultured epidermal autograft (CEA). Methods Patients with congenital melanocytic nevi that were not expected to be closed by primary closure will be recruited for the present study. The target number of nevi is 10. The full-thickness nevus of the target is removed and pressurized at 200 MPa for 10 minutes. The pressurized and inactivated nevus is sutured to the original site. A small section of the patient’s normal skin is taken from around the nevus region and a CEA is prepared after a 3-week culturing process. The CEA is then grafted onto the engrafted inactivated nevus at four weeks after its retransplantation. The primary endpoint is the engraftment of the CEA at 8 weeks after its transplantation and is defined as being engrafted when the engraftment area of the inactivated nevus is 60% or more of the pretransplantation nevus area and when 80% or more of the transplanted inactivated nevus is epithelialized. Results The study protocol was approved by the Institutional Review Board of Kansai Medical University (No. 1520-2, January 5, 2016: version 1.3). The study opened for recruitment in February 2016. Conclusions This protocol is designed to show feasibility in delivering a novel treatment combining the engraftment of inactivated autologous nevus tissue and CEA. This is the first-in-man clinical trial of this treatment, and it should be a promising treatment of patients suffering from GCMN. Trial Registration University Hospital Medical Information Network: UMIN000020732; https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000022198 (Archived by WebCite at http://www.webcitation.org/6jLZH2vDN)

Melanin pigments in the melanocytic nevus regress spontaneously after inactivation by high hydrostatic pressure

We report a novel treatment for giant congenital melanocytic nevi (GCMN) that involves the reuse of resected nevus tissue after high hydrostatic pressurization (HHP). However, the remaining melanin pigments in the inactivated nevus tissue pose a problem; therefore, we performed a long-term observation of the color change of inactivated nevus tissue after HHP. Pressurized nevus specimens (200 MPa group, n = 9) and non-pressurized nevus tissues (control group, n = 9) were subcutaneously implanted into nude mice (BALB/c-nu) and then harvested 3, 6, and 12 months later. Color changes of the nevus specimens were evaluated. In the 200 MPa group, the specimen color gradually regressed and turned white, and brightness values were significantly higher in the 200 MPa group than in the control group after 6 months. This indicated that melanin pigments in the pressurized nevus tissue had spontaneously degraded and regressed. Therefore, it is not necessary to remove melanin pigments in HHP-treated nevus tissue.

Combined use of fenestrated‐type artificial dermis and topical negative pressure wound therapy for the venous leg ulcer of a rheumatoid arthritis patient

We report a case of circumferential venous leg ulcer in a rheumatoid arthritis patient. Mesh skin grafting was performed in another hospital, but the graft failed and the patient was referred to our hospital. This ulcer was treated by the combination therapy of a fenestrated‐type artificial dermis with negative pressure wound therapy (NPWT) and secondary mesh grafting using our ‘grip tape technique’. NPWT was started at −100 mmHg and continued until the formation of dermis‐like tissue. A section stained using haematoxylin and eosin and an anti‐αSMA (α smooth muscle actin) immunohistological section of the biopsy from dermis‐like tissue showed an abundant infiltration of fibroblasts and capillary formation beneath the fenestration of the silicone sheet. Threefold mesh skin grafting was subsequently performed and it was taken up completely. The fenestrated‐type artificial dermis in combination with NPWT produced good results without infection in the treatment of complex wounds. In addition, our ‘grip tape technique’ was useful to apply polyurethane foam to the entire surface of the lower leg.

Preparation of Partial-Thickness Burn Wounds in Rodents Using a New Experimental Burning Device.

ObjectiveThe manual application of hot water or hot metal to an animals skin surface is often used to prepare burn wound models. However, manual burn creation is subject to human variability. We developed a new device that can control the temperature, time, and pressure of contact to produce precise and reproducible animal burn wounds and investigated the conditions required to prepare various burn wounds using our new device. MethodsWe prepared burn wounds on F344 rats using 3 contact times 2, 4, and 10 seconds using a stamp heated to 80°C. We observed the wound-healing process macroscopically and histologically and evaluated the burn depth using a laser speckle contrast-imaging device, which evaluated the blood flow of the wound. ResultsThe changes in the burned area over time, tissue perfusion of the burn wounds, histological evaluation of the burn depth by hematoxylin-eosin and azocarmine and aniline blue staining, and the epithelialization rate (the ratio of the epithelialized area to the wound length) were evaluated on histological sections. Results indicated that the burn wounds prepared with contact times of 2, 4, and 10 seconds corresponded to superficial dermal burns, deep dermal burns, and full-thickness burns, respectively. ConclusionsWe demonstrated that partial- and full-thickness burn wounds can be precisely and reproducibly created with our new automated burning device.

Development of a novel bioabsorbable implant that is substituted by adipose tissue in vivo

Recently, adipose tissue has been regenerated by combining scaffolds, growth factors, and/or adipose‐tissue‐derived stromal cells. However, the safety of growth factors and adipose‐tissue‐derived stromal cells has not been confirmed in cancer patients. We reported the regeneration of adipose tissue in the internal space of a polypropylene mesh containing a collagen sponge (CS), without using any growth factors or cells. We herein explored the formation of adipose tissue, using the bioabsorbable implant containing CS, in rats. We prepared the implants without and with CS, using threads of either poly‐l‐lactide‐co‐ε‐caprolactone or poly‐l‐lactic acid (PLLA), and measured their strengths. The procedure was performed in the rat inguinal region. In the control group, no operative procedure was performed. In the sham‐operation group, skin incision without implantation was performed. The other groups received CS alone and the 2 implants with and without CS. The areas of formed tissue and adipose tissue inside the implants and the remnants of CS were evaluated. All implants maintained the internal space before implantation. At 6 and 12 months after implantation, the internal space was maintained and the formation of adipose tissue was promoted in the 2 PLLA groups. At 6 months, the internal space was maintained, and more adipose tissue was formed in the PLLA‐with‐CS group than in the PLLA group. Porcine collagen was absorbed within 3 months. The PLLA implant with CS is a novel bioabsorbable implant that is replaced with autologous adipose tissue after implantation.

Cultured Human Epidermis Combined With Meshed Skin Autografts Accelerates Epithelialization and Granulation Tissue Formation in a Rat Model.

Introduction As the take rate of cultured epidermal autografts in burn wound treatment is variable, widely expanded meshed auto skin grafts are often used in combination with cultured epidermal autograft to increase the take rate and achieve definitive wound coverage. However, a long time (3–4 weeks) required to prepare a cultured epidermis sheet is a disadvantage. Allogeneic cultured epidermis can be prepared in advance and cryopreserved to be used in combination with auto meshed skin grafts for treating third-degree burns. Nevertheless, the human cultured epidermis (hCE) has not been proved to accelerate wound healing after meshed skin grafting. Here, we investigated the effect of hCE on wound healing in a rat model of meshed skin grafting. Materials and Methods Human cultured epidermis was prepared from human neonatal foreskin and assessed by the release of growth factors into the culture medium using enzyme-linked immunosorbent assay. Skin wounds were inflicted on male F344 rats and treated by the application of widely meshed (6:1 ratio) autogenous skin grafts with or without hCE (n = 8 rats per group). Wound area, neoepithelium length, granulation tissue formation, and neovascularization were evaluated on day 7 postgrafting. Results Human cultured epidermis secreted IL-1&agr;, Basic fibroblast growth factor, platelet-derived growth factor-AA, TGF-&agr;, TGF-&bgr;1, and vascular endothelial growth factor in vitro. In rats, hCE accelerated wound closure (P = 0.003), neoepithelium growth (P = 0.019), and granulation tissue formation (P = 0.043), and increased the number of capillaries (P = 0.0003) and gross neovascularization area (P = 0.008) compared with the control group. Conclusions The application of hCE with meshed grafts promoted wound closure, possibly via secretion of growth factors critical for cell proliferation and migration, suggesting that hCE can enhance the healing effect of widely expanded skin autografts.