Chizuru Jinno

The Rapid Inactivation of Porcine Skin by Applying High Hydrostatic Pressure without Damaging the Extracellular Matrix

We previously reported that high hydrostatic pressure (HHP) of 200 MPa for 10 minutes could induce cell killing. In this study, we explored whether HHP at 200 MPa or HHP at lower pressure, in combination with hyposmotic distilled water (DW), could inactivate the skin, as well as cultured cells. We investigated the inactivation of porcine skin samples 4 mm in diameter. They were immersed in either a normal saline solution (NSS) or DW, and then were pressurized at 100 and 200 MPa for 5, 10, 30, or 60 min. Next, we explored the inactivation of specimens punched out from the pressurized skin 10 × 2 cm in size. The viability was evaluated using a WST-8 assay and an outgrowth culture. The histology of specimens was analyzed histologically. The mitochondrial activity was inactivated after the pressurization at 200 MPa in both experiments, and no outgrowth was observed after the pressurization at 200 MPa. The arrangement and proportion of the dermal collagen fibers or the elastin fibers were not adversely affected after the pressurization at 200 MPa for up to 60 minutes. This study showed that a HHP at 200 MPa for 10 min could inactivate the skin without damaging the dermal matrix.

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.

Verification of the Inactivation of Melanocytic Nevus in vitro Using a Newly Developed Portable High Hydrostatic Pressure Device

High hydrostatic pressure (HHP) technology is a physical method for inactivating tissue. We reported that nevus specimens were inactivated after HHP at 200 MPa and that the inactivated nevus could be used as autologous dermis for covering skin defects. In this study, we verified the inactivation of nevus specimens using a newly developed portable HHP device which will be used in a clinical trial. Nevus tissue specimens were obtained from 5 patients (mean age 7.2 years, range 1-19). We cultured fibroblasts and nevus cells from the tissue specimens and then evaluated their inactivation after HHP at 200 MPa by confirming the attachment of the suspensions and by the live/dead staining of the suspensions, through the dissociation of the cells on chamber slides and by the live/dead staining of the remaining cells. The cells were also quantitatively evaluated by WST-8 assay. We then confirmed the inactivation of the nevus specimens after HHP using explant culture. Our results indicated that fibroblasts and nevus cells were inactivated after HHP at 200 MPa, with the exception of a small percentage of green-colored cells, which reflected the remaining activity of the cellular esterases after HHP. No cells migrated from the nevus specimens after HHP at 200 MPa. We verified the inactivation of fibroblasts and nevus cells cultured from nevus specimens, and in the nevus samples themselves after pressurization at 200 MPa using this device. This device could be used in clinical trials for giant congenital melanocytic nevi and may thus become useful in various medical fields.

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 evaluation of the engraftment and the blood flow of porcine skin autografts inactivated by high hydrostatic pressure.

We previously reported that exposure to a high hydrostatic pressure (HHP) of 200 MPa could completely inactivate porcine skin without damaging the extracellular matrix. In this study, we used an autologous porcine skin graft model and explored whether the skin inactivated by HHP could be engrafted without inflammation to the residual cellular components. Twenty-one full-thickness skin grafts of 1.5 × 1.5 cm in size were prepared from a minipig (n = 2). Grafts were either nonpressurized or pressurized to 100, 150, 200, 300, 500, or 1000 MPa (n = 3) and randomly implanted on the fascia and removed at 1 and 4 weeks after grafting. All grafts showed complete engraftment at the macroscopic level and microcirculation was detected by a full-field laser speckle perfusion imager. The epidermis was removed and skin appendages were not observed in the grafts pressurized to more than 200 MPa. Azan and Elastica van Gieson staining showed no sign of dermal collagen fiber degeneration, while elastin fibers were observed. The fibroblasts and capillaries were observed to have infiltrated to dermis in all groups without severe inflammation. In conclusion, we showed that skin inactivated by HHP up to 1000 MPa could be engrafted successfully without removing cellular remnants.

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.

The superiority of the autografts inactivated by high hydrostatic pressure to decellularized allografts in a porcine model

We are developing a novel skin regeneration therapy in which the inactivation of nevus tissue via high hydrostatic pressure (HHP) is used in the reconstruction of the dermis in combination with a cultured epidermal autograft. In this study, we used a porcine skin graft model to explore whether autologous skin including cellular debris inactivated by HHP or allogeneic skin decellularized by HHP is better for dermal reconstruction. Grafts (n = 6) were prepared for five groups each: autologous skin without pressurization group (control group), autologous skin inactivated by 200 MPa group, autologous skin inactivated by 1000 MPa group, allogeneic skin decellularized by 200 MPa group, and allogeneic skin decellularized by 1000 MPa group. All of the grafts at 1, 4, and 12 weeks showed complete engraftment macroscopically. The mean areas of the grafts of the control group (p < 0.01) and autologous 200 MPa group (p < 0.01) were larger than that of the allogeneic 1000 MPa group at four weeks after implantation. The thickness of the control group and autologous 200 MPa group was comparable, and that of the autologous 200 MPa group was significantly thicker than that of the allogeneic 200 MPa group (p < 0.01). This suggests that the autologous dermis was superior to the allogeneic decellularized dermis as a skin graft, and that HHP at 200 MPa provided a better outcome than HHP at 1000 MPa.