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

Abstract

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.

Nail Damage (Severe Onychodystrophy) Induced by Acrylate Glue: Scanning Electron Microscopy and Energy Dispersive X-Ray Investigations

“…In recent years, EDX/SEM systems have become more and more attractive for the nanoscale investigation of biological structures in various medical fields (pathology of the heart [6,7], kidney [8,9], skin [10,11], and hair [12,13,14]).…”

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

“…We previously reported that HHP at 200 MPa for 10 min can inactivate mammalian cells, skin and melanocytic nevus tissue; however, those were not inactivated at HHP lower than 150 MPa [Mahara et al, 2014;Liem et al, 2015;Morimoto et al, 2015]. Therefore, we choose pressures of 100 and 200 MPa in the present study.…”

“…We previously reported that HHP at 200 MPa for 10 min can inactivate mammalian cells and that it is capable of inactivating skin and melanocytic nevus tissue in light brown to black patches or plaques that contain nevus cells [Mahara et al, 2014;Jinno et al, 2015;Liem et al, 2015;Morimoto et al, 2015]. Giant congenital melanocytic nevi (GCMN), which are defined as nevi of more than 20 cm in diameter [Watt et al, 2004;Tannous et al, 2005;Zaal et al, 2005;Turkmen et al, 2010;Arad and Zuker, 2014], are reported to occur in approximately 1 in 20,000 newborns and have the risk of transforming into malignant melanomas in approximately 1-3% of individuals with GCMN [Tannous et al, 2005;Zaal et al, 2005;Arad and Zuker, 2014].…”

“…We reported that all of the cells in nevus specimens were inactivated after HHP at 200 MPa and that the inactivated nevus could itself be used as autologous dermis for covering skin defects after its removal Liem et al, 2015]. The combination of the inactivated nevus with a cultured epidermal autograft could be a promising treatment for GCMN.…”

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

“…We also reported that the human-cultured epidermis took and survived on the pressurized skin and nevus after pressurization at 200 MPa [7,9]. Skin consists of the epidermis, which acts as a barrier against infection and water loss, and the dermis, which supports and supplies nutrition to the epidermis.…”