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

Abstract

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.

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].…”

“…The pressurization process takes only 10 min and it is desirable to pressurize the nevus during the operation to minimize the operation time and the operative stress to patients. However, conventional HHP devices are too big and heavy, and require a specialized place of installation; thus, they are not suitable for use in the operating room setting Morimoto et al, 2015].…”

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 combining inactivation of the nevus via HHP (200 MPa) for the reconstruction of the dermis with CEA for the reconstruction of the epidermis . The advantage of this therapy is its use of autologous dermis, in which all cells including nevus cells have been inactivated using HHP . Despite concerns that the remaining debris from the inactivated cells may induce an inflammatory reaction, we have already shown that autologous porcine skin inactivated using HHP up to 1000 MPa was successfully engrafted for up to 4 weeks after grafting .…”

“…It is usually difficult to reconstruct skin defects after the removal of a GCMN, due to the shortage of available autologous skin grafts; therefore, it is also difficult to prevent the subsequent development of melanoma . To overcome this problem, we are developing a novel reconstruction treatment using the removed nevus tissue inactivated by high hydrostatic pressure (HHP) at 200 MPa for dermal reconstruction . We used a cultured epidermal autograft (CEA) via Green's method, which has been established since the 1970s, for the epidermal reconstruction in this treatment strategy.…”