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.

Fig 1. Comparison of the viability of pressurized skin.

The mean absorbance of DMEM was set as an arbitrary zero point. The absorbance levels of NSS-0, DW-0, NSS-100, DW-100 and NSS-150 were significantly higher than that of DMEM (p<0.01). The absorbance levels of DW-150, NSS-200, DW-200, NSS-1000 and DW-1000 were significantly lower than those of NSS-0, DW-0, NSS-100, DW-100 and NSS-150 (p<0.01). The absorbance level of NSS-0 was significantly higher than those of NSS-100, DW-100 and NSS-150 (p<0.01). The absorbance level of NSS-150 was significantly lower than those of NSS-100 and DW-100 (p<0.01).

Fig 2. Micrographs of fibroblasts on Day 14.

Yellow arrows indicate pressurized skin and blue arrowheads indicate fibroblasts. Scale bar: 100 μm.

Fig 3. Comparison of the viability of pressurized skin after cultivation on Day 14.

The absorbance levels of NSS-0, DW-0, NSS-100, DW-100, NSS-150 and DW-150 were significantly higher than that of DMEM (p<0.01). The absorbance levels of NSS-200, DW-200, NSS-1000 and DW-1000 were significantly lower than those of NSS-0, DW-0, NSS-100, DW-100, NSS-150 and DW-150 (p<0.01).

Fig 4. Micrographs of HE sections of pressurized skin.

Yellow arrowheads indicate the remained epidermis. Scale bar: 100 μm.

Fig 5. Micrographs of immunohistochemical staining of type IV collagen.

Red arrowheads indicate the basement membrane of epidermis and green arrowheads indicate the basement membrane of capillaries. Scale bar: 100 μm.

Fig 6. Micrographs of immunohistochemical staining of vWF.

Red arrowheads indicate stained capillaries. Scale bar: 100 μm.

Fig 7. SEMs of pressurized skin.

Scale bar: 100 μm.

Fig 8. HE-stained sections of implanted pressurized skin without CE on Day 14.

Black arrowheads indicate the epidermis of the implanted pressurized skin and red arrowheads indicate the dermis of the pressurized skin. Scale bar: 100 μm.

Fig 9. HE-stained sections (upper figures) and fluorescent micrographs (lower figures) of implanted pressurized skin with CE on Day 14.

Yellow arrowheads show CE that took on pressurized skin on HE sections and the CE was PKH-positive in fluorescent micrographs. Scale bar: 100 μm.