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

Liem PH, Morimoto N, Mahara A, Jinno C, Shima K, Ogino S, Sakamoto M, Kakudo N, Inoie M, Kusumoto K, Fujisato T, Suzuki S, Yamaoka T - PLoS ONE (2015)

Bottom Line: We have reported that high-hydrostatic-pressure (HHP) technology is safe and useful for producing various kinds of decellularized tissue.Skin samples pressurized at more than 200 MPa were inactivated in both NSS and DW.CE took on skin pressurized at 150 and 200 MPa after implantation, whereas it did not take on skin pressurized at 1000 MPa.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan; Department of Plastic and Aesthetic Surgery, Pham Ngoc Thach University of Medicine, Ho Chi Minh City, Vietnam.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0133979.g009: 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.

Mentions: HE-stained sections of implanted pressurized skin without CE on Day 14 showed that the epidermis in NSS-0, NSS-100 and DW-0 was still intact after implantation (Fig 8). The epidermis did not regenerate in the other groups in which it had been removed before implantation. CE prepared from PKH26-stained keratinocytes was implanted into those groups and took on pressurized skin in all groups except for NSS-1000 and DW-1000 (Fig 9). The infiltration of recipient cells to pressurized skin was confirmed and no apparent inflammatory response was observed (Fig 8 and Fig 9). The epidermis was not observed in NSS-1000 and DW-1000, and PKH-positive keratinocytes were scattered on the dermis and did not form epithelium in these two groups.


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

Liem PH, Morimoto N, Mahara A, Jinno C, Shima K, Ogino S, Sakamoto M, Kakudo N, Inoie M, Kusumoto K, Fujisato T, Suzuki S, Yamaoka T - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520601&req=5

pone.0133979.g009: 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.
Mentions: HE-stained sections of implanted pressurized skin without CE on Day 14 showed that the epidermis in NSS-0, NSS-100 and DW-0 was still intact after implantation (Fig 8). The epidermis did not regenerate in the other groups in which it had been removed before implantation. CE prepared from PKH26-stained keratinocytes was implanted into those groups and took on pressurized skin in all groups except for NSS-1000 and DW-1000 (Fig 9). The infiltration of recipient cells to pressurized skin was confirmed and no apparent inflammatory response was observed (Fig 8 and Fig 9). The epidermis was not observed in NSS-1000 and DW-1000, and PKH-positive keratinocytes were scattered on the dermis and did not form epithelium in these two groups.

Bottom Line: We have reported that high-hydrostatic-pressure (HHP) technology is safe and useful for producing various kinds of decellularized tissue.Skin samples pressurized at more than 200 MPa were inactivated in both NSS and DW.CE took on skin pressurized at 150 and 200 MPa after implantation, whereas it did not take on skin pressurized at 1000 MPa.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan; Department of Plastic and Aesthetic Surgery, Pham Ngoc Thach University of Medicine, Ho Chi Minh City, Vietnam.

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.

No MeSH data available.


Related in: MedlinePlus