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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

Micrographs of fibroblasts on Day 14.Yellow arrows indicate pressurized skin and blue arrowheads indicate fibroblasts. Scale bar: 100 μm.
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pone.0133979.g002: Micrographs of fibroblasts on Day 14.Yellow arrows indicate pressurized skin and blue arrowheads indicate fibroblasts. Scale bar: 100 μm.

Mentions: After the outgrowth culture of the pressurized skin, fibroblasts were observed in NSS-0, DW-0, NSS-100, DW-100, NSS-150 and DW-150, but no cells were confirmed in NSS-200, DW-200, NSS-1000 and DW-1000 on Day 14 (Fig 2). After cultivation, the viability of pressurized skin samples in DW-150 was revitalized, but the samples pressurized at more than 200 MPa still showed no viability (Fig 3).


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)

Micrographs of fibroblasts on Day 14.Yellow arrows indicate pressurized skin and blue arrowheads indicate fibroblasts. Scale bar: 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133979.g002: Micrographs of fibroblasts on Day 14.Yellow arrows indicate pressurized skin and blue arrowheads indicate fibroblasts. Scale bar: 100 μm.
Mentions: After the outgrowth culture of the pressurized skin, fibroblasts were observed in NSS-0, DW-0, NSS-100, DW-100, NSS-150 and DW-150, but no cells were confirmed in NSS-200, DW-200, NSS-1000 and DW-1000 on Day 14 (Fig 2). After cultivation, the viability of pressurized skin samples in DW-150 was revitalized, but the samples pressurized at more than 200 MPa still showed no viability (Fig 3).

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