Abstract
We previously reported that human nevus tissue was inactivated after high hydrostatic pressure (HHP) higher than 200 MPa and that human cultured epidermis (hCE) engrafted on the pressurized nevus at 200 MPa but not at 1000 MPa. In this study, we explore the changes to the epidermal basement membrane in detail and elucidate the cause of the difference in hCE engraftment. Nevus specimens of 8 mm in diameter were divided into five groups (control and 100, 200, 500, and 1000 MPa). Immediately after HHP, immunohistochemical staining was performed to detect the presence of laminin-332 and type VII collagen, and the specimens were observed by transmission electron microscopy (TEM). hCE was placed on the pressurized nevus specimens in the 200, 500, and 1000 MPa groups and implanted into the subcutis of nude mice; the specimens were harvested at 14 days after implantation. Then, human keratinocytes were seeded on the pressurized nevus and the attachment was evaluated. The immunohistochemical staining results revealed that the control and 100 MPa, 200 MPa, and 500 MPa groups were positive for type VII collagen and laminin-332 immediately after HHP. TEM showed that, in all of the groups, the lamina densa existed; however, anchoring fibrils were not clearly observed in the 500 or 1000 MPa groups. Although the hCE took in the 200 and 500 MPa groups, keratinocyte attachment was only confirmed in the 200 MPa group. This result indicates that HHP at 200 MPa is preferable for inactivating nevus tissue to allow its reuse for skin reconstruction in the clinical setting.
Highlights
High hydrostatic pressure (HHP) technology allows cells or tissues to be inactivated without the use of chemicals such as detergents
We previously reported that all of the cells in nevus tissue were inactivated after HHP at pressures of higher than 200 MPa, as well as in normal human skin, and that the Human cultured epidermis (hCE) survived on the pressurized nevus after pressurization at 200 and 500 MPa but not at 1000 MPa
We further explore the changes of the epidermal basement membrane of human nevus and elucidate the cause of the difference in hCE engraftment by the immunohistochemical staining of laminin-332 [13] which is the key component in epidermal attachment and type VII collagen and transmission electron microscopy (TEM) [14,15,16]
Summary
High hydrostatic pressure (HHP) technology allows cells or tissues to be inactivated without the use of chemicals such as detergents. We previously reported that HHP treatment for 10 min at pressures of higher than 200 MPa can completely inactivate human and porcine skin [4, 5]. The dermal collagen fibers of human skin, which was pressurized at up to 1000 MPa, showed no apparent damage under scanning electron microscopy (SEM), and the epidermal basement membrane could be detected by the immunohistochemical staining of type IV collagen [5, 6]. We tried to reconstruct skin using a combination of skin specimens which were subjected to pressures of higher than 200 MPa, which had no viable cells, including keratinocytes, and cultured epidermal autografts which had been used clinically in the treatment of BioMed Research International patients with extended burns [7, 8]. Human cultured epidermis (hCE) engrafted and survived on human skin pressurized at 200 MPa but failed to take on the human skin pressurized at 1000 MPa [5]
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