The Alteration of the Epidermal Basement Membrane Complex of Human Nevus Tissue and Keratinocyte Attachment after High Hydrostatic Pressurization

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.

Figure 1

Micrographs of the immunohistochemical staining of type VII collagen. The open arrowheads indicate the remaining original epidermis of the nevus specimens in the control and 100 MPa groups. The closed arrowheads indicate the stained type VII collagen. The type VII collagen was clearly stained in the control, 100 MPa, and 200 MPa groups and weakly stained in the 500 MPa group but was not stained in 1000 MPa group. Scale bar = 100 μm.

Figure 2

Micrographs of the immunohistochemical staining of laminin-332. The closed arrowheads indicate the stained laminin-332 in the basement membrane. Laminin-332 was clearly stained in the control, 100 MPa, and 200 MPa groups and weakly stained in the 500 MPa group but was not stained in the 1000 MPa group. Scale bar = 100 μm.

Figure 3

TEM micrographs of the dermoepidermal junction of the pressurized nevus. The closed arrowheads indicate the lamina densa in the basement membrane, which was observed in all specimens. The open arrowheads indicate the anchoring fibrils, which were observed in the control and 200 MPa groups but not in the 500 MPa or 1000 MPa groups. Scale bar = 2 μm.

Figure 4

Micrographs of the immunohistochemical staining of type VII collagen in the nevus specimens with CE after implantation. The open arrowheads indicate the implanted CE in the 200 MPa and 500 MPa groups. The closed arrowheads indicate the stained type VII collagen. The type VII collagen was clearly stained in the 200 MPa group but not in the 500 MPa or 1000 MPa groups. Scale bar = 100 μm.

Figure 5

Micrographs of the immunohistochemical staining of laminin-332 in the nevus specimens with CE after implantation. The closed arrowheads indicate the stained laminin-332. Laminin-332 was clearly stained in the 200 MPa and 500 MPa groups but not in the 1000 MPa group. Scale bar = 100 μm.

Figure 6

CLSM images of keratinocytes on the pressurized nevus specimens after 6 days of culturing. Actin fibers and nuclei were stained with rhodamine phalloidin and DAPI, respectively. Only the 200 MPa group was clearly stained. Scale bar = 50 μm.

Figure 7

Micrographs of the HE-stained sections of the pressurized nevus specimens after 6 days of culturing. The original epidermis of the nevus specimens remained intact and seeded keratinocytes were not observed in the control group. The original epidermis was removed in the 200, 500, and 1000 MPa groups. The attachment of seeded keratinocytes on the pressurized nevus was only observed in the 200 MPa group. The open arrowheads indicate the original epidermis. The closed arrowheads indicate the seeded keratinocytes. Scale bar = 50 μm.

Figure 8

Micrographs of immunohistochemical staining of type VII collagen in the nevus specimens after 6 days of culturing. The closed arrowheads indicate the stained type VII collagen. The anchoring fibrils were clearly stained in the control and the 200 MPa groups and were weakly and locally stained in the 500 MPa group but were not stained in the 1000 MPa group. Scale bar = 50 μm.

Figure 9

Micrographs of immunohistochemical staining of laminin-332 in the nevus specimens after 6 days of culturing. The closed arrowheads indicate the stained laminin-332. Laminin-332 was clearly stained in the control and 200 MPa groups and weakly and locally stained in the 500 MPa group but was almost completely unstained in the 1000 MPa group. Scale bar = 50 μm.