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International Journal of Bioprinting 3D bioprinting of full-thickness skin with a rete ridge structure

surface of the dermis using the plow equipped in the third expressed at high levels at the bottom of the rete ridge
head (Figure 2B, inset). Subsequently, we preset-extruded region, indicating a localized enrichment of COL17 in the
a strand with a fissure along the furrow to ensure the lowermost aspect of the rete ridge topography (Figure 3C,
maintenance of the fissure structure. Figure 2F presents the white dotted box). In addition, we explored the expression
OCT photograph of the furrow (Figure 2E, upper panel) of ALPHA6 and BETA1 integrins in the rete ridge FTSE.
and the extruded stand with fissure along the furrow We observed elevated expression of ALPHA6 and BETA1
(Figure 2E, lower panel). The fissure printed along the integrins, precisely localized at the top corner of the rete
furrow exhibited a deeper and narrower formation, closely ridges (Figure 3C, white arrowheads).
resembling the cross-section of the strand at the exit of the We investigated the cellular proliferation within the rete
nozzle. Notably, the fissure was maintained after washing ridge FTSE by assessing the expression of the Ki67 marker.
out the sacrificial alginate. In this study, we created three +
fissures per model (Figure 2F). There was a notable difference in Ki67 cell density between
the bottom of the rete ridge and the adjacent flat surface.
3.3. Rete ridge FTSE analysis The bottom of the rete ridge exhibited a higher density of
+
Conventional and rete ridge FTSEs were fabricated by Ki67 cells, indicating an area of increased cellular activity
differentiating keratinocytes on the surface of the dermis. and proliferation (Figure 3C, red dotted box). Moreover,
+
Keratinocytes were seeded onto the surface of each model the higher Ki67 cell density at the bottom of the rete ridge
and allowed to attach for one day before undergoing further correlated with our earlier observation of thicker
differentiation under air-liquid-interface (ALI) culture and well-stratified layers beneath the stratum corneum in
conditions. For conventional FTSEs, keratinocytes were this region (Figure 3A, bottom red dotted box). However,
uniformly distributed only on the surface of the dermis despite our rigorous analysis, we did not observe a
(Figure 3A, upper panels). In contrast, in the rete ridge significant difference in the expression of K10, K14, and
skin model, cells were attached not only to the surface of filaggrin between the rete ridge region and adjacent flat
the dermis but also to the narrow fissure. On day 3 of ALI region, suggesting that both regions exhibit comparable
culture (Figure 3A, middle panels), the model exhibited a levels of epidermal differentiation and protein expression.
progression in cellular organization, indicating the onset of
differentiation and the formation of the epidermal tissue. 3.4. UV irradiation on FTSE
We confirmed that the rete ridge FTSE was well-filled We irradiated UVB on rete ridge and conventional
with keratinocytes extending into the fissure, whereas the FTSEs and observed the histology and protein expression
conventional FTSE, lacking such a structure, maintained a (Figure 4). In conventional FTSEs, we observed that the
flat shape. On day 7, distinct stratification of the cell layer integrity of the stratum corneum was compromised and the
suggested a successful response of both rete ridge and expression levels of COL17, ITGA6, and ITGB1 decreased
conventional FTSEs to ALI culture conditions. On day upon exposure to UV irradiation, regardless of its intensity.
7, the H&E results (Figure 3A, bottom panel) indicated However, in rete ridge FTSEs, we observed different
significant differentiation, with a well-established stratified results based on the intensity of UV and the depth of the
2
epidermal structure. Although there was no significant model. When exposed to 25 mJ/cm of UV irradiation,
difference observed in the stratum corneum layer the integrity of the stratum corneum located at the top of
between the rete ridge and conventional FTSEs, intriguing the skin model was compromised in a manner resembling
distinctions emerged in the underlying layers. In the rete that of conventional FTSEs. However, the structure of the
ridge region, the layers beneath the stratum corneum stratum corneum within the rete ridge remained intact.
2
appeared noticeably thicker and well-organized. These Upon exposure to 50 mJ/cm UV, we observed a more
features were quantified through the interdigitation index pronounced disruption of the stratum corneum integrity.
using the H&E results on day 7. Figure 3B displays the Nevertheless, the stratum corneum in the valley part of
interdigitation index, representing the ratio of DEJ length the rete ridge remained intact. These differences were also
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to straight line length, of rete ridge and conventional observed in protein expression; although COL17, ITGA6,
FTSEs. Owing to the narrow and deep fissure structure, and ITGB1 expression decreased upon UV exposure in the
the interdigitation index of rete ridge FTSEs exhibited adjacent flat region, it remained constant within the rete
a higher value than conventional FTSEs. However, the ridge structure.
interdigitation index value of rete ridge FTSE decreased We investigated the expression of K10 and Ki67 after
over time, though it still maintained the rete ridge structure UV irradiation. Figure 5A displays the results of IF staining
compared to conventional FTSEs until day 7. for K10 and Ki67. UV exposure induced damage to K10 in
We investigated the protein expression in the skin both rete ridge and conventional FTSEs. However, under
2
model through IF staining (Figure 3C). COL17 was exposure to 25 mJ/cm UV irradiation, preservation of
Volume 10 Issue 5 (2024) 494 doi: 10.36922/ijb.3961