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

Subsequently, the slides were incubated at 4°C for 18 h with the experimental outcomes. Using the validated CFD
specific primary antibodies against proteins of interest, method, we designed a precursor cartridge with a simple
including collagen 17 (COL17, ab184996; Abcam, United cross-sectional architecture capable of creating narrow
Kingdom [UK]), integrin beta 1 (ITGB1, ab24693; Abcam, fissures, and fluid flow simulations were performed. The
UK), integrin alpha 6 (ITGA6, ab181551; Abcam, UK), detailed dimensions of the simulation model are illustrated
cytokeratin 10 (K10, ab9026; Abcam, UK) cytokeratin in Figure 1A.
14 (K14, ab7800; Abcam, UK), Kiel 67 (Ki67, ab16667; The simulation result indicated that the sacrificial bioink
Abcam, UK), and filaggrin (FLG, 905804; Biolegend, USA). (indicated by red particles) in the printed strand would be
Following primary antibody incubation, the samples were
washed thoroughly and then incubated with goat anti- over-extruded, leading to the formation of large fissures
mouse Alexa Fluor 488, (ab150113; Abcam, UK) and goat (Figure 1B). Therefore, we conducted further simulations
anti-rabbit lgG Alexa Fluor 594 (ab150080; Abcam, UK). by iterating the architecture of the precursor cartridge until
Nuclei were counterstained with a nuclear marker, 4°C,6- achieving the desired structure size. Through this trial-and-
diamidino-2-phenylindole. Finally, the stained samples error process in the simulation, a precursor cartridge was
were observed using an upright microscope (BX53; designed, as illustrated in Figure 1C, with the simulated
Olympus, Japan) equipped with a digital camera (DP73; width and depth of the fissure in the extruded strand being
Olympus, Japan). 164 and 594 μm, respectively. Finally, the simulation result
was confirmed by comparison with the experimental result
2.11. Interdigitation index (Figure 1D). Figure 1E indicates that the width and depth
The interdigitation index was calculated by analyzing H&E of the fissure in the experimental results were similar to
images using ImageJ (version 1.53t; National Institute of those obtained in the simulation results.
Health, USA). Briefly, using the segmented line tool, the
boundary between the DEJ of the FTSEs was traced from 3.2. Printing process for rete ridge FTSEs
one end of the image to the other and divided by a straight Figure 2A illustrates a schematic overview of the key
line connecting the same start and endpoints. steps in the printing process, offering a comprehensive
insight into our methodology. To fabricate the rete ridge
2.12. Statistical analysis skin model, four printing heads were used in this step
Statistical analysis was performed using Microsoft Excel. (Figure 2B). From left to right, the first and second
Two-tailed t-tests were used for comparisons, and p < 0.05 heads were designated for dermis printing; however, the
was considered significant. All data are presented as mean precursor cartridge, designed to create fissure structures
± standard deviation. in the extruded strand, was loaded only in the second
printing head. Therefore, only the dermis bioink was
3. Results loaded in the first head, whereas the dermis bioink and

3.1. Precursor cartridge design sacrificial bioink were loaded in the second head. The third
The cross-sectional design of the precursor cartridge head was equipped with a plow and used to form furrows
determines the cross-sectional shape of the extruded on the dermis layer. The final head was a high-temperature
strand. However, as aforementioned, the cross-sectional printing head for PCL, responsible for constructing the
shape of the extruded strand may sometimes differ from frame of the model.
that of the precursor cartridge. Therefore, to determine The PCL frame was initially printed onto the surface of
the optimal architecture of the precursor cartridge, we the culture dish using the high-temperature printing head.
performed CFD simulations. Initially, using the non- Subsequently, the dermis bioink was extruded inside the
Newtonian fluid model of the bioink described in Figure PCL frame using the first head to construct a 2000 µm thick
S2, Supporting Information and Table S1, Supporting dermis layer. Preset extrusion printing to form fissures
Information, we validated the CFD method with a simple was then performed on the surface of the printed dermis
core-shell model (details are described in the Supporting layer. Figure 2C illustrates preset extrusion 3D bioprinting
Information). Briefly, alginate was loaded into the core using a precursor cartridge, which was filled with bioink
compartment of the precursor cartridge, whereas the and sacrificial material. The cartridge was inserted into
SdECM was loaded into the shell compartment, and a the barrel, and the bioink and sacrificial material were
strand was generated by preset printing. Concurrently, simultaneously extruded.
CFD was conducted for the same model. A comparison
between the experimental and CFD results regarding the Fluid with a low Reynolds number can flow while
proportion of the alginate within the cross-section of the maintaining a pattern of precursor cartridge; maintaining
strand indicated that the CFD method effectively predicted a strut shape with the bioink on a hydrophilic matrix is

Volume 10 Issue 5 (2024) 491 doi: 10.36922/ijb.3961