Page 341 - v11i4

International Journal of Bioprinting GradGelMA 3D-bioprinted vascular skin

G, horseradish peroxidase secondary antibody (1:5000) using 3–3% and 3–5% GelMA hydrogel pairings in the
(Abcam, United States) at room temperature for 2 h. left–right regions of a 6-well plate. In each well, the left-
Signal detection was performed using an enhanced hand region was filled with 3% (w/v) GelMA hydrogel
chemiluminescence detection kit (Servicebio, China), and containing HUVECs at a concentration of 1 × 10⁶ cells/mL,
images were captured using the ChemiDoc XRS+ system while the right-hand region was filled with blank GelMA
(Bio-Rad, United States). Band intensities were quantified hydrogels at either 3% (w/v) or 5% (w/v), depending on
using ImageJ software. the test condition. The culture medium was uniformly
added over the hydrogels, and the plate was placed in an
2.10. Hematoxylin and eosin and Masson staining, incubator. The migration behavior of HUVECs during
immunohistology, and image analysis the subsequent cultivation process was observed under
The samples were fixed in 4% paraformaldehyde for the microscope.
48 h and washed thrice for 5 min in PBS, followed by
processing according to standard methods of paraffin wax 2.12. Single concentration line fusion into the film
embedding, dewaxing, and tissue sectioning. The samples plane printing
were cut into 5-μm-thick sections using a microtome The GelMA with a concentration of 20% (w/v) was printed
(RM2016, Leica, China) for hematoxylin and eosin (H&E) into samples with groove textures and smooth planar films
and Masson (BHBT, China) staining. The sections were by extrusion. After photocuring, HaCaT cell suspension
immersed in xylene (I-20 min, II-20 min, and III-15 min) was added to the surface and cultured in a 37°C 5% carbon
and sequentially immersed in ethanol solutions (100% dioxide incubator. Cell adhesion on the sample surface was
twice, 85%, and 75%) for 5 min. Then, the sections were recorded on the first and fifth days. The samples on the fifth
immersed in a hematoxylin solution for 5 min, washed day were used for cytoskeleton and nuclear staining.
with tap water for a minute, and dipped in 1% acid alcohol
for 10 s. They were further immersed in a bluing solution 2.13. Multi-concentration print layer fusion
for 5 min and then in an eosin Y solution for 2 min. After The 20% (w/v) GelMA and 5% (w/v) GelMA bio-inks were
washing with tap water for a minute, the sections were prepared for layer printing, which was divided into full
sequentially immersed in a series of ethanol solutions crosslinked printing (FCP) and partial crosslinked printing
(75%, 85%, 95%, and 100% twice) for 5 min in each (PCP). For FCP, the bottom layer of 20% (w/v) GelMA
solution for dehydration. After two immersions in xylene ink was photocured for 100 s to achieve a higher degree
for 5 min each, the specimens were sealed with Neutral of crosslinking. In contrast, for PCP, the bottom layer of
Balsam. Masson staining was performed following the 20% (w/v) GelMA ink was photocured for 10 s to limit
crosslinking, while the upper layer of 5% (w/v) GelMA
instructions in the manual. Samples were visualized using ink was photocrosslinked for 100 s. Frozen sections of the
an optical microscope (DMiLLED, Leica, China).
samples were made, and the cross sections were observed
The sections (5 μm thick) were treated for epitope under a microscope. Meanwhile, the samples were soaked
recovery and then incubated in 10% goat serum in PBS in the medium for 96 h, and after being taken out, a tensile
to block non-specific staining for immunohistological test was performed using the method described previously.
analysis. Skin sections were stained with rabbit antihuman
(Abcam, United States) diluted in a 1:100 ratio in a 2.14. Printing of vascularized skin substitutes
1× PBS solution containing 1% bovine serum albumin at The 3% (w/v) GelMA bio-ink containing HUVECs (1 ×
6
4 °C overnight, followed by washing and labeling with 10 cells/mL) and blank 5% GelMA bio-ink were prepared.
Alexa Fluor 488 goat anti-rabbit antibody (1:500; Abcam, The extrusion method was used to construct circular
United States) for an hour at room temperature. The dermal substitutes with microvascular network structures
sections were then counterstained with DAPI. The images in the areas of the letters U, J, and ZJU. After 14 days, F-actin
of the samples were observed using confocal laser scanning staining was performed as described previously. Bio-
6
microscopy. HUVECs suspension (1 × 10 cells/mL) was inks containing HFF cells (1 × 10 cells/mL) in 5% (w/v)
6
mixed with 3% GelMA hydrogels and cultured for 1, 7, GelMA and blank 20% (w/v) GelMA were prepared. The
and 14 days at 37 °C and 5% carbon dioxide, followed by dermal layer and epidermal base were constructed using a
staining with platelet endothelial cell adhesion molecule-1 layer-by-layer printing approach. After 14 days of culture
(PECAM-1/CD31) immunofluorescence and F-actin in a Transwell, H&E and Masson staining were performed
fluorescence as described above. as described. Then, a 5% (w/v) GelMA layer containing
HFF cells as the reticular layer, a 3% (w/v) GelMA layer
2.11. Cell migration containing HUVECs as the papillary layer, and a blank
To assess HUVEC migration in response to GelMA 20% (w/v) GelMA layer as the epidermal basal layer were
7
concentration gradients, a test model was constructed sequentially built from the bottom up. HaCaT cells (1 × 10
Volume 11 Issue 4 (2025) 333 doi: 10.36922/IJB025090069