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International Journal of Bioprinting 3D-bioprinted hydrogel for pulp regeneration

rehydrated through graded ethanol solutions. Then, the process. Compared to extrusion and inkjet printing,
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sections were H&E-stained according to the manufacturer’s DLP-based printing could prevent the lysis of the
instructions. For immunohistochemistry (IHC) staining, encapsulated stem cells in hydrogels due to the high
primary antibodies against dentin sialophosphoprotein mechanical stresses caused by the nozzles, and also rescue
(DSPP; Santa Cruz Biotechnology, Dallas, Texas, USA) and the viability of encapsulated stem cells, which can be
CD31 (Abcam) were used to evaluate the odontogenesis decimated due to prolonged printing in a line-by-line or
and angiogenesis. IHC images in each group were drop-by-drop fashion. 29,30 Upon unconfined compression,
randomly captured at 20× magnification for quantitative DPGC showed a lower mechanical strain for a certain
measurement. DSPP- and CD31-positive expression rates compression stress, compared with the bulk hydrogel
were measured using Image J software. (Figure 2C). For instance, under 50% deformation of
the DPGCs before fracture, the average compression
2.19. Statistical analysis stress on bulk hydrogel constructs was 72.5 kPa, while
Data are presented as the means ± SD from at least three the average stress on DPGCs was less than 39.8 kPa. In
independent experiments. Independent sample t-test
and one-way analysis of variance (ANOVA) followed fact, the mechanical strengths of materials can be divided
by Tukey’s test were used for inter-group comparisons. into macroscopic mechanical strength and microscopic
Statistical significance was analyzed using GraphPad Prism contact strength. It should be noted that this experiment
7 (GraphPad Software Inc., Boston, MA, USA). P < 0.05 explored the macroscopic mechanical property, while
was considered as a criterion of statistical significance. mechanotransduction is mainly affected by mechanical
cues from the surrounding microenvironment of cells
3. Results at the microscale. Given the undiluted dextran phase
and the same crosslinking degree, cells experienced the
3.1. Characterization and optimization of DLP-based same microscopic mechanical properties of surrounding
3D-bioprinted DPGC environments within DPGCs or bulk hydrogels. The
To assure the appropriate mechanical property of shape stability evaluation demonstrated that, over the
DPGC, 15% (w/v for all expressions unless otherwise course of 2 h and 24 h, and then 48 h, the structural shape
indicated) GelMA solution was selected in this study of the hydrogel constructs in all groups still retained its
as the base for all bioink formulations to be mixed with original form during the study (Figure 2D and Figure S2
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dextran solution in the volume ratio of 2:1. In order to in Supplementary File). Considering the pore size
determine the proper concentration of dextran solution, (dextran droplet size) and mechanical property of the
the different size distributions of the dextran droplets in DPGC, 10% concentration of dextran was chosen as
the GelMA-Dextran emulsion were visualized in confocal the appropriate setting for the subsequent cell culture
fluorescence field photographs (Figure 2Ai–iii and Figure and in vivo experiments in the experimental group. The
S1 in Supplementary File). When the concentration of the microscopic morphology of DPGC in the 10% dextran
dextran phase was lower than 5% in the mixture, phase group was evaluated by using SEM. As shown in Figure 2E,
separations were poorly formed; when the concentration DPGC exhibited a 3D hierarchical microporous sponge-
of the dextran phase was higher than 15%, the inversion like structure with highly interconnected micropores.
of the two phases occurred. Considering the structure
stability of the microporous constructs, no statistical 3.2. Stemness properties and YAP nuclear
analysis was performed for the distribution of dextran transduction of hDPSCs under the influence of
droplets in the 15% dextran group. The average size 3D-bioprinted DPGC
(16.5 ± 0.7 μm) of the 5% dextran group was lower than Stemness properties play a pivotal role in the functions
that (49.1 ± 2.4 μm) of the 10% dextran group. The size of stem cells, both in vitro and in vivo. Therefore, the
distribution of the dextran droplets became more extensive, pluripotent stem cell markers (OCT4, NANOG, and
and the average size increased to 49.1 ± 2.4 μm as the SOX2) of the hDPSCs cultured in DPGCs and bulk
concentration of dextran increased to 10% (Figure 2B). GelMA hydrogel constructs (control group) and on TCPs
These data indicated that the pore size (the dextran droplet were evaluated. The expression levels of OCT4, NANOG,
size) of the GelMA-Dextran emulsion could be optimized and SOX2 of hDPSCs encapsulated in DPGC significantly
through this method to offer an advanced bioink for the increased compared to that of the other two groups at 3
subsequent 3D bioprinting procedure. and 5 days (Figure 3A and B). In addition, the expression
After the emulsions were photo-crosslinked by UV of OCT4, NANOG, and SOX2 increased in the DPGC
light, DPGC with a hierarchically microporous structure and the control group, which exhibited a gradually
was prepared through a DLP-based 3D bioprinting increasing trend overall and peaked at day 5. These results

Volume 10 Issue 3 (2024) 306 doi: 10.36922/ijb.1790