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

spreading area in DPGC increased 2.2-fold compared to have demonstrated the distinctive ability of DPSCs
that in the bulk constructs (Figure 6B). The morphology to secrete neurotrophic factors that regulate neurite
of hDPSCs encapsulated in constructs was further outgrowth and neuroprotection. 47,48 Therefore, we
evaluated by SEM. As shown in Figure 6C, similar to the further evaluated the neuroinductive capacity of DPSCs
fluorescent images, the SEM images show that the hDPSC encapsulated in the 3D-bioprinted DPGC. PC12 cells
adjacent to pores exhibited elongated and extended spread (rat pheochromocytoma cells) were chosen in the study,
morphology, with relatively more cellular protrusions as they possess neuron-like physiological properties.
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closely connecting to the microporous structure, whereas PC12 cells displayed a distinctly neuron-like morphology
the hDPSC in the control construct maintained a near with longer neurite elongation from the cell bodies in
spherical morphology, giving rise to less intimate cell– the DPGC group, as shown in Figure 8A. The length of
construct interaction. As the main downstream effector of the axon extending from the bodies of cells was further
the Hippo signaling pathway, YAP mechanotransduction analyzed with ImageJ software (Figure 8B). In the DPGC
plays a vital role in a number of biological outcomes group, PC12 cells exhibited significantly enhanced axonal
dictated by mechanical strains. 43-45 It was reported that YAP outgrowth with the measured axon length of 106.0 ±
activity relies on actin cytoskeletal tension and regulates 29.26 μm, compared to the control group (37.71 ± 15.54
the fate of stem cells. In this study, the hydrogels made by a μm), indicating that the CM in the DPGC group was
void-forming process not only provide a porous structure more capable of inducing neurite elongation.
for cell spreading, but also exhibit stress relaxation. These
mechanical cues increase YAP nuclear accumulation in Overall, these results reflect that 3D-bioprinted DPGC
the DPGC group, while confinement provided by dense possibly promotes cellular paracrine activity, formation
hydrogel leads to YAP turn-off. of blood vessels, and rapid growth of axons through the
hierarchical, interconnected porous structure.
In short, hDPSCs encapsulated in the DPGCs through
the DLP-based bioprinting process demonstrated 3.5. In vivo pulp-like tissue regeneration promoted
increased cell growth, migration, and spreading. These by 3D-bioprinted DPGC
results demonstrated that the biological properties of Having an appropriate microstructure is one of the crucial
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3D-bioprinted hierarchical DPGC lend themselves to features of tissue-engineered constructs. Hydrogel
induce and support the behaviors of hDPSCs through their constructs can be well designed and printed by a DLP-
highly interconnected porous structure. based printer to match tissue defects with complex shape,
and offer an artificial extracellular matrix and proper
3.4. Pro-angiogenesis and neurogenesis evaluation macrostructural support to promote tissue regeneration.
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of DPSCs-laden 3D-bioprinted DPGC in vitro We used digital scanned data of the TDM to fabricate
Rapid pro-angiogenesis is deemed as a precondition for the conformal DPGC with the microporous structure
dental pulp regeneration to support nutrient and oxygen according to the irregular shape of root canal (TDM
exchange, recruitment of multiple cells, and circulating cavitary contour) using DLP-based 3D printer (Figure
factor delivery. Hence, the modulation and evaluation 9Ai–iii). As shown in Figure 9Aiv, after being inserted
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of angiogenesis are critical procedures for hDPSC-based into the TDM to mimic the real environment of the root
therapies. Based on the above-mentioned finding of canal, the 3D-printed DPGC was precisely matched
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stemness properties and YAP in this study, the angiogenic with the irregularly shaped root canal of the TDM.
effects of the hDPSC-encapsulated DPGCs and bulk Subsequently, the endodontic repair efficacy of DPGC
GelMA hydrogel constructs were investigated (Figure 7A). was further explored in vivo. hDPSCs-laden DPGCs were
In vitro tube formation experiment (Figure 7B–D) combined with TDMs before subcutaneous implantation
showed that the CM in the DPGC group exhibited a into immunodeficient mice for 8 weeks (Figure 9B). Based
stronger ability to induce tubule formation in HUVECs. on the H&E staining images, dental pulp-like structures
Specifically, the parameters of tubule formation (branches, containing rich microvessel formation (black arrow) and
junctions, nodes, and total length) showed similar trends, an odontoblast-like layer (yellow triangle) can be found in
with the higher values detected in the DPGC group. For the DPGC group (Figure 9C). In marked contrast, only a
example, the total number of nodes of newly formed blood small amount of neotissue was observed near the open ends
vessels was 254.7 ± 29.7 in the DPGC group, which was of the TDM combined with the bulk hydrogel construct
significantly higher than the number in the control group (control group), and there was almost no soft tissue
(118.8 ± 23.8). formed in the empty TDM group. Interestingly, unlike in
Neurogenesis is considered one of the significant the DPGC group, the neotissue mostly distributed at the
challenges in endodontic regeneration therapy. . Studies open ends of the TDM and little neotissue was formed in
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Volume 10 Issue 3 (2024) 312 doi: 10.36922/ijb.1790