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

Figure 1. Schematic illustration. (A) The 3D-bioprinted DPGC was fabricated using DLP-based bioprinting technique. (B) The 3D-bioprinted DPGC with
highly hierarchical, interconnected pores could promote cell–construct interaction, including (i) facilitating cell spreading; (ii) improving the proliferation,
migration and stemness of hDPSCs via YAP nuclear location; and (iii) enhancing angiogenesis and odontogenesis. (C) Consequently, more persistent
hDPSCs respond to the regulation of the highly hierarchical, interconnected porous structure to facilitate dental pulp regeneration.

(Sigma Aldrich, St. Louis, MO, USA), dextran (average 2.2. Cell isolation and culture
Mw = 500,000; J&K Scientific, Beijing, China), phosphate-
buffered saline (PBS; Sangon Biotech, Shanghai, China), 2.2.1. Isolation and culture of human dental pulp stem
alpha-Minimum Essential Medium (α-MEM; VivaCell, cells (hDPSCs)
Shanghai, China), fetal bovine serum (FBS; Gibco, This study was approved by the institutional review boards
Waltham, MA, USA), penicillin–streptomycin (P/S; of Daping Hospital, Third Military Medical University. As
described previously, human dental pulp tissues were
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VivaCell, Shanghai, China), collagenase type I (Solarbio, isolated from the extracted healthy permanent teeth after
Beijing, China), dispase II (Solarbio, Beijing, China), obtaining informed consent of donors (aged 18–25 years).
trypsin-EDTA (VivaCell, Shanghai, China), Endothelial The isolated dental pulp tissues were rinsed repeatedly
Growth Medium-2 (EGM-2, Lonza, Basel, Switzerland), with PBS, and then digested in 3 mg/mL collagenase
Cell Counting Kit-8 (CCK-8, MCE, New Jersey, USA), type I and 4 mg/mL dispase II for 30 min at 37°C in a
Live/Dead assay kit (KeyGEN BioTECH, Jiangsu, China), humidified atmosphere containing 5% CO Subsequently,
2.
Alexa Fluor 488 Phalloidin (Thermo Fisher, Waltham, MA, digested hDPSCs were cultured in α-MEM medium
USA), iFluor 555 Phalloidin (Abcam), 4’,6-diamidino-2- supplemented with 10% FBS and 1% P/S. At a confluence
phenylindole (DAPI; Thermo Fisher, Waltham, MA, USA), of 80%, human dental pulp stem cells (hDPSCs) were
and 4% paraformaldehyde (PFA; Boster, Wuhan, China). trypsinized with trypsin-EDTA and subcultured. hDPSCs
Gelatin methacryloyl (GelMA) and lithium phenyl-2,4,6- of passages 3–6 were used in subsequent studies. About
trimethyl-benzoylphosphinate (LAP) were synthesized 10 days after the dental pulp tissue culture, hDPSCs were
according to procedures described in a previous study. completely expanded in culture flasks and maintained
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Animals utilized in this study were purchased from mesenchymal morphology during passage (Figure S3A in
Charles River Laboratories (Beijing, China). Balb/C-NU Supplementary File).
mice were acclimatized to the environment of the animal 2.2.2. Human umbilical vein endothelial cells
facility for at least 7 days prior to the experiments. The (HUVECs) culture
animal experimental procedures were approved by the Human umbilical vein endothelial cells (HUVECs) were
Institutional Animal Care and Use Committee (IACUC) of purchased from Procell (China) and expanded in EGM-2.
the Army Medical University (AMUWEC20212172). Cells of passages 3–6 were used in the experiments.

Volume 10 Issue 3 (2024) 302 doi: 10.36922/ijb.1790