Hdpscs

Before dentin-pulp-like organoid generation, hDPSCs (Lonza Inc., Basel, Switzerland, PT-5025) were cultured in maintenance medium until passage 5. Next, dentin-pulp-like organoids were fabricated with four different protocols to find the optimal culture duration for the maintenance medium (MM) and odontogenic differentiation medium (ODM). Culture durations for the four groups are shown in Figure 1A. All organoids were observed under a light microscope and harvested at 21 days. Their characteristics were analyzed by a quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF), and histology. After optimization of culture and differentiation, micro-compute tomography (micro-CT) and electron microscopy (EM) were performed for structural analyses. Next, organoids were dissociated and reorganized into dentin-pulp-like organoids to evaluate their forming structure ability. We also assessed their biologic activities after adding Biodentine^® (FDA-approved commercially available stimulator of odontoblastic differentiation) during organoid culture and differentiation.

We purchased human dental pulp stem cells (hDPSCs) collected from human sound third molars (Lonza, Basel, Switzerland). These cells have been reported to contain mesenchymal-like cells. Surface antigens on these cells have been confirmed for cluster designation (CD) 29+, CD73+, CD90+, CD105+, CD166+, CD34-, CD45-, and CD133- by flow cytometry. Cells were collected (TrypLE Select, Life Technologies, Carlsbad, CA, USA) when they reached 80% confluence. For the following assay, hDPSCs (passages 2–4) were cultured in α-Minimum Essential Media (α-MEM; Life Technologies, Carlsbad, CA, USA) supplemented with penicillin-streptomycin 10 µg/mL (Sigma-Aldrich, St. Louis, MO, USA). In the in vitro assays, 1% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) was used.

hDPSCs (Lonza) were cultured in Minimum Essential Medium alpha (MEM‐α; Gibco, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) (Gemini Bio‐Products), and 1% penicillin/streptomycin (Thermo Fisher Scientific) was used as the cell growth medium (GM). Cells (passage 4) were used in the bioinks.
The cell‐laden constructs were cultured in a 6‐well culture plates supplemented with GM and incubated at 37 °C in 5% CO₂. The medium was replaced every 2 days. To induce osteogenic differentiation of the hDPSCs, 100 μM dexamethasone (Sigma‐Aldrich), 10 mM β‐glycerophosphate (Sigma‐Aldrich), and 50 μM ascorbate‐2‐phosphate (Sigma‐Aldrich) were mixed with the GM. The fabricated cell‐laden structures were cultured in osteogenic differentiation medium (DM) after 7 days of culture. The DM was changed every 2 days.

Mouse bone marrow stromal‐derived MSCs were obtained from a commercial supplier (D1 MSC, CRL‐12424, ATCC). The cells were cultured in high‐glucose GlutaMAX Dulbecco's Modified Eagles Medium (DMEM, ThermoFisher) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were cultured at sub‐confluency (70% maximum) to maintain stemness, passaged (up to P10 maximum), and medium was changed every 2 days.
Similarly, human bone marrow‐derived MSCs (hBM‐MSCs, PCS‐500‐012, ATCC) and human dental pulp stem cells (hDPSCs, PT‐3927, Lonza) were obtained from commercial suppliers and cultured using established protocols with culture medium purchased from the respective suppliers. hBM‐MSCs were cultured in MSC basal medium (PCS‐500‐030, ATCC) supplemented with 7% FBS, rh IGF‐1 (15 ng/ml), rh FGF‐b (125 pg/ml), l‐alanyl‐l‐hlutamine (2.4 mM), and 1% P/S. hDPSCs were cultured using a DPSC BulletKit (PT‐3928 and PT4516, Lonza). Both primary human cell types were cultured at sub‐confluency (maximum 70%) to maintain stemness, passaged (up to P5 maximum), and medium was changed every 2 days.

Commercially available human dental pulp stem cells (hDPSCs, Lonza Group, Basel, Switzerland) were used in the experiments to assess cell viability, angiogenic factor release and alkaline phosphatase (ALP) activity. The hDPSC growth medium was prepared as per the instructions outlined by the manufacturer. The cells were seeded in a 75 cm² culture flask containing culture medium and kept in an incubator at 37 °C under standard conditions with 5% CO₂. Upon reaching 80% confluency on the 5th day, the cells were subcultured.
During the passaging process, the medium was removed from the flasks and the cells were sequentially washed with 4 ml of HEPES followed by 2 ml of trypsin EDTA (Thermo Fisher Scientific, USA). After 5 min of incubation, the cells were successfully detached from the flask’s base. The detached cells were washed with 4 ml of trypsin neutralization solution (TNS) and then transferred into new sterile Falcon tubes. The tubes were centrifuged at 1500 rpm for 5 min and cultivated in fresh culture flasks. Cell cultures at the third passage were used for all experiments.

The hDPSCs (cat# PT-5025, Lonza Bioscience, Basel, Switzerland) were cultured in DPSC SingleQuot Growth Medium (cat# PT-4516, Lonza Bioscience, Basel, Switzerland). For osteogenic differentiation, cells were seeded in collagen-coated 48-well plates at a density of 2 × 10⁴ cells/well. After 24 h of seeding, the medium was changed with osteogenic medium [a-MEM, 2% fetal bovine serum (FBS), 10 mM b-glycerophosphate, 50 μg/ml ascorbic acid, 100 nM dexamethasone] with or without apigenin (cat# 520-36-5, Sigma Aldrich, Saint Louis, MO, United States). The hDPSCs were cultured with three different sets: osteogenic medium only (negative control), osteogenic medium with 0.05% dimethyl sulfoxide (DMSO; vehicle control), and osteogenic medium with various concentrations of apigenin (experimental). The culture medium was changed three times per week (Figure 1A).

Human dental pulp stem cells (hDPSCs, Lonza, PT-5025, Basel, swiss) were cultured in M-DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin G, and 100 μg/mL streptomycin at 37 °C in a 5% CO₂ incubator. For the experiments, hDPSCs were cultured in standard odontogenic induction medium, namely, M-DMEM supplemented with 10% FBS, antibiotics, 10 nM dexamethasone (Sigma–Aldrich, D4902, St Louis, MO, USA), 50 ng/mL BMP-2 (Sigma–Aldrich, B3555, St Louis, MO, USA), 20 ng/mL TGF-β1 (Bio Legend, 580704, San Diego, CA, USA), and 5 ng/mL FGF-2 (Bio Legend, 571504, San Diego, CA, USA) for 3 or 5 days at 37 °C in a 5% CO₂ incubator. For drug exposure, MS-275 (Apexbio, A8171, Houston, TX, USA) was initially dissolved in dimethyl sulfoxide (DMSO), then the concentration was adjusted with PBS, and it was added to the media. The medium was changed every 2–3 days. The hDPSCs from passages 3 to 5 were used for the experiments. DMSO (Sigma-Aldrich, D5879, St Louis, MO, USA) was added to the control at an additional 1/5000 to offset the effect of DMSO on the experiment.