Live dead staining kit

According to the manufacturers’ instructions, we fabricated hydrogels of the collagen gel (3 mg·mL⁻¹, Nitta Gelatin, Japan), matrigel (8 mg·mL⁻¹, Corning, NY, USA), and GelMA (5% and 10% of mass/volume, EFL, Suzhou, China). For 2D culture, confocal dishes were precoated with 100 µL collagen gel and matrigel solution followed by 30 min incubation at 37 °C, while precoated 100 µL GelMA solution followed by 40 s light-curing for gel transition. Then 3 × 10⁴ h-DPSCs were seeded on the surface of gels and images were captured using an optical microscope on day 1 and day 3. After phalloidine immunostaining, the cytoskeleton of h-DPSCs was observed by a confocal microscope on day 3. For 3D culture, 1 × 10⁶ h-DPSCs were resuspended into 1 mL gel solution. Cell viability was detected using a Live/Dead staining kit (KeyGEN BioTECH) on days 1, 3, and 6. Live cells with green fluorescence and dead cells with red fluorescence were identified by a confocal microscope and quantified by ImageJ. Moreover, five fields of every single image were randomly selected (the cell number of each field was about 6) to analyze the cell spreading from day 1 to day 6 by calculating the aspect ratio (the ratio between the short axis and the long axis) of h-DPSCs.

Gelatin, from cold‐water fish skin, was purchased from Sigma‐Aldrich (Shanghai, China). Methacrylic anhydride, ε‐caprolactone and polyethylene glycol (PEG) (Mw = 4000) were purchased from Macklin (Shanghai, China). Tin (II) 2‐ethylhexanoate and sodium laurylsulfonate were purchased from Aladdin (Shanghai, China). Ethylacetate, acetone, petroleum ether and dichloromethane were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Lithium phenyl(2,4,6‐trimethylbenzoyl) phosphinate (LAP) was purchased from Shanghai Yinchang New Material Biological Co., Ltd. Methylamidated OP3 peptide (YCEIEFCYLIR) was purchased from Jiangsu Ji Tai Peptide Industry Science and Technology Co., Ltd. Streptomycin sulfate, penicillin, fetal bovine serum and trypsin were purchased from Gibco (Shanghai, China). Cell counting kit 8 was purchased from Bioss antibodies (Beijing, China). Live/Dead staining kit was purchased from KeyGEN BioTECH (Jiangsu, China). TRITC Phalloidin was purchased from YEASEN (Shanghai, China). Rabbit Anti‐RUNX2 antibody, Alizarin Red S Staining Kit, DAPI staining, and Anti‐Osteocalcin Rabbit pAb were purchased from Servicebio (Wuhan, China). Rat bone marrow mesenchymal stem cells were purchased from ChuangSeed Biomaterials.