Collagen gel

Antibodies used include anti-β1 integrin (ab52971, Abcam), anti-Rac1 (ab33186, Abcam), anti-E-cad (ab1416, Abcam), and anti-MUC-1 (ab109185, Abcam). Other reagents included β1 integrin inhibitor AIIB2 (Developmental Studies Hybridoma Bank, United States), type I collagenase (Solarbio, China), collagen gel (Nitta Gelatin, Japan), culture media (Gibco), and Lipofectamine 2000 and TRIzol (Invitrogen). siRNAs were synthesized by GenePharma, China, and the sequences were as follows: siRNA-β1 integrin: forward, 5′-GUU UAA UGU CUG GUG CUU TT-3′; reverse, 5′-AAG CAC CAG ACA UUA AAC TT-3′; siRNA-Rac1: forward, 5′-UAA AGA CAC GAU CGA GAA AUU-3′; reverse, 5′-UUU CUC GAU CGU GUC UUU AUU-3′; and siRNA-ctrl: forward, 5′-UUC UCC GAA CGU GUC ACG UTT-3′; reverse, 5′-ACGUGACACGUUCGGAGAATT-3′.

To examine the hard tissue formation capacity in vivo, PDLSCs expanded using SFM or FCM were subcutaneously transplanted under the dorsal skin of 6-week-old female nude mice (BALB/C-nu/nu; Nihon Clea, Tokyo, Japan). The cell/scaffold construct consisted of a mixture of approximately 1  ×  10⁶ cells and 400 µL collagen gel (Nitta Gelatin, Osaka, Japan) with 40 mg hydroxyapatite (HA)/β-TCP-scaffold (Ceraform^®; NGK Spark Plug, Aichi, Japan). All transplants were collected for histological analysis at 16 weeks post-transplantation (n = 6 per group).
Histological and immunohistochemical examinations were performed as previously described [21 (link)]. Primary antibodies were used as follows: mouse monoclonal anti-human-specific vimentin (1:10,000; Merck KGaA), rabbit polyclonal anti-osteopontin (OPN; 1:1000; Abcam, Cambridge, MA, USA), and rabbit polyclonal anti-cementum attachment protein (CAP; 1:100; LifeSpan Biosciences, Seattle, WA, USA). For negative controls, the primary antibody was omitted during immunostaining.

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.