Msc medium

All research involving human stem cells was approved by the Ethics Committee of the Affiliated Stomatological Hospital of Nanjing Medical University. Premolars were collected from six healthy orthodontic patients (12–16-year old) under approved guidelines set by the Jiangsu provincial dental hospital with informed consent. Teeth were disinfected with 75% ethanol and then washed with phosphate-buffered saline (PBS). hPDLSCs were isolated, cultured, and identified as previously described. Briefly, primary hPDLSCs were separated from the periodontal ligament in the middle third of the root. The periodontal ligament tissue was subsequently digested in a solution of 3 mg/mL collagenase type I (Worthington Biochemical Corp., Lakewood, NJ, USA) and 4 mg/mL dispase (Roche Diagnostics Corp., Indianapolis, IN, USA) for 1 h at 37°C. Primary hPDLSCs were grown in a humidified incubator under 5% CO₂ at 37°C in Gibco® α-MEM (with GlutaMAX™) supplemented with 15% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Passaged primary hPDLSCs were cultured in MSC medium (ScienCell, San Diego, CA, USA) and the medium was changed every 3 days. The obtained hPDLSCs were characterized as previously described [21 (link)].

Adipose tissue was collected from two patients undergoing liposuction in Plastic Surgery Hospital. Both donors were around 40 years old, similar height and weight, and had no other systemic diseases. The written informed consents were given from the donors and the study was approved by the Ethics Committee of the Plastic Surgery Hospital. Adipose tissue was incubated with type I collagenase solution, and its surface markers were analyzed using MSC analysis Kits (BD Biosciences, Franklin Lakes, NJ, at passage USA). Then the cells were maintained in Mesenchymal Stem Cell (MSC) Medium (ScienCell, Carlsbad, CA, USA) and used in passages 3–5 (P3–5). Therefore, ADSCs from two donors were independently used in the following experiments.

Agarose solution (2%) was melted and then slowly added to a MicroTissues 3D Petri Dish (Sigma). Then, the agarose was solidified into the cell culture mold. MSC (ScienceCell) were inoculated in the agarose mold. After 12 h culture in the mold with MSC medium (ScienceCell), the cells were agglomerated into cell spheres.

G1-CS hydrogel (prepared as described in a previous report (Zhang et al., 2019a )), MSC medium (ScienCell Research Laboratories, Carlsbad, CA, USA), fetal bovine serum (Gibco Life Technologies, Carlsbad, CA, USA), penicillin-streptomycin (Gibco, 15140122), TRIzol (Invitrogen, Carlsbad, CA, USA), SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), cDNA synthesis kit (Bio-Rad), 4 % paraformaldehyde (Servicebio, Wuhan, China), CD31 antibody (Abcam, Cambridge, UK), vWF antibody (Abcam), CD34 antibody (Abcam), CD90 antibody (Abcam), FLK-1 antibody (Abcam), GIT1 antibody (Abcam), and GAPDH antibody (Abcam). Secondary antibody (Abcam), FITC-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA), DAPI (Beyotime Institute of Biotechnology, Jiangsu, China), Alkaline Phosphatase staining kit (Servicebio), PBS (Gibco), Opti-MEM medium (Gibco), AAV-GIT1 and Plasmids-GIT1 [constructed by Obio Technology (Shanghai) Company, Shanghai, China], BD Matrigel (BD Bioscience, San Jose, CA, USA), and Microfil MV-122 (Flow Tech Inc., Carver, MA, USA).