Mesenchymal stem cell medium

HBMMSCs were purchased from ScienCell Research Laboratories (Cat. number 7500; Carlsbad, CA, USA). The HBMMSCs were maintained with Mesenchymal Stem Cell Medium (ScienCell, Carlsbad, CA, USA) at 37°C in a humidified atmosphere of 95% air and 5% CO2. For the osteogenic differentiation of HBMMSCs, the cells were cultured in osteogenic medium containing 10 mmol/L β-glycerol phosphate, 50 μg/mL ascorbic acid, and 10−7 mol/L dexamethasone.

Human umbilical cord mesenchymal stem cells (hucMSCs, donated by Wingor Biotechnology Co, Ltd) were cultured with media mixed with mesenchymal stem cell medium (ScienCell) and DMEM/F12 (Gibco), supplemented with 5% fetal bovine serum (FBS; ScienCell). The identification of hucMSCs was determined as previously described 16 (link). To prepare MSC-exos, hucMSCs (Passage 2-6) were cultured with FBS-free media for 48 h, and the supernatants were subsequently collected for MSC-exos extraction. MSC-exos were isolated by gradient centrifugation and filtration 16 (link). In brief, the supernatants were centrifuged at 2000×g for 20 min, 13,500×g for 30 min, filtrated with a 0.22-μm filter and ultracentrifuged at 200,000×g for 120 min (Beckman Coulter Optima L-80 XP) at 4 °C. The exosome pellets were resuspended in sterile phosphate buffer saline (PBS) and filtered using a 0.22-μm filter once again. Purified MSC-exos were resuspended in PBS and stored at -80 °C.

Both hBMSCs and HUVECs were purchased from Sciencell (Carlsbad, CA, United States). The cells were seeded at 5,000 cells/cm² in culture bottles treated with bovine plasma fibronectin (Sciencell). ECM culture medium (Sciencell) containing 5% fetal bovine serum (Gibco, Grand Island, NY, United States), 1% penicillin mixture, and 1% endothelial cell growth factor (Sciencell) was used for HUVECs. hBMSCs were cultured in mesenchymal stem cell medium (Sciencell) containing 5% fetal bovine serum, 1% penicillin mixture, and 1% mesenchymal stem cell growth factor. The cells were placed in a constant temperature incubator with 5% CO₂ at 37°C to subculture. The third to fifth passages of cells were used in the experiments.

Fibrinogen (F3879, Sigma, United States) dissolved in phosphate-buffered saline (PBS, 0.01 M, pH 7.40) at a concentration of 200 mg/ml, thrombin (T4393, Sigma, United States) dissolved in PBS at a concentration of 100 U/ml, and genipin (G4796, Sigma, United States) dissolved in dimethyl sulfoxide (D2650, Sigma, United States) at a concentration of 400 mg/ml, were prepared for the glue fabrication. This component concentration of Fib-T-G gel was optimized by testing the biocompatibility and mechanical properties with different concentrations of genipin. The Fib-T-G gel contained final concentrations of Fibrinogen 140 mg/ml, thrombin 28 U/ml, and genipin 6 mg/ml–these concentrations exhibited the best consistency for biocompatibility and adhesive strength for the following experiment in our pilot study, which was added as Supplementary Data. hMSCs (Passage 2, 15901; purchased from ScienCell Research Laboratories, Carlsbad, CA) routine cultured in Mesenchymal Stem Cell medium (7501, Sciencell, United States) which included DMEM supplemented with 5% fetal bovine serum (FBS, 0025, Sciencell, United States), 1% penicillin-streptomycin (PS, 0503, Sciencell, United States) and Mesenchymal Stem Cell Growth Supplement (MSCGS) (7552, Sciencell, United States) up to passage 6, were used in all the experiments in this study.

Zn(NO₃)₂·6H₂O and Na₂SO₄ were purchased from Sinopharm. Pyrazine was acquired from Alfa Aesar. PVP, Mn-SOD, catalase, complete Freund’s adjuvant, and L-ascorbic acid were acquired from Sigma–Aldrich. MnTCPP⁺ monochloride, TCPP⁺ monochloride, FeTCPP⁺ monochloride, gallic acid and reduced glutathione were provided by J&K Scientific. N,N-dimethylformamide (DMF) was bought from Shanghai Lingfeng Chemical Reagent Co., Ltd. CeO₂ nanoparticles were purchased from Jiangsu XFNANO. SOD assay kit, catalase assay kit, Hoechst, LPS, and alizarin red S staining kit were acquired from Beyotime. Dulbecco’s modified eagle’s medium (DMEM), PBS, fetal bovine serum (FBS), and penicillin/streptomycin were obtained from YoBiBio. Annexin V-FITC, PI and CCK-8 assay were provided by 7seabiotech. Mesenchymal stem cell medium was bought from ScienCell. FITC, DCFH-DA and rhodamine phalloidin were provided by Solarbio. Paraformaldehyde (PFA) was obtained from Ribiology.

For CFU-F cultures, single cells were seeded into 96-well plates (1 single-cell/well) containing Mesenchymal Stem Cell Medium (ScienCell, US) supplemented with 1% Penicillin/Streptomycin solution, and incubated at 37 °C with 5% CO2. We then changed half of the medium every 3–4 days. When cultured for 2 weeks, the cells were fixed and stained with crystalline violet staining solution.