Primary rat bone mesenchymal stem cells (BMSCs) supported by the medical school of Ningbo University were expanded in low-glucose Dubecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco). The medium was changed every other day. The cells were passaged upon above 70% confluence and only four-passage BMSCs were used for all experimental studies.
For in vitro cell injection studies, trypsinized BMSCs with a cell density of 106 cells/mL were suspended in 1.5 wt% CHMA and 6 wt% OHA pre-polymer solutions containing specified concentrations of LAP and CaCl2. The cell-laden solutions were mixed at 1v:1v and placed in the barrel of a 1 mL syringe fitted with a 25-gauge needle. The mixture was allowed to gel for 5 min before injecting into a 24-well plate using a syringe pump (LSP02-2B; LONGER Instruments) at a flow rate of 0.1 mL/min. Cell viability was determined using LIVE/DEAD viability/cytotoxicity kit (Biovision) at 30 min post-injection and at days 1, 3, and 5 post-injection (n = 5), according to manufacturer's instructions. The live (calcein AM labeled) and dead (ethidium homodimer labeled) cells were visualized using a Laser Scanning Confocal Microscope (LSCM, Leica TCS-SP8, Germany). Viability is reported as the percentage of cells with positive calcein staining (n = 3).
For in vitro cell injection studies, trypsinized BMSCs with a cell density of 106 cells/mL were suspended in 1.5 wt% CHMA and 6 wt% OHA pre-polymer solutions containing specified concentrations of LAP and CaCl2. The cell-laden solutions were mixed at 1v:1v and placed in the barrel of a 1 mL syringe fitted with a 25-gauge needle. The mixture was allowed to gel for 5 min before injecting into a 24-well plate using a syringe pump (LSP02-2B; LONGER Instruments) at a flow rate of 0.1 mL/min. Cell viability was determined using LIVE/DEAD viability/cytotoxicity kit (Biovision) at 30 min post-injection and at days 1, 3, and 5 post-injection (n = 5), according to manufacturer's instructions. The live (calcein AM labeled) and dead (ethidium homodimer labeled) cells were visualized using a Laser Scanning Confocal Microscope (LSCM, Leica TCS-SP8, Germany). Viability is reported as the percentage of cells with positive calcein staining (n = 3).