2.1 Experimental animals
A total of 5 healthy male New Zealand rabbits (aged 4~6 weeks, weight 400~800 g) were obtained from the Animal Experiment Center of Xiamen University. All rabbits were in good condition and had no related diseases based on examination.
2.2 Isolation and culture of rBMSCs
The 5-week-old male New Zealand rabbits were sacrificed with an intraperitoneal injection of an excess of 10% chloral hydrate (Sinopharm Chemical Reagent, China) and the hindlimbs of the rabbit were removed and immersed in 75% alcohol for 3 minutes. Then the muscles of femur and tibia were removed and the bone cavity was washed by PBS mixed with an appropriate amount of heparin sodium (0.04 mg/mL, Beijing Solarbio Science & Technology, China) for bone marrow. The suspension was collected and adjusted to the cell concentration of 2×108 to 1×109 /mL. A total of 5 mL rabbit mesenchymal cell separation fluid (Tianjin HaoYang HuaKe Biological Technology, China) was added to a 15 mL centrifuge tube, and then the single cell suspension was placed onto the separation liquid level, at 450g centrifugation for 20 minutes. Cells in the second layer were collected in a Petri dish containing MSC complete medium [DMEM/F12 (HyClone, USA, the glucose concentration of 16.67 mmol/L) for F12 group or DMEM/Low Glucose (HyClone, USA, the glucose concentration of 5.56 mmol/L) for LG group or DMEM/High Glucose (HyClone, USA, the glucose concentration of 25 mmol/L) for HG group + 10% FBS (Biological Industries, Israel) + 1% streptomycin/penicillin (HyClone, USA) + 50 μmol/L β-mercaptoethanol (Beijing Solarbio Science & Technology, China)]. The petri dish was placed in a cell culture incubator at 37°C with 5% CO2 for 3 days, and then the medium was changed every 2 days.
As for the optimizational culture method (HG-LG) group, rBMSCs were isolated and cultured with DMEM/High glucose for the fomer 6 days, then they were digested with trypsin and cultured with DMEM/Low glucose.
2.3 Detection of MSC surface markers
The 3rd passage cells of the 4 groups were collected respectively, then CD45 (Acris GmbH, Germany), CD44 (BD Biosciences, USA), CD34 (Thermo Fisher, USA), CD29 (Millipore Corporation, USA), goat anti-mouse secondary antibody (Multi Sciences, China) were used to detect their MSC surface markers according to the instructions [19]. Fluorescence activated cell identify was performed with flow cytometry (Beckman USA) and data were analyzed with CytExpert (Tree Star, Ashland, OR, USA).
2.4 Multi-directional differentiation potential of rBMSCs
Osteogenic induction: The 4 groups of rBMSCs were seeded in 6 cm dishes with 3×105 in each one and cultured at 37°C with 5% CO2 for 24 hours. The original medium was then discarded and replaced for osteogenic induction with one containing 10-2 mol/L of β sodium glycerophosphate (Dalian Meilun Biotech, China), 10-8 mol/L of dexamethasone (Dalian Meilun Biotech, China), and 3×10-4 mol/L vitamin C (Dalian Meilun Biotech, China). The medium was changed every 2 days for 2 weeks. Then calcium nodules in different groups were detected by Alizarin Red staining (Beijing Leagene Biotech, China) according to the instructions.
Chondrogenic induction: The 4 groups of rBMSCs were seeded in 6 cm dishes with 3×105 in each one and cultured at 37°C with 5% CO2 for 24 hours. The original medium was then discarded and replaced for chondrogenic induction with one containing 10 μg/L of rabbit TGF-β1 (Dalian Meilun Biotech, China), 10-7 mol/L of dexamethasone, 5×10-5 mol/L vitamin C, 6.25×10-3 g/L of insulin. The medium was changed every 2 days for 2-3 weeks. The induction was terminated and stained with Alison Blue Dye (Beijing Leagene Biotech, China) according to the instructions.
Adipogenic induction: The 4 groups of rBMSCs were seeded in 6 cm dishes with 3×105 in each one and cultured at 37°C with 5% CO2 for 24 hours. The original medium was then discarded and replaced for adipogenic induction with adipogenic induction medium A containing 2×10-4 mol/L of indomethacin, 10-6 mol/L of dexamethasone, 5×10-4 mol/L of 3-isobutyl-1-methylxanthine (China National Pharmaceutical Group Chemical Reagent, China) and 10-2 g/L insulin (Dalian Meilun Biotech, China), and changed to adipogenic induction medium B containing 10-2 g/L insulin after 2 days, and after 1 day, it was replaced with liquid A, and it was repeated. After 2 weeks, the induction was terminated and stained with Oil Red O (Beijing Leagene Biotech, China) according to the instructions.
The positive rate of dyeing (PRD) was calculated and compared by Image Pro Plus software.
2.5 Detection of cell proliferation ability
The cells to be detected were seeded in well plates with 2×103 cells/well, and then a total of 10 μL CCK-8 (Transgen Biotech, Beijing, China) reagents were added into each well and incubated at 37°C with 5% CO2 for 2 hours after culture for 1, 2, 3 and 4 days, the absorbance at 450 nm (A450) were determined at wave length of 450 nm with Microplate Reader (Bio-Rad, USA) according to the instructions.
2.6 Detection of cell adhesion ability
The cells to be detected were seeded in a 9 cm culture dish at a ratio of 80 cells per petri dish. After 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours, the number of adherent cells (number of cell colonies) was counted respectively [20]. Cell adherence rate = (number of adherent cells /80) × 100%.
2.7 Introduction of green fluorescent protein (GFP)
Seed cells were labeled with GFP to monitor the survival of seed cells in TEB scaffold, this was inspired from the methods reported by Yuriko Kakimoto [21]. 105 primary rBMSCs were put into a 10 cm culture dishes with 8 mL culture medium, the lentivirus with GFP gene were put into each dish. The multiplicity of infection was adjusted to 100 and the density of polybrene was adjusted to 5 μg/mL. The medium was changed after 24 hours and then changed every 2nd day. The fluorescence expression of cells were observed by an inverted fluorescence microscope and these GFP-rBMSCs were collected and sorted by a Flow Separation Cell Meter (Beckman USA) and cultivated for amplification.
2.8 Preparation of deproteinized defatted cancellous bone scaffold (DDCBS)
Our research team has previously proved that DDCBS obtained from calf femoral heads had good porous structure, and the DDCBS implantation could effectively promote bone defect repair in New Zealand rabbits [22]. In this study, the DDCBS obtained from calf femoral heads was used as scaffold for TEB construction. The cancellous bone of the calf femur was prepared into a cylindrical cancellous bone of 8 mm in diameter, deproteinized with 20% hydrogen peroxide at 37°C for 72 hours, and then degreased with ether at room temperature for 48 hours to prepare for DDCBS, as described before. Scanning electron microscope was used to observe the structure of the DDCBS (Fig 1).
2.9 Construction of TEB
The seed cells were adjusted to 3×105 /mL, 10 mL of cell suspension and a piece of TEB were put into a 15 mL centrifuge tube for mixed culture. The culture was carried out for 7 days with a rotary cell culture apparatus at 20 r/min (Sup. Fig 1). Then the TEB to be detected were placed into another 15 ml centrifuge tube and an appropriate amount of PBS was added to fully infiltrate the stent. The fluorescence intensity of each group was observed and compared by living fluorescence imaging system.
2.10 Statistical analysis
Results were expressed as mean ± standard deviation. Prism 5.0 (GraphPad Software Inc.) was used to make statistical analysis. Statistical comparisons for different groups were performed using one-way analysis of variance (ANOVA). P < 0.05 were considered statistically significant.