Isolation and identification of Fetal BMSCs
Isolation and culture of Fetal BMSCs
Institutional animal care committee approval was obtained for the study. Artificial fertilization was used to breed white goats (the goat was provided by the breeding base of Chongming Three Star Town, Shanghai, China, which were licensed by the Animal Experiment Committee of Medical School of Shanghai Jiaotong University). All experimental procedures were according to the institutional guidelines for the care and use of laboratory animals in Medical School of Shanghai Jiaotong University. Then the cesarean section was performed after 3 months pregnancy (the normal pregnancy of the goat is 5 months). Before operation, the goats were fasting for 2 days, and forbidden to water for 12 h. The goats was sedated by atropine half an hour before the operation. Tracheal intubation was performed after general anesthesia (intramuscular injection of xylazine). After skin preparation and iodophor disinfection, we cut the skin by electric knife, separated the subcutaneous fascia, opened the abdominal cavity, and took out the uterus. Finally, the fetal goats were taken out by cutting the uterus along base.
The limb bones of fetal goats were isolated in the sterile environment, and the ends of the limbs were cut off. Then the long bone marrow cavities were repeatedly aspirated with 5 mL syringes until the cavities appeared white. After that, the fresh bone marrow tissue was seeded onto 10 cm culture dishes with 10 mL of low glucose DMEM (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Hyclone), and 1% penicillin and streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). The culture dishes were incubated in a humidified environment (5% CO2, 37 °C) and the culture medium was changed every 3 days [15]. When the cells confluence reached 80%, 0.25% trypsin/1 mM EDTA (Thermo Fisher Scientific) was used to digest the MSCs (marrow stromal cells) for the passage until the third generation (P3).
Characterization of cells dynamics and identification of stem cells properties
Growth curve
When passaged to P3, the cells were seeded in 96-well plates at 1000 cells/well, then placed in a conventional incubator and incubated with cck-8 for 3 h on day 1, 3, 5, 7, 9, 11. With a micro-plate reader, the OD values of per hole and control hole were measured at 450 nm wavelength, and the difference value of the two was the final OD value. According to the final OD value, the cell growth curve was drawn.
Colony forming efficiency
The experimental procedure was the same as previously reported [10]. The Fetal BMSCs of P3 generation were evenly seeded into dishes at a cell density of 200 cells/dish. The colonies were formed after 21 days of culture.
Three induced differentiation ability
BMSCs (passage3) were passaged into 6-well plates at a cell density of 3 × 105 cells/well. When the cells confluence reached 80%, the culture medium was replaced by induction medium [16]. For each plate, two wells were stained with Alizarin red S after 10 days’ osteogenic induction (10 mM β-glycerophosphate, 0.1 μM dexamethasone, and 50 μM ascorbic acid). Two wells were stained with Oil red O after 3 weeks’ lipid induction (5 μg/mL insulin, 200 μM indomethacin, 1 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine). Two wells were stained with Toluidine blue after 3 weeks of chondrogenic induction (DMEM, 10% fetal bovine serum, 0.1 μM dexamethasone, 0.17 mM ascorbic acid, 1 mM sodium pyruvate, 0.35 mM l-proline,1% insulin-transferrin sodium-selenite, 1.25 mg/mL bovine serum albumin, 5.33 μg/mL linoleic acid, and 0.01 μg/mL transforming growth factor-β).
Construction of TEBG (tissue engineering bone graft)
Preparation of DBM
As previously described, the goat femur was decalcified and deproteinized to form decalcified bone matrix (DBM) [17]. The diameter of the goat skull defect was 20 mm, and according to that, the DBM scaffolds were fabricated by cutting the DBM into small discs (diameter 20 mm, thickness 2–3 mm).
Seeding cells
The DBM scaffolds were soaked in 75% alcohol for 5 h, and cleaned by PBS for 3 times. After dried up, the P3 generation BMSCs were then seeded on these DBM scaffolds at a cell concentration of 20 million/mL. After incubated in a humidified environment (5% CO2, 37 °C) for 4–5 h, the L-DMEM culture medium was carefully added along the dish wall until the scaffolds were covered by culture medium. On the second day, the culture medium was replaced by osteogenic induction medium, and the medium was changed every 3 days for 14 days.
Exam of cell viability by FDA/PI staining
After 7 days and 14 days of osteogenic induction, the cells of the TEBG were stained by fluorescein diacetate/propidium iodide (FDA/PI). The dyeing procedure was as previously described, FDA stained viable cells green and PI stained dead cells red [18].
Observation of TEBG before implantation by SEM
After 14 days of osteogenic induction, the TEBG was prefixed in 2.5% glutaraldehyde for 24 h at 4 °C. After being rinsed three-times in PBS, the specimens were immersed in 1% osmic acid for 2 h at 4 °C. And then rinsed three-times again in PBS. After drying, the specimens were sputter-coated with gold (BAL-TEC, Philips, Eindhoven, The Netherlands), and examined finally with a scanning electron microscope (PhilipsXL-30, The Netherlands) [10].
BETG repair experiments in vivo
Repair of goats skull defects
Anaesthesia
The young group (2–3 years old normal adult female goat) and the aged group (10–12 years old, normal old female white goat) each had 6 goats, which were licensed by the Animal Experiment Committee of Medical School of Shanghai Jiaotong University. All animal work in this study was performed according to the institutional guidelines for care and use of laboratory animals in Medical School of Shanghai Jiaotong University. The preoperative fasting was 2 days, and water was forbidden for 12 h. Tracheal intubation was performed after general anesthesia (intramuscular injection of xylazine). Before the operation, the goat horn was sawed by a hand saw, leaving the roots, and bleeding was stopped by bone wax.
Defect model
The skin was cut along the midline of the craniofacial midline with electric knife. The subcutaneous fascia and muscle were separated and the bleeding was stopped by electrocoagulation. Then the top of the skull was exposed. The skull periosteum was stripped, and two skull defects of diameter 20 mm were drilled symmetrically at the top of the skull along midline. The skull bones that were drilled off were pried carefully, and residual skull fragments were removed by a bone nipper. The endocranium was retained.
Defect repair
After 14 days’ osteogenic induction, the TEBG was implanted into the left defect, while the Control DBM scaffold was implanted into the right defect as a control group. Then the muscle was sutured and the incision was closed after the TEBG and DBM were implanted in the defects. Three days after the operation, penicillin was injected for anti-infection.
CT scan and analysis
1.5 months, 3 months and 6 months after surgery, we anesthetized the goats and performed CT examination on the skull defects. After scanning, the skull was reconstructed with three-dimensional reconstruction software (VG studio Volume Graphics GmbH, Germany), and the bone volume (mm3) and bone volume/tissue volume ratio (BV/TV) were calculated. All these data were presented as mean ± SD.
Histology examination
6 months later, all the goats were over-anesthetized and euthanized in accordance with the animal experimental ethical standards of Medical School of Shanghai Jiaotong University. The specimens was separated and fixed by paraformaldehyde for 72 h. After decalcification of 0.6 M diluted hydrochloric acid, the specimens were sliced. As reported in previous studies [10, 19], the specimens were stained by H&E, VG and Masson respectively.
