Effects of physiological aging factor on bone tissue engineering repair based on 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

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].

Repair of goats skull defects

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.

Cell morphology

The Fetal BMSC had “triangular” shape, with no obvious nuclei (aging characteristics), and its cytoplasm was homogeneous and dark (Fig. 1a).

Growth curve

In the proliferation test, the cells entered the logarithmic growth period from the 3rd day, and entered the platform stage between the 9th and 11th day. The growth curve appeared S shape, which was consistent with the growth characteristics of stem cells (Fig. 1b).

Colony formation units rate

Fetal BMSCs could form (49.2 + 4.3%) colonies, more than 30% (Fig. 1c).

Three induced differentiation ability

After the fetal BMSCs were induced by osteogenic differentiation, many red positive nodules were observed under alizarin red staining, with a larger number and larger area (Fig. 1d). This result suggested that the osteogenic capacity of fetal BMSCs was good. After the fetal BMSCs were induced by adipogenic differentiation, lipid droplets in the cells were dyed red by Oil red O staining (Fig. 1e). This result suggested that fetal BMSCs had a good lipid performance. After the fetal BMSCs were induced by chondrogenic differentiation, highly aggregated cell mass was dyed blue by toluidine blue staining (Fig. 1f). This result suggested that the fetal BMSCs had a good cartilage performance, containing rich glycosaminoglycan and collagen. From the above data, we can see the fetal BMSCs had the potential for multidirectional differentiation.