Isolation and culture of WJ-MSCs
The protocol for isolation and culture of rabbit umbilical cord mesenchymal stem cells was the same as that published in our previous paper12. Briefly, pregnant rabbits (27th day of pregnancy) were narcotized using 3% pentobarbital sodium (Germany Merck Biotechnology Co., Ltd.). The umbilical cords were harvested under sterile conditions and dissected to expose the Wharton’s Jelly (WJ). The Wharton’s Jelly was cut into small parts and digested with 1 mg/ml collagenase II (Sigma-Aldrich) at 37°C for 30 minutes. The digestion was terminated by low glucose Dulbecco’s Modified Eagle’s Medium (LG-DMEM, Gibco, USA), containing 10% fetal bovine serum (FBS, Biological industries, Israel) and 100 U/mL of penicillin with 100 mg/ml streptomycin (Gibco, USA). The digested solution was centrifuged, the cells in the sediment were resuspended using the complete culture medium, and then plated on Petri dishes at a density of 4 × 104/cm2 in an incubator at 37°C under a 5% CO2 atmosphere. Media replacement was performed every 72 hours and the cells were allowed to grow until cell cultures reached 70% to 80% confluence.
Construction of ECM gel and adipogenic WJ-MSC complex
Cells at passage 6 were used for the following experiments. Cells were cultured in adipogenic media inducing solution A (dexamethasone 1 μM, IBMX 0.5 mM, insulin 10 μg/ml, and indomethacin 200 μg added to complete medium) for 72 hours, and then replaced with solution B (10 μg/ml insulin added to the complete medium) for another 72 hours. After 15 days, the success of adipogenic induction was identified by Oil Red O staining. Then the adipogenic WJ-MSCs (aMSCs) were digested and resuspended in the complete culture media at 4°C at a density of 1 x 106 cells/ml. The tissue-engineered fat complex was prepared by mixing with ECM gel (Sigma-Aldrich) and the cell suspension with the ratio of 1:1. The complex was kept at 4°C until use (the time period was less than 1 hour).
Thirty-six New Zealand rabbits were randomly divided into three groups: control group, ECM group, and ECM+aMSCs group. The treatments were as follows: Control group was treated with 1 ml saline after laminectomy, the ECM group was treated with1 ml ECM gel, and the ECM+aMSCs group was treated with 1 ml of the tissue-engineered complex with ECM gel as scaffold and aMSCs as the seed cells.
The New Zealand rabbits weighed approximately 3.0-3.5 kg and were injected intraperitoneally with 3% pentobarbital sodium (Germany Merck Biotechnology Co., Ltd.) at a dose of 1-1.5 ml/kg for anesthesia. Next, the rabbit's back was depilated and disinfected with iodophor. The skin at the L5 vertebrate was cut, the subcutaneous tissue was exposed, and the superficial fascia and supraspinous ligament was incised to expose the L5 laminae. Then the spinous process and laminae were removed by rongeur, and the epidural fat was carefully eliminated to expose the spinal dura sac. Then the corresponding groups were injected with corresponding materials with injection syringe. The wounds were closed with 4-0 suture. Penicillin sodium (80,000 units/kg) was used to prevent infection with intramuscular injection. Then, the New Zealand rabbits were returned to the rabbit cage for routine feeding.
At the 4th, 8th, and 12th weeks after operation, three New Zealand rabbits in each group were subjected to MRI examinations to observe the formation of sagittal and transverse epidural fibers and fat at the laminectomy area.
After MRI, a little soft tissue near the laminae defect was collected in a 1.5-ml cryotube and stored in liquid nitrogen. The rabbits were euthanized by excess injection of 3% sodium pentobarbital. The L5 lumbar spine combined with the upper and lower lumbars were cut en bloc, fixed with 10% formaldehyde for 1 week, and then sent to Wuhan Google Biotechnology Co., Ltd. Shanghai Branch for decalcification treatment. EDTA (10%) was used for decalcification for 4-8 weeks until the vertebral bone could be cut with a slicing knife.
After decalcification, the specimens were embedded in paraffin. Hematoxylin and eosin (H&E) staining was used to count the fibroblasts at 400X magnification (Fig. 1). The ratio of the area of blue collagen fibers to the total area of tissues in the field of view at 200X magnification was used as a measure to estimate the degree of fibrosis using Masson’s trichrome staining. Oil Red O staining was used to observe the formation of epidural fat.
Quantitative real time polymerase chain reaction (qRT-PCR)
qRT-PCR was used to detect the relative expression of local inflammatory factor IL-6 and fibrosis factor TGF-β in each group at each time and β-actin was used as internal reference. All tissues in the cryotubes were decomposed with the Trizol® reagent (Invitrogen, USA) to obtain RNA, and reverse transcriptase (Takara, Japan) was added to form cDNA. Quantitative real-time PCR (RT-PCR) was performed using the 7900 Real-Time PCR System (Applied Biosystems) with the Power SYBR® Green PCR Master Mix (Takara, Japan) to explore the relative expression of genes. The primers (Shanghai, China) were sequenced as shown in Table 1. Relative gene expression was calculated using the equation: ΔCt=Ct (Rest genes) -Ct (β-actin); ΔΔCt=ΔCt (experimental groups) -ΔCt (control group); Fold change=2-ΔΔCt.
The results of each set of experiments were repeated three times, and statistical analysis was performed using the GraphPad prism 6.0 software. The multiple T test was mainly used and P <0.05 was considered statistically significant.