Experimental animals
Forty SPF SD rats about 3-week-old were purchased from the Experimental Animal Center of Guangdong Medical College (Certificate No.2018A029; batch number, 20190102). All rats were housed in the Experimental Animal Center of Zhejiang University of Traditional Chinese Medicine and the experiments were approved and agreed. All the traditional Chinese medicinal materials in this study were provided by Zhejiang University of Traditional Chinese Medicine.
Cartilage defect model
Forty SD rats were fed adaptively for 1 week, and were divided into 4 groups randomly. Each group contains 10 rats, group A was treated as control; group B was treated with simple herbal formulation; group C was treated with complex ingredients and Group D was treated with herbal formulation combined with stem cell 3D biological scaffold. All rats were placed on a sterile table and anesthetized with 3% pentobarbital sodium (1 ml/kg), and the rats were gently fixed on the table, and the knees were routinely prepared for the disinfection. For the cartilage defect surgery, a longitudinal incision was cut in the medial knee, and the patellar ligament to the lateral side was removed, so that femoral intercondylar concave was exposed. Under sterile conditions, the femoral condyle trochlear joint surface is drilled with a defect in 3.5 mm diameter and 3.0 mm in depth to make full-thickness cartilage defects, and the blood was cleared off.
Drug intervention
Herbal formulation: The whole recipe is composed of 18 g radix rehmanniae, 18 g yam, 12 g dogwood, 18 g wolfberry, 18 g spleen, 6 g aconite, 6 g cinnamon, and 6 g licorice. According to previous study, we used the best extraction process consisting of 1.5 h for one extraction, extracted twice, added 10 times water to prepare herbal formulation. Quality control should meet the standards based on 2005 edition of the People's Republic of China Pharmacopoeia. Appearance indicators for the beverage should be earthy yellow, strong fragrance, even and no visible foreign bodies. Physical and chemical identification should be as following, the pass rate for the particle size was about 96.6±2.28%. The average moisture content measured by infrared moisture analyzer was about 7.37±0.02%. The average content of catalpol is about 6.39mg/g, and the average content of 5-hydroxymethylfurfural is about 0.265mg/g. According to the human and rat dose conversion formula, and the daily gavage dosage in rats should be about 2.75 g/ml of crude drug, and the rats should be administered once a day in the morning and evening for 8 weeks.
According to the cartilage defects of SD rats, 3D bio-scaffolds were made by three-dimensional micro-CT scanning. Harvest the transfected cells, and digest cells with trypsin, and centrifuge at 1000 rpm for 3 min, and discard the supernatant, and add the cells to the 3D biological scaffold. Scanning electron microscope was used to observe the distribution and morphological changes of cells on the scaffold.
Bone debris and blood clots was thoroughly removed using the saline. Groups C and D were gently embedded with BMSCs3D bio-scaffolds transfected with TGF-beta1, but groups A and B were not treated. The patellar ligament was reduced and the knee capsule and skin were sutured. After the operation, penicillin was injected intramuscularly for 100,000 u for 1 week. The limbs were not fixed, and they were free to move.
H&E staining
The rats were given the low flow of CO2 and then decapitated to harvest the tissues. After 4% PFA fixation for 3d, the tissues were placed in 100 g/LEDTA for about one month to decalcified the tissues, and then the tissues undergo several steps of dehydrations consisted with xylene and series of gradient ethanol. The tissues were placed microtome to section into 6 µm thickness, H&E staining was performed based on the protocol and the staining were analyzed under the microscope.
Mankin evaluation
The fixed cartilage tissue was sectioned and stained with toluidine blue, and under the light microscope, the Mankin score was evaluated, the total score is 14 points, the higher the score is, the more severe the cartilage damage is. Mankin score of each group of rats was recorded and analyzed.
Determination of Young's Modulus of Elasticity in Cartilage
The cartilage tissues were collected and melted at room temperature. Using a sharp knife to remove the articular cartilage and reveal the subchondral bone. Using one omnipotent testing machine and miniature sensors to determine the load-displacement curve, stress-strain curve and compression modulus.
Western blot
The rat cartilage was collected, and minced with scissors, and the total protein was extracted. The protein levels were run by 10% SDS-polyacrylamide gel electrophoresis, and then transfer the protein to the PVDF membrane using semi-dry method. Afther the blocking of the transferred proteins into PVDF membrane, primary antibodies were incubated (1:1000, rabbit Col2a, PA5-111424, Thermo; rabbit aggrecan, PA1-1746, Thermo) overnight at 4 degree, and secondary antibodies goat anti-rabbit-HRP was used and ECL was applied to exposure the lots in the membrane using ChemiDoc Touch (Biorad) and GADPH was used as the internal control.
RT-PCR experiments
The rat cartilage tissues were collected and total RNA was extracted using Trizol (Invitrogen) based on the protocol. After the evaluation of RNA concentration, about 10 ug RNA was used to reverse into cDNA using Invitrogen SuperScript III Reverse Transcriptase (18080093, Thermo). To measure the expressions of Col2a (Forward: 5’-GGG TCA CAG AGG TTA CCC AG-3; Reverse: 5’-ACC AGG GGA ACC ACT CTC AC-3’) and aggrecan expressions () using ABI PCR 7500, as well as PowerUp SYBR Green Master Mix (A25742, Thermo) was used as the mix, and PCR program was as following: 95 ℃ pre-denatured for 2 min; using 95 ℃ for 8 s, 58 ℃ for 35 s, 72 ℃ for 40 s, 40 Cycles. Relative value of target gene expression RQ=2-ΔΔCt. GAPDH was used as a control.
Statistical method
Statistical analysis was performed using SPSS 17.0 statistical software package. The experimental data are presented in (x±s), and the data was analyzed using two-way ANOVA and the comparison between multiple groups were analyzed using independent sample t test. The p value less than 0.05 indicated as significance.