Thirty patients with confirmed OA (18 males and 12 females; age range: 49-70 years, mean age: 62 ± 5.24 years) undergoing total knee arthroplasty at First Affiliated Hospital of Kunming Medical University were recruited. Cartilage tissues from tibial and femoral osteotomy (modified Mankin score of 0 or 1) were collected during surgery. The study was approved by the Ethics Committee of First Affiliated Hospital of Kunming Medical University, and the informed consent was obtained from all patients.
P1 chondrocytes isolation
Chondrocytes were isolated from cartilage tissues following a previously described approach30. Briefly, after collection, cartilage tissues were cut in 1mm × 1mm × 1mm size in the clean bench and were then transferred into 6-well plates. Samples were then treated with 0.25% trypsin and type II collagenase for digestion (Gibco, USA), and centrifuged in a centrifuge tube. After washing with sterile PBS, cells were seeded into a 25 cm2 culture flask (5mL/bottle) at a cell density of 1 × 105/mL, and cultured in a humidified incubator containing 5%CO2/95% air at 37ºC. The medium was changed every 3 days. When cells reached 80%-90% in confluence, the subculture was performed.
P1 chondrocytes were randomly assigned to four groups: blank control group, TN14003 group, T140 group, and AMD3100 group. Each group was cultured in high-glucose DMEM containing 10% fetal bovine serum and double antibody (100 U/mL penicillin + 0.1 mg/mL streptomycin). The TN14003 group was treated with 1000nmol/L TN14003 and 100ng/mL SDF-1; the T140 group with 1000nmol/L T140 and 100ng/mL SDF-1; the AMD3100 group with 1000nmol/L AMD3100 and 100ng/mL SDF-1; only 100ng/mL SDF-1 was added to the blank control group. All drugs were mixed in DMEM for 1 hour, and then with SDF-1 (Peprotech, USA). The cells of the four groups were cultured for 1, 2, 4, 6, 8, and 10 days. After each time point, cells were examined using MMT assay (cell viability), flow cytometry (cell apoptosis), ELISA, Quantitative real-time PCR; each method is described in detail in the following paragraph. The medium was changed every 4 days. Also, cell morphology and the number of cells were observed under an inverted phase-contrast microscope (Olympus, Japan). After treatment, cells were stained with Col II.
A total of 1 × 104/mL cells from each group with their corresponding drugs were plated in 96-well plates and incubated for 1, 2, 4, 6, 8, and 10 days. After each time point, 20μl of sterile MTT dye (5 mg/mL; Amresco, USA) was added to each well and incubated for another 4h at 37°C. After removal of the medium, 200 μl of DMSO (DMSO, Amresco, USA) was added to each well and properly mixed for another 10 min. The absorbance at 570nm was determined using a microplate reader (BioTek, USA). IC50 values were calculated from the linear regression of the plot.
A total of 1 × 105/mL cells from each group with their corresponding drugs were plated in 6-well plates and incubated for 2, 4, 6, 8, and 10 days. After each time point, All cells were collected and treated with AnnexinV/PI staining (Invitrogen, USA). After each time point, cell were collected in the centrifuge tubes and centrifuged at 1000 rpm for 5 min. After that, the supernatant was discarded. Then, the cells were resuspended in PBS and the cells were resuspended in 1 × 106/mL. The cells were centrifuged at 1000rpm for 5 min. The supernatant was discarded and 5 μL of the diluted 1 × conjugate was pipetted. 5 μL of PI and Annexin V-FITC staining were added respectively, cells were mixed at room temperature away from light for 15 min, then 400 μL of diluted 1 × liquid was added, and cells were mixedin the dark place with ice bath. Cell suspension were filtered with 300 mesh nylon mesh before detection. After detection, the testing was under 488 nm excitation wavelength and 525 nm emission wavelength was used.The apoptotic rate of the four groups was detected on a flow cytometer (Partec, Germany).
A total of 1 × 105/mL cells from each group with their corresponding drugs were plated in 6-well plates and incubated for 2, 4, 6, 8, and 10 days. After each time point, 500ul medium was collected and quickly cryopreserved in -80 °C refrigerator. the concentration of MMP-3 and MMP-13 in four groups at each time point were detected by ELISA kit (R & D Systems) according to the manufacturer’s protocol, respectively. Briefly, the micro ELISA plate has been pro-coated with an antibody specific to MMP-3 and MMP-13, and the appropriate medium samples were added in each microplate well, followed with incubation for 90 min at 37 °C. After washing with wash buffer, the corresponding secondary antibody was applied. After incubation for 60 min, the plates were washed again. The optical density was measured spectrophotometrically at a wavelength of 450 nm. The concentration of MMP-3 and MMP-13 was calculated from a standard curve.
Quantitative real-time PCR
A total of 1 × 105/mL cells from each group with their corresponding drugs were plated in 6-well plates and incubated for 2, 4, 6, 8, and 10 days. After each time point, the total RNA was extracted using TRNzol-A+ Reagent Total RNA Extraction Kit and were then reversely transcribed in cDNA using a PrimeScript® RT reagent Kit Perfect Real-Time kit, following the manufacturer’s instructions. After the reverse transcription was completed, the obtained cDNA was stored at -80 °C or -20 °C for PCR detection. The NCBI GeneBank database was searched to find the gene sequences of human Col II and ACAN. The General Biosystems (Anhui) Corporation Limited was commissioned to design and synthesize each gene primer. The primer sequence of each gene and the length of the amplified DNA fragment are shown in Table 1. The PCR reaction conditions were as follows: Stage 1: pre-denaturation, Reps: 1, 95 °C for 15 min; Stage 2: PCR reaction, Reps: 40, 95 °C for 10s, 60 °C for 30s; Stage 3: melting curve analysis, Reps: 1, 95 °C for 15s, 60 °C for 20s, 95 °C for 15s. The relative quantitative analysis method was used. The CT value represented the number of cycles at which the target amplified product reached the set threshold. The amplification fold change of the target gene in the test specimen relative to the control=2 -△△CT. The formula was as follows: △ CT = CT (target gene) -CT (reference gene); △△ CT = △ CT (experimental group)-△ CT (control group).
In this study, the blank control group in the 2-day group of the OA cartilage group was used as a reference, and the relative quantitative 2-△△CT method was used, while the fold change for each group was 2 -△△CT. Data in the experimental group were analyzed by SPSS (version 20) software. The values of each group were expressed as mean ± standard deviation (± S), and analysis of variance was adopted; α = 0.05 was used as the significant level.
Spontaneous OA Animals and grouping
As the Hartley guinea pig is an accepted model for spontaneous Osteoarthritis, the Osteoarthritis Research Society International (OARSI) has developed guidelines on the histologic examination of knee joints from these animals42. So, we used Hartley guinea pigs because these animals spontaneously develop degenerative cartilage changes in the knee joint that mimic those of human OA. A total of 96 male Hartley guinea pigs, 6-month-old, were obtained from Institute of Zoology, Chinese Academy of Sciences (No. TBA287640-334). All the animals were housed in an environment with a temperature of 20-25 ºC, a relative humidity of 50%-60%, and a light/dark cycle of 12 hr. All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Kunming medical University animal care and conducted according to the IACUC guidelines.
Hartley guinea pigs with spontaneous OA were then randomly assigned to four groups (24 pigs/group): TN14003 group, T140 group, and AMD3100 group and control group. Four groups were subcutaneously implanted Alzet osmotic pumps into the back, and each group was pumped with TN14003, T140, and AMD3100, respectively at a concentration of 180 g/mL daily; the blank control group was injected with saline. After 12 weeks of conventional breeding, guinea pigs were sacrificed by intraperitoneal injection with pentobarbital sodium (30mg/kg), and articular cartilages from knees were collected; morphological detection of cartilage (H&E staining and safranin O staining), and the Modified Mankin grading system were performed.
The data were analyzed by SPSS (version 20) software package. All data were presented as mean ± standard deviation (± S). The difference between groups was analyzed by Analysis of Variance, Mann-Whitney U test, and χ2 test when applicable. A P-value of < 0.05 was considered statistically significant.