Patients and samples
This study was approved by the Ethics Committee of Shanxi Medical University [Approval number: SXMUE(2019004)], and informed consentwas obtained from each donor. In the present analysis, westudied a group of 65 patients (11 males and 54 females) with an average age of 60.13 years (61.54 ± 11.5). Five young patients suffered from amputation due to severe trauma, and 60 patients received total knee replacement. All cartilage specimens were derived from the tibial plateau and remained sterile, and part of the tissue was cut for histological sections and stained with saffron O to determine the OARSI grade of the cartilage tissue.
Human tibial plateau chondrocytes
A sterile scalpel was used to cut the corresponding layers of cartilage, and chondrocytes were obtained after tissue clipping and collagenasetype II digestion.The isolated chondrocyteswere cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal calf serum (FCS) at 37°Cin a humidified atmosphere of 95% air and 5% CO2. The chondrocytes were cultured to the third generation(P3) for experiments.
IHC staining was used for detection of collagen II (Abcam, ab34712,1:200),Aggrecan(Abcam, ab186414,1:500), collagen X(Abcam, ab58632,1:100),MMP-13 (Abcam, ab39012,1:200) and PCNA(Abcam, ab92552,1:200) in chondrocytes inclusters.We quantitatively scored theIHC results accordingto the percentage of positive chondrocytes and the staining intensity, asdescribed below.We rated the intensity of staining on a scaleof 0 to 3: 0, negative; 1, weak; 2, moderate; and 3, strong. Weassigned the following proportion scores: 0 if 0% of the chondrocytes showed positive staining, 1 if 0% to 1% of the chondrocytes werestained, 2 if 2% to 10% were stained, 3 if 11% to 30% werestained, 4 if 31% to 70% were stained, and 5 if 71% to 100%were stained. We then combined the proportion and intensityscores to obtain a total score (range: 0-8), as described previously.38 The results were assessed by 2 experienced pathologistsin a blinded manner.
Polymerase chain reaction (PCR)
Total RNA was extracted from cartilage tissue and chondrocytes by using TRIzol reagent (Thermo Fisher Scientific). The quality and quantity of total RNA samples were tested using a NanoDrop 2000C spectrophotometer(Thermo Fisher Scientific). A preparation of RNAwith an A260/A280 ratio of 1.8~2.0 was used for analysis of mRNA expression. Individual RNA samples (1 μg per sample) were reverse transcribed into cDNAusing the PrimeScript RT Master Mix kit (Takara, Shiga,Japan) according to the manufacturer’s instructions. Therelative expression levels of target gene mRNA to the control 18SrRNA transcripts were determined by RT-PCR using SYBR Premix Ex TaqTM (Takara) and the specific primers in the IQ5 Multicolor Real-Time PCR Detection system(Bio-Rad Laboratories, Hercules, CA, USA). The sequences of the primerswere forward 5’-TGGACGATCAGGCGAAACC-3’andreverse 5’-GCTGCGGATGCTCTCAATCT-3’ for collagen II; forward 5’-ACTCTGGGTTTTCGTGACTCT-3’ andreverse 5’-ACACTCAGCGAGTTGTCATGG-3’ for aggrecan;forward 5’-ATGCTGCCACAAATACC
-CTTT-3’ andreverse 5’-GGTAGTGGGCCTTTTATGCCT -3’for collagen X;forward 5’-CAGGAATT
-GGTGATAAAGTAGAT-3’ andreverse 5’-CTGTATTCAAACTGTATGGGTC-3’for MMP13; forward 5’-TTGCACTGAGGTACCTGAACTT -3’ andreverse 5’-CCTTCTTCATCCTCGATCTTG-3’for PCNA.
The chondrocyte samples were lysed in RIPA lysis buffer containing PMSF, protease and phosphatase inhibitors (Keygen). A certain amount of protein was mixed with loading buffer,boiled for 10 minutes and subjected to SDS-PAGE followed by transfer to PVDF membranes. The blots were probed with primary antibodies, including anti-collagen II(Abcam, ab34712,1:3000), anti-Aggrecan(Abcam, ab186414,1:3000), anti-collagen X(Abcam, ab58632,1:1000), anti-MMP-13 (Abcam, ab39012,1:3000) and anti-PCNA(Abcam, ab92552,1:1000) after being blockedwith 5% fat-free dry milk in TBST.The relative levels of the target proteinto the control β-actin expressionwere determined by western blot analysis .The bound antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using the enhanced chemiluminescence reagent. Thedata were analysed by densitometric analysis using IMAGEJ software.
The cartilage tissue was made into a cylinder that was 6 mm in diameter using a perforator.An elastic seal membrane was used to wrap around the cartilage column and control the seal membrane to be approximately 5mm above the cartilage surface.Contrast medium(Iohexol, Yangtze River Pharmaceutical Company,China) was added to the sealing film sealed membrane to permeate only from the surface of the cartilage.The specimens were subjected to micro-CT scanning at 0 h, 1 h, 3 h and 5 h.
SPSS 20.0 (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. All data in this study
were expressed as the mean ± standard deviation (SD). A P value of less than 0.05 was considered