1.In vitro experiments
1.1. Isolation and culture of chondrocytes from OA patients
Cartilage tissue was obtained from discarded knee joints of osteoarthritis patients (n=10) who underwent total knee replacement surgery at the First Affiliated Hospital of Harbin Medical University. Cartilage tissues were washed three times with phosphate-buffered saline (PBS), and then cut into 1 mm2, digested in 2mg/ml collagenase type II (Sigma, St. Louis, MO, USA) 37℃ overnight. After that, the cells were filtered through a cell strainer. Primary cells were used from the P0-P1 passage.
1.1.2 Culture of human normal chondrocytes
Human normal articular chondrocytes were obtained from the C28/I2 human normal chondrocytes line(Otwo Biotech, Inc, Cat#HTX2308;China)and cultured in DMEM medium with the addition of 10% FBS and 0.5% penicillin-streptomycin, at 37 °C and 5% CO2.
1.1.3 Culture of HMVEC
Human microvascular ECs (HUVECs) (OTWO, Inc, Cat#HTX1922;China), were grown in endothelial cell medium (ECM)(ScienCell, Cat#1001; USA) supplemented with 25ml fetal bovine serum (FBS)(ScienCell, Cat#0025; USA), 5 ml Endothelial Cell growth supplement (ECGS)(ScienCell, Cat#1052; USA), 5 ml penicillin/streptomycin solution(P/S)(ScienCell, Cat#0503; USA), and used for tube formation and migration experiments. HUVECs were cultured to confluence, followed by incubation in serum-reduced ECM (1% FBS) for 24 hours prior to stimulation with THBS-3 for an additional 24 hours.
1.2 Small interfering RNA and transfection experiment
The small interfering RNAs (siRNAs) and transfection reagent were purchased from RIBOBIO Co., Ltd. (RiboBio, China)according to manufacturer’s instructions.
Transfection target sequence (Table 1).
1.3 Western blotting
Following a triple wash with PBS, cells underwent protein extraction. Proteins were separated on 10% or 12% SDS-PAGE gels. Subsequently, proteins were transferred onto polyvinylidene difluoride membranes (Thermo-Fisher, Hampton, NH) and probed with primary antibodies against THBS-3 (Proteintech, Cat#19727-1-AP;USA), Aggrecan (Abbkine, Cat#ABP54013;China), ADAMTs-5 (Abcam, Cat#AB41037;USA), MMP-13(Bioword Technologyl, Cat#BS1231;China),BMP-2(Abbkine, Cat#ABP0179;China), FGF-2 (Abbkine, Cat#ABP53280;China), ANG-2(Abcam, Cat#AB155106;USA), VEGF-A (Abcam, Cat#AB214424;USA), PDGF-B(Abcam, Cat#AB178409;USA), TGF-β1 (Abcam, Cat#AB179695;USA), p-smad2/3 (Abbkine, Cat#ABP0048;China)and GAPDH (ABclonal, Cat#A19056;China),overnight at 4°C. The blots were then subjected to incubation with a secondary antibody (LI-COR, Cat#926-32211;USA) at room temperature for 1 hour. Visualization was achieved using a LI-COR Imaging System (Biosciences, USA), and subsequent quantification was carried out with Image-Pro Plus software.
1.4 Real-time quantitative PCR.
Total RNA from chondrocytes were extracted with Trizol reagent (Invitrogen, Cat#R1100; USA) according to manufacturer’s instructions. cDNA was then synthesized from 1 µg of total RNA using the 4*DN Master Mix with gDNA Remover and 5*RT Master Mix II ( TOYOBO ,Cat#FSQ-301; Japan) of the resultant cDNA, 25 ng was used for qRT-PCR. The qRT-PCR mix contains 5 μl iTaq Universal (BIO-RAD, Cat#1725124; ,USA), 0.5 μl of each primer, cDNA 1ul and Nuclease-free H2O with a total volume of 10 μl. Samples were amplified for 40 cycles using the Applied Biosystems® 7500 Real-Time PCR System (ThermoFisher Scientific). The 2^-ΔΔCT method was employed for calculating relative gene expression levels, with normalization against the housekeeping gene GAPDH.
Primer sequences(Table 2)
1.5 Tube Formation Assay
Endothelial cells were seeded at a density of 2 × 10*4 cells per well in μ-Slide Angiogenesis (ibidi, Cat#81506; GER) pre-coated with 50 μL of Matrigel in each well (Corning, Cat#354230; USA). The plates were subsequently incubated at 37°C under various treatment conditions. After 6 hours of incubation, the cells were imaged and analyzed using an inverted microscope (Leica) and Image-Pro Plus software.
1.6 Migration assay
The wound scratch assay was employed to assess the migration of BMECs under various concentrations of human recombinant THBS-3 proteins. In brief, 5.0 × 10*5 cells were seeded per well in a Six-well culture plate and incubated for 24 hours at 37°C until reaching confluence. Subsequently, a scratch was created using a sterile p200 pipette tip, and the cell debris was washed three times with PBS. Following this, the cells were incubated in DMEM supplemented with 5% fetal bovine serum (FBS; Gibco, USA) under the various concentrations of human recombinant THBS-3 proteins. Images of the wounds were captured immediately after scratching and again 24 hours later. Image J software was used to measure the change in the scratched areas. The migration rate was calculated as follows: migration area (%) = (A0 – An)/A0 × 100, where An and A0 represent the remaining area and initial area of the wound, respectively.
2 In vivo experiments
2.1 VII type collagenase-induced OA mouse models
For the collagenase-induced OA model, 12-week-old male C57BL/6 (Animal Center of Harbin Medical University) mice were anesthetized, and then injected the VII type collagenase on the left knee. 2’OMe+5’Chol modified THBS-3 siRNA labeled with 5FAM were purchased from Ribo Company (RiboBio, China). Mice(n=18) were randomly divided into the following groups: Control(n=6), CIOA(n=6), CIOA+siTHBS-3(n=6). At 7 days post-injection, 5nM siTHBS-3 (OA + siTHBS-3 group) were injected intra-articularly once a week into the mice knee joints. Mice were killed at week 7 for bone and cartilage analysis. All the animals were kept in a controlled environment that ensured they were free from specific pathogens. They were subjected to a 12-hour light and 12-hour dark cycle and were provided with a standard chow diet. All animal experiments were approved by the Harbin Medical University Committee on the Use and Care of Animals.
2.2 Micro-CT
Subchondral bone explants from the mouse specimens were initially fixed overnight in a 10% formalin solution. Subsequently, these explants were examined using Microcomputed Tomography (Micro-CT) imaging (Quantum GX, Perkin Elmer, Waltham, USA).
2.3 Histology.
Knee joints were fixed in 4% paraformaldehyde for 24 hours and then decalcified over an 8-week period using a 10% EDTA solution at pH 7.4. The samples were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek) or paraffin. The medial portion of the samples was longitudinally oriented and sectioned into 4 μm slices. The knee joint's medial compartment was subjected to staining procedures, including hematoxylin-eosin (H&E staining), Safranin O and fast green staining as well as immunohistochemistry. Images were captured using a microscope (Olympus).
4.Statistical Analysis
The data is presented as the mean ± standard error of the mean (SEM). For comparisons between two groups, a two-tailed Student's t-test was employed. In the case of multiple group comparisons, a one-way analysis of variance (ANOVA) was utilized. Initially, the homogeneity of variance was assessed, followed by post hoc multiple comparisons to evaluate differences between groups. Statistical significance was defined as p < 0.05.