2.1 Reagent
Fetal bovine serum (FBS), penicillin/streptomycin, Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), and 0.25% trypsin were purchased from Gibco BRL (NY, USA). Collagenase I, CoCl2, hydrogen peroxide (H2O2), N-Acetyl-L-cysteine (NAC), and MG132 were also purchased from Sigma-Aldrich (MI, USA). Cycloheximide (CHX) was purchased from Aladdin (Shanghai, China). HIF-1a overexpression plasmid and the overexpression control plasmid were produced and purchased from Genechem (Shanghai, China). Preparation of the osteogenic induction medium: To DMEM complete media, we added 10 mmol/L β-Glycerol phosphate disodium salt (Solarbio, Beijing, China), 0.1 µmol/L dexamethasone (Solarbio, Beijing, China), and 50 mg/L ascorbic acid (Solarbio, Beijing, China), used after sterilizing with a 0.22 µm colander (Becton Dickinson, Franklin Lakes, NJ). Prepare the low-oxygen inducing medium with a concentration of 200 µM CoCl2: 0.0065 g CoCl2 was added into 250 ml DMEM complete medium, shaken and mixed, and used in a 0.22 µm filter (Becton Dickinson, Franklin Lakes, NJ, USA) after degerming (Balogh et al. 2019).
2.2 Animals and ethical approval
All surgical interventions and postoperative animal care were performed according to the National Research Council’s Guide for the Care and Use of Laboratory Animals and were approved by the Southeast University’s Animal Research Ethics Committee (No. 20220620010). The experimental design aimed to minimize the number of animals used and sacrificed. Male Sprague–Dawley (SD) rats (6 weeks old, 250–300 g) were purchased from the Shanghai Family Planning Research Institute Laboratory Animal Management Department (Shanghai, China). The rats were housed in a humidity-controlled room at 25°C, under a 12 h light/dark cycle, with free access to food and water.
2.3 Establishment of a diabetic rat model
The rats were fed a high-sugar and fat diet for 2 months. The rats were weighed after overnight fasting. Further, 1% streptozotocin dosed at 40 mg/kg was prepared with a citric acid buffer solution based on fasting weight and quickly administered into the abdomen, which was completed within 30 min. After 3 days, blood glucose concentration in the rat tail vein was measured using a glucose meter. More details are outlined in a previous study (Shi et al. 2019). Figure 7A shows the flowchart of the animal experiments.
2.4 Establishment of a rat model of calcific tendinopathy
After successfully establishing the diabetic rat model(Shi et al. 2019), the rats were divided into four groups. They were Non-DM + NS (healthy rats injected with NS in bilateral Achilles tendon), DM + NS (diabetic rats injected with NS in bilateral Achilles tendon), DM + H2O2 (diabetic rats injected with 800 µmol/L H2O2 in bilateral Achilles tendon), and DM + H2O2 + NAC groups (diabetic rats injected with a mixture of 800 µmol/L H2O2 and 10 mM NAC into the bilateral Achilles tendon). Each group comprised eight rats. All rats received a single injection of 0.2 mL solution into the unilateral Achilles tendon twice a week for 8 weeks. Figure 7A shows a flowchart of the animal experiment.
2.5 Extraction and culture of TSPCs isolated from healthy rats
In this study, a mature and perfect extraction method developed by our research group was used for TSPCs extraction (Shi et al. 2021). The steps were as follows: Four 6-week-old healthy SD rats were selected, weighing 270–330 g. After the rats were euthanized, the middle part of the Achilles tendon was dissected from both sides, and the connective tissue around the Achilles tendon was carefully removed. Type I collagenase (3 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) digested collagen for 1.5 h. A 70 µm filter (Becton Dickinson, Franklin Lakes, NJ) formed a single-cell suspension, and the cells were inoculated at a low density (50 cells/cm2) and cultured for 7–10 days. After clonal formation, pancreatic enzyme digestion was mixed and labeled as primary cells (P0). P3–5 cells were used in follow-up experiments. The culture medium was changed every 3 days during the experiment.
2.6 In vitro cell culture condition
All TSPCs cells were extracted from healthy SD rat Achilles tendons. Based on the development and progression of calcific tendinopathy, the TSPCs environment is divided into three stages: 1. Low oxygen and low glucose (LO–LG) levels represent the environment of tendon cells in healthy individuals without diabetes. We simulated TSPCs using a low-glycemic complete medium containing 200 µM CoCl2 (Balogh et al. 2019). 2. Low oxygen and high glucose (LO–HG): In the early stages of diabetes, the blood glucose level increases; however, the tendon does not show vascularization, thickening, erythrocyte increase, or tissue oxygen concentration change. To simulate the intervention, a high-glucose complete medium containing 200 µM CoCl2 was used. 3. High oxygen and high glucose (HO–HG) represent the progressive stage of diabetes when tendons have increased blood vessels and thickening, red blood cells, and tissue oxygen concentration. The cells were placed in a high-glucose complete medium in an incubator with a 20% oxygen concentration for the intervention simulation.
2.7 RNA-sequence assay
TSPCs were divided into three groups, with three samples in each group, and they were placed in three different environments of LO–LG, LO–HG, and HO–HG for continuous culturing for 2 weeks. The medium was discarded, and 5 ml PBS (RNase-free, room temperature) was added to the cell culture bottle. The bottle was washed once, and an appropriate amount of cell lysate was added and repeatedly sucked until no cell clumps were seen. The lysed cells were transferred to a 1.5 ml capping tip-bottomed centrifuge tube (RNase-free). Biomarker Technologies (BMK, Beijing, China) were tested on dry ice. After the sequencing data were unloaded, the BMK cloud bioinformatics analysis procedure was used for data analysis (www.biocloud.net). Mapped data were obtained after sequence alignment with a specified reference genome. Library quality assessment, structure-level analysis, differential expression analysis, gene functional annotation, and functional enrichment were performed (Zhan et al. 2015).
2.8 Quantitative real-time polymerase chain reaction (qRT-PCR) assay
Total RNA was extracted from TSPCs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration and purity were measured using a spectrophotometer (NanoQTM; Beijing, China). The template RNA had an OD260/OD280 ratio ranging from 1.9 and 2.0. According to the manufacturer’s user guide, complementary genes were synthesized by reverse transcription of 2 µg template RNA in a two-step RT-PCR SuperMix Kit (TransGen Biotech, China) (Zhao et al. 2012). Subsequently, a reverse transcription polymerase chain reaction was performed using the ABI7500 system (Thermo Fisher Scientific, USA). Finally, the mRNA expression of the gene was detected by 2-ΔΔCt. See Supplementary Table S1 for the primer sequences used.
2.9 Western blot (WB) assay
We evaluated the expression of Vascular Endothelial Growth Factor(VEGF), Hypoxia inducible factor-1a(HIF-1α), Osteopontin(OPN), Runt-related transcription factor 2(RUNX2), Ubiquitin, and other proteins in the whole-cell lysate products. Cells from each group were collected, and radio-immunoprecipitation assay protein lysate (Invitrogen, Carlsbad, CA, USA) was added to the ice for full cracking. The cells were centrifuged at 4°C and 12,000 rpm, and the supernatant was collected. Using a BCA Protein Analysis Kit (Beyotime Biotechnology, Shanghai, China), the protein concentration in the supernatant was thoroughly mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein loading buffer (5X) (Beyotime Biotechnology, Shanghai, China) at a 4:1 volume ratio. The resulting protein was heated at 100°C for 10 min for denaturation. Using 4–20% SDS-PAGE (ACE Biotechnology, Beijing, China), the same amount of protein (30 mg system) was transferred to the polyvinylidene fluoride (PVDF) membrane (Sigma, USA). The remaining experimental steps referred to previous experiments (Y. W. Zhang et al. 2022). ImageJ software was used to analyze the relative band strengths. The antibody information is listed in Supplementary Table S2.
2.10 Co-immunoprecipitation (Co-IP) assay
TSPCs were cultured for 3 days in a 10 cm-diameter cell culture dish in a high-oxygen environment. The cell culture medium was removed, washed once with PBS, and cells were lysed with 500 µL of cell lysate (Invitrogen, Carlsbad, CA, USA). Approximately 1 µg of protein samples was obtained and mixed with 200 µl of immunoglobulin G (IgG) precipitation solution. The mixture was incubated with Protein A + G Agarose (Beyotime Biotechnology, Shanghai, China), containing 20 species of the same general IgG. The incubation was conducted at 4°C with gentle shaking for 1 h. The supernatant was collected for immunoprecipitation after centrifugation at 2,500 rpm for 5 min. Two micrograms of ubiquitin or HIF-1a primary antibody were added and slowly shaken overnight at 4°C. The next day, 40 µL of fully resuspended Protein A + G Agarose was added and slowly shaken at 4°C for 3 h. Following instantaneous high-speed centrifugation, the supernatant was carefully removed, washed, and precipitated with 500 µL PBS five times. After the final washing, the supernatant was removed and added to the Vortex of 20–40 µL 1X SDS-PAGE loading buffer for re-suspension precipitation, and the sample was centrifuged to the bottom of the tube at instantaneous high speed. After treatment at 100°C for 5 min, some or all samples were subjected to SDS-PAGE.
2.11 Transfection of TSPCs assay
A day before transfection (18–24 h), approximately 200,000–700,000 cells per well were inoculated into a six-well plate for a culture so that the cell density reached approximately 70–80% on the following day. Before transfection, 2 mL of fresh culture solution was added to each well of a six-well plate containing the cells. A clean, sterile centrifuge tube was obtained. Next, 125 µl of DMEM low- or high-glucose media without antibiotics and serum was added to cells from each well of the six-well plate to be transfected. Furthermore, 2.5µg of HIF-1a carrying GV657 vector (GV657-HIF-1A) plasmid (GeneChem, Shanghai, China) was transfected with the empty vector GV657 as the negative control, gently blown, and mixed with a gun. Next, a transfection reagent was added to the 4 µL Lipo8000™ (Beyotime Biotechnology, Shanghai, China). After preparation, the samples were stored at room temperature for 6 h for stabilization. At 125 µl of the Lipo8000™ transfection agent-DNA mixture per well of a six-well plate, drops were evenly added to the entire well and gently mixed. Approximately 48 h after continuous culture, fluorescence microscopy was used to detect the transfection effect, and the transfected cells were successfully used for follow-up.
2.12 Alkaline phosphatase (ALP) staining and quantification assay
After 1 week of cell culture in each group, the culture medium was removed, and the cells were washed once with PBS. An ALP staining kit (Solarbio, Shanghai, China). According to the manufacturer’s instructions, the ALP-fixing solution was added for 3 min and distilled water was used for cleaning. The prepared ALP solution was placed in a wet box, incubated for 20 min in the dark, and cleaned with distilled water. A nuclear solid red staining solution was added and re-dyed for 3 min. Cleaning with PBS, observation, and detection was performed under a light microscope. An ALP detection kit (Nanjing Jiancheng Bioengineering Institute) was used for quantitative detection. Relevant reagents were added according to the manufacturer’s instructions, and an enzyme marker measured the absorbance value of 520 nm of each well. Finally, the ALP activity was calculated using a correlation formula.
2.13 Alizarin red staining (ARS) and quantification assay
After 3 weeks of cell culture in each group, the culture medium was removed, and the cells were washed once with PBS. ARS staining kits (Beyotime Biotechnology, Shanghai, China) were used according to the manufacturer’s instructions to join the fixed liquid for 20 min, followed by washing three times with PBS. An appropriate amount of ARS solution was added, and the cells were evenly covered and stained at room temperature for 30 min. The cells were washed thoroughly with distilled water, viewed, and photographed under a microscope. To quantify ARS in 96-well plates, we added 100 mmol/L cetylpyridine chloride (C9002; Sigma) solution (100 µ/L), and cetylpyridine chloride solution as blank. Optical density was measured at 405 nm using a Hitachi U-2800A spectrophotometer (Y. Chen et al. 2020).
2.14 Transmission electron microscope inspection assay
TSPCs were cultured continuously for 2 weeks in LO–LG, LO–HG, and HO–HG environments. The cells were washed once with PBS, hung with a cell spatula, centrifuged at 1,500–3,000 RPM for 5–10 min, and two to three rice-sized samples were collected from the cells at the bottom of the tubes. The supernatant was discarded. Electron microscopic fixation solution (Servicebio, Wuhan, China), precooled at 4 ºC was slowly added along the tube wall, and the structure of organelles was observed by transmission electron microscope (Tecnai Spirit G2, FEI, USA).
2.15 ROS assay
Three days following each cell culture, the nutrient solution was removed, and the cells were washed with PBS using the active oxygen detection kit (Beyotime Biotechnology, Shanghai, China) to detect the contents of intracellular ROS, in accordance with the manufacturer’s instructions to join the appropriate dilution volume of good DCFH-DA, which was added to the positive control well. The cells were then incubated at 37 ºC for 20 min. The cells were washed thrice with a serum-free cell culture solution to remove dichloro-dihydro-fluorescein diacetate that did not enter the cells completely. Finally, inverted fluorescence microscopy (Leica DM IL LED Fluo, Leica Microsystems, Germany) or flow cytometry (USA, Beckmancoulter, USA) was used to detect the fluorescence intensity in each group.
2.16 Lipid peroxidation MDA assay and total antioxidant capacity assay
The MDA (Beyotime Biotechnology, Shanghai, China) and total antioxidant capacity (T-AOC) assay kits (Beyotime Biotechnology, Shanghai, China) were used according to the manufacturer’s instructions. The detection reagents were gradually added, and the lipid oxidation capacity and T-AOC of each group of cells were calculated according to relevant formulas.
2.17 Immunocytofluorescence (ICF) assay
The cell medium in each group was removed, and the cells were washed with PBS thrice. Cold polyformaldehyde (4%; Biosharp, Hefei, China) was fixed for 15 min, washed three times with PBS for 5 min each time, and the bed was shaken. Triton X-100 (0.25%) (Sigma-Aldrich, USA) was used to break the film for 15 min, washed thrice in PBS for 5 min each time, and the bed was shaken. The immune dyeing sealing fluid (Beyotime Biotechnology) was incubated for 60 min. Primary antibody (primary antibody diluent mixture) was added for incubation, and the bed was shaken overnight at 4°C. The primary antibody was collected, washed thrice with PBS for 5 min each time, and the bed was shaken. Secondary antibodies, Alexa Fluor 488 and 594 (Proteintech, Wuhan, China), were incubated at room temperature for 60 min (in the dark). The secondary antibody was recovered, washed three times with PBS, and the bed was shaken for 5 min each time. 4′,6-diamidino-2-phenylindole(DAPI) (Sigma-Aldrich, USA) was incubated for 10 min (in the dark). The bed was washed three times with PBS for 5 min each. Detection was performed using an inverted fluorescence microscope (Leica DM IL LED Fluo; Leica Microsystems, Germany).
2.18 Micro-computed tomography (Micro-CT) assay
After anesthesia, bilateral Achilles tendons were used for micro-CT detection (SkyScan 1176, Bruker, Karlsruhe,Germany) (Y. Chen et al. 2020) to evaluate the calcification of bilateral Achilles tendons in rats. The X-ray tube settings were 60 kV and 134 µA, and images were acquired at 50 µm resolution. A 0.5° rotation step through a 360° angular range with 50 ms exposure per step was used. The images were reconstructed using the Hiscan reconstruct software (Bruker, Karlsruhe, Germany). The Hiscan Analyzer software (Bruker, Karlsruhe, Germany) was used to analyze the ossified area parameters, including bone volume and CT values (Y. Chen et al. 2020; Dong et al. 2021).
2.19 Hematoxylin and eosin (H&E) staining assay
Paraffin sections were dewaxed in water, stained with hematoxylin (Nanjing Yorm Biotechnology, China) for 5 min, and washed with running water to remove excess dye. The sections were stained with eosin (Nanjing Yorm Biotechnology, China) for 1 min and washed with tap water to remove excess dye. The slices were then immersed in anhydrous ethanol for 2 min and xylene for 5 min. Each section was removed, an appropriate amount of neutral gum was added to the section, and the slide was covered with a scanner (VS200, OLYMPUS, Japan) and scanned at 20X objective.
2.20 Masson staining assay
The paraffin sections were dewaxed in water, immersed in Bouin’s solution (Kohypath, Shanghai, China), and fixed overnight at room temperature. The slices were immersed into Masson staining Kit A solution (Nanjing Yorm Biotechnology, China), stained at room temperature for 10 min, washed with tap water, and stained blue. Sections were immersed in Masson staining Kit B solution, stained at room temperature for 8 min and washed with tap water. The sections were divided into Masson staining kit C solution, differentiated at room temperature for 5 min, and excess liquid was drained. The sections were immersed in Masson’s staining kit D solution, stained at room temperature for 3 min, and washed with tap water. Next, the slices were added to 0.2% glacial acetic acid and washed thoroughly. The slices were then immersed in anhydrous ethanol for 2 min and xylene for 5 min. Each section was removed, an appropriate amount of neutral gum was added and the slide was covered with a scanner (VS200, OLYMPUS, Japan) and scanned at 20X objective.
2.21 Immunohistofluorescence (IHF) assay
The paraffin sections were dewaxed in water, and the high-temperature-resistant plastic dye stand containing the sections was placed in a repair box filled with citric acid antigen repair solution (pH 6.0) and heated at medium-high temperatures for 30 min. The solution was cooled naturally to room temperature and soaked in PBS thrice for 5 min each. In each group, 3% of BSA in closed drops (Beyotime Biotechnology) was added for uniform coverage, followed by a 30 min incubation at room temperature. After the BSA sealer was removed, an appropriate amount of the primary antibody working liquid diluted with PBS was added to the tissue, and the sections were placed flat in a wet box and incubated overnight at 4°C. The slices were removed the following day and reheated at room temperature for 30 min. Soak in PBS three times for 5 min each time. An appropriate amount of the secondary antibody was added to the tissue and incubated at room temperature for 1 h. Subsequently, each section was soaked in PBS three times for 5 min each time. The sections were immersed in an autofluorescence quencher A solution and incubated at room temperature for 30 min. Next, each section was soaked in PBS three times for 5 min each time. An appropriate amount of DAPI solution (Sigma-Aldrich, USA) was added to the tissues and incubated at room temperature for 10 min in the dark. Each section was soaked in PBS three times for 5 min each time. An appropriate amount of anti-quenching sealing glue was added to the tissue to cover the glass. Each section was removed, an appropriate amount of neutral gum was added and the slide was covered with a scanner (VS200, OLYMPUS, Japan) and scanned at 20X objective.
2.22 Immunohistochemistry (IHC) assay
The paraffin sections were dewaxed in water, and the high-temperature-resistant plastic dye stand containing the sections was placed in a repair box filled with citric acid antigen repair solution (PH6.0) and heated at medium-high temperatures for 30 min. The solution was cooled naturally to room temperature and soaked in PBS thrice for 5 min each. All other experimental procedures have been described in previous studies (Ni et al. 2013).
2.23 Statistical analysis.
SPSS version 26 (IBM, Chicago, IL, USA) was used for statistical analysis. Graphical representations were generated using GraphPad Prism 8.4 (GraphPad Software, San Diego, CA, USA). Experimental data were described using mean ± standard deviation. All data were tested for normality and equal variance. Intergroup data were analyzed using the t-test or one-way variance analysis. Pearson’s test was used for correlational analysis. Statistical significance was set at P < 0.05.