Animal model
The animal experiment was grouped into three cohorts, sham group(Sham), Achilles tendon puncture group (Tendon puncture group, TPG), 30% total body surface area (TBSA) partial thickness burn injury combined Achilles tendon puncture group (HOG), respectively. Partial thickness scald injury was performed as Peterson JR et al. described[17]. In brief, 8 weeks, male, C57BL/6 mice in sham group, the Achilles tendons were exposed alone. In TPG, after Achilles tendon exposed, a 27G needle was punctured into the Achilles tendon body perpendicularly at different parts from proximal to distal and this process was repeated three times. In HOG, apart from the Achilles tendon punctured, mice also received about 30% TBSA burn injury with exposure to 60℃ for 18 s (Fig. 1a) [18].
Cell harvest and in vitro culture assays
Achilles tendon stem cells (SCs) were harvested from the different groups. Stem cells (SCs) from Achilles tendon were isolated based on the research Xu et al. reported [8], and modified slightly. In short, freshly harvested tendon was washed with sterile phosphate-buffered saline (PBS; Gibco, New York, USA), and then tendon tissue was placed into 30mm dish. The tissue was cut into fragments and digested in 3 mg/ml type I collagenase (Sigma-Aldrich, St. Louis, USA) for 45 minutes. Then, the digested fragments were cultivated in 35 mm dish (Corning, New York, USA) in α-minimum essential medium (α-MEM, Gibco) containing 100 U/mL penicillin (Gibco), 100 µg/mL streptomycin (Gibco), and 10% fetal bovine serum (FBS; Gibco, Australian). In in vitro experiment, the complete culture medium was changed every 3 days. Cell passage was performed until the cell fusion rate reached about 80~90%. SCs at passage 2-5(P2-P5) were used for further experiment. For osteogenic differentiation, complete osteo-inductive medium was replaced every 2 days when the percentage of cell fusion dipped to 60–70%. Osteo-inductive medium contained α-MEM and 10% FBS supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM β-glycerophosphate, 50 µg/mL L-ascorbic-2-phosphate and 10 nM dexamethasone (all from Cyagen). TNF-α(10 ng/ml) and IL-1β ༈5 ng/ml༉ was added to the medium of SCs to simulate inflammation environment in vitro[19].
Micro CT
Micro CT imaging of HO of Achilles tendon was obtained from all animals by means of the high-resolution micro-CT equipment (Siemens, Munich, Germany). CT scan settings: 80 kV, 500µA, and 1100-ms exposure. The bone mineral in the soft tissues was quantified for HO bone volume using standard protocols as Peterson et al. described[20].
Immunofluorescent Staining
MSCs were seeded into a 96-well culture plate designed for confocal microscopy and cultivated in α-MEM containing 10% FBS. When the confluence of cells reached 80~90% (day 0), different condition was added respectively for intervention. After 7d culture or 14d osteogenesis inducing, adherent cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 for 15 min and blocked with 10% goat serum for 40 min at room temperature. The cells were then incubated at 4°C with an antibody against P2X7R (1:200, Abcam, ab48871) overnight. The samples were washed with PBST, and then incubated in the dark place at 25℃ with Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (1:500; Abcam, ab150077) for 2 h. Finally, the cells were stained in a dark room at 25℃ with DAPI for 30 min. Images were captured by a confocal laser scanning microscopy (A1R S1; Nikon, Tokyo, Japan).
ALP staining
ALP activity in SCs was analyzed with ALP staining. In brief, cells were seeded at 1⊆104 cells/well in 96-well plates. ALP staining was performed after 14 days of osteo-inductive culture using an ALP staining kit (Beyotime Biotechnology, China) according to the instructions. The quantitative analysis of ALP staining was performed with the help of an ALP assay kit (Nanjing Jiancheng Bioengineering Institute, China).
ARS staining
According to the manual instructions, Alizarin red staining (ARS, Cyagen, Suzhou, China) was completed which was used to evaluate the osteogenic differentiation of cells. To quantify the staining for statistical analysis, the mineralized nodes were dissolved in 2% cetylpyridinium chloride for 20 min, and then OD values were measured at 560 nm wavelength[21].
qRT-PCR assays
Total RNA was extracted using RNAeasy Kit (Vazyme, RC112-01) and 1 µg of total RNA was used to generate cDNA using HiScript II Q RT SuperMix (Vazyme, R223-01) for qPCR. Quantitative Real time PCR (qRT-PCR) was performed on cDNA samples diluted twentyfold in double-distilled water using SYBR Green Master Mix (Vazyme, Q711). The CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) was used. To amplify them under the following conditions: denaturation at 95°C for 5 min; 40 cycles at 95°C for 10 s, 55°C for 20 s, and 72°C for 20 s, with melt curve analysis from 65 to 95°C in increments of 0.5°C. The gene-specific qRT-PCR primers were specific for mouse. Relative mRNA gene expression levels were measured by qRT-PCR. Relative quantification for qRT-PCR was calculated using the 2 − ΔΔCT method. The sequences of primers used in the present study are shown in supplementary Table. 1.
Statistical Analysis
All results are calculated by the mean and standard deviation (mean ± S.D.). There are more than three samples. Experiments for each cell were performed independently for three times. GraphPad Prism 9 software was utilized for statistical tests. The Student's t-test was used for analysis of two unpaired groups, and one-way ANOVA followed by Tukey’s posttest for analysis of multiple groups. Statistical significance was established at p < 0.05.
Ethics statement
The ethics committee of the general hospital of People Liberate Army of China and Harbin Medical University approved this experiment. All methods were performed in accordance with the relevant guidelines and regulations from the same.