TDSC isolation and cell culture
Achilles tendons (AT) were harvested from one of the hindlimbs of Sprague–Dawley (SD) rats, anesthetized with 0.5% pentobarbital sodium (0.3 mL/100 g, Sigma-Aldrich) by intraperitoneal administration. Pieces of the ATs 0.5 × 1.0 cm2 were isolated, cut into 0.25 cm3 pieces and digested with type I collagenase (2 mg/mL; Sigma-Aldrich) in DMEM supplemented with 2% fetal bovine serum for 16 h at 37°C and collected using a 70-mm cell strainer. PBS was used to wash the cells by centrifugation at 800 × g for 5 min, then reseed the cells at 1 × 104 cells/cm2 in monolayer cultures in complete high glucose DMEM (HG-DMEM) supplemented with 20% fetal bovine serum, 100 mg/mL streptomycin, and 100 U/mL penicillin. After 24 h of initial culture, transfer the nonadherent cells to fresh culture flasks to exclude the rapidly-adherent, fibroblast-like cells. At day 7, TDSCs were trypsinized and mixed together as passage 0, after which they were passaged four or five times before use for experiments.
Flow cytometry assay
TDSCs at passage 4 were harvested by digestion with 0.25% trypsin/ ethylenediaminetetraacetic acid (EDTA) as described above. 1 mg of fluorescein isothiocyanate (FITC) fluorescent dyes conjugated with anti-rat monoclonal antibodies (CD44, CD45, and CD90) was used to incubate around 1 × 106 cells separated by centrifugation, after that 4% paraformaldehyde was used to fix the cells at room temperature for 20 minutes. Then 200 µL PBS (pH = 7.4) was used to wash the cells, after that, cells were removed from the fixing solution, and resuspended in PBS for flow cytometry. FC 500 Flow Cytometry Analyzer (Beckman Coulter, Brea, USA) was used to perform the analysis of the phenotypic results.
Laser Intervention Method
A 532 nm Nd:YAG laser (Oculight; Iridex, Mountain View, USA) with a diameter of 5 mm was used. During the irradiation, the distance between the laser emitter and the cell layer was kept at 6 cm for all cell groups. Sterile foil was used to create an enclosed space to prevent interference caused by light dispersion and refraction. After irradiation, continue to incubate the cells, and the culture medium was replaced every 1–2 days.
Cell Titer-glo (Ctg) Assay
96-well plates were used to seed the stem cells at 1 × 104 cells/well, and then the cells were assigned to one of seven energy density laser groups (0 J/cm2, 1.5 J/cm2, 3 J/cm2, 6 J/cm2, 9 J/cm2, 15 J/cm2 and 24 J/cm2). The irradiation times for these seven groups were: 0 s, 30 s, 60 s, 2 min, 3 min, 5 min, and 8 min, respectively. The CTG assay was performed at 24 and 48 h after a single exposure.
Crystal Violet Assay
Cells were seeded into 96-well plates at 1 × 104 cells/well and cultured at 37°C in a constant temperature incubator. After 6, 12, or 24 h, remove the culture medium, and 4% paraformaldehyde (PFA) solution was used to fix cells for 15 min at room temperature (RT), after that, cells were washed with PBS, and then stain each well with 100 µL of 0.2% Crystal Violet solution in PBS for 15 min at RT. Deionized water was used to wash the dye solution from the plates, and the dye was then solubilized in 100 µL of 1% sodium dodecyl sulfate (SDS) solution in PBS. A microplate reader was used to measure the optical density (OD) at the emission wavelength of 570 nm to compare the proliferation of the two groups of cells.
Quantitative Real-time Reverse Transcription-polymerase Chain Reaction (Rt-pcr)
The differentiation-related gene expression after 24 and 48 h of 532 nm laser single irradiation were analyzed by RT-PCR. An RNA purification kit (Corning, Corning, NY, USA) was used to extract total RNA. Reverse transcription (RT) step was conducted using a Prime Script RT Reagent Kit (Takara Bio Inc., Shiga, Japan) to reverse transcribe RNA into cDNA. Real-time PCR was quantified using SYBR Premix Ex Taq (Takara Bio Inc.) in a Light Cycler® 96 System (Roche, Basel, Switzerland). GAPDH was set as an internal reference. The specific primers for differentiation-related genes (Invitrogen, Carlsbad, CA, USA) used in this study are listed in Table 1. The result was expressed as the relative expression ratio of the target sample and the control group for each sample, calculated using the 2−ΔΔCT method.
Lysis Buffer (RIPA, Beyotime, Shanghai, China) was used to collect total protein from cultured cells, then the proteins were supplemented with 1% protease inhibitor (Roche Applied Science), and a total of 40 µg protein were loaded for electrophoresis. Then a 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) was prepared to separate the proteins. After that, the proteins were transferred to a nitrocellulose membrane (Merck Millipore). Membranes were blocked for 1 h with 5% nonfat milk at RT, and blots were probed with the following antibodies: anti-Nr4a1 (1:1,000, ab13851, Abcam, Cambridge, UK), anti-Scx (1:1,000, ab185940, Abcam), and anti-tenomodulin (1:1,000, ab203676, Abcam) at 4°C overnight. The membrane was incubated with anti-rabbit IgG (1:1,000, ab205718, Abcam) for 1h at RT after washing in Tris-buffered saline containing Tween.
Gene Chip Microarray Assay And Analysis
After 48h of 532 nm single laser irradiation, cells of 532 nm laser group and control unirradiated cells were dissolved in Trizol (Life Technologies, USA) to obtain total RNA isolation. The EXON Gene Chip instrument (Affymetrix, Thermo Fisher Scientific) was used to quantified RNA concentration, and an Agilent Bioanalyzer 2100 (Agilent Technologies, USA) was used to assess RNA integrity. Raw data were extracted by the Gene Chip Command Console software version 4.0 (Affymetrix) according to the manufacturer’s instructions. The Gene software version 13.1 (Agilent Technologies) was used to perform Basic analysis. Expression Console software version 1.3.1 (Affymetrix) was used to perform RNA normalization. Ultimately, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene ontology (GO) analysis were applied to clarify the molecular functions of up-regulated genes and relevant pathways.
Tendon Injury Model
Thirty-six SD rats (male, 6-weeks-old) were obtained from the Laboratory Animal Services Centre. All the animals were kept in an environment with a temperature of 25 ± 1ºC, a relative humidity of 65 ± 5%, and a light/dark cycle of 12/12 hr. Animals were given water and sterilized food ad libitum. All animal studies (including the rat euthanasia procedure) were carried out in compliance with the regulations and guidelines of Shanghai Jiao Tong University institutional animal care, the AAALAC and the IACUC.
Thirty-six rats were divided into two groups: a control group and a 532 nm laser group. For the tendon injury model, we adopted a rat model of Achilles-tendon injury following a previously-reported method . The Achilles tendon was separated from the plantaris and soleus tendons and injury were surgically induced by semi-cutting, followed by primary suture repair. For rats in the laser group, the tendon was irradiated with a 532 nm laser and then sutured, while for those in the control group the tendons were directly sutured. Tendon tissue samples were collected 7 days after modeling.
Hematoxylin And Eosin (H&e) Staining
4% paraformaldehyde was prepared to fix Pieces of tendons, then the samples were dehydrated with 30% sucrose and embedded in OCT (Sakura Finetek, Torrence, USA). Before section, the samples were frozen at -80°C, then cut cryosections at 5 µm thickness. After that, H&E (Sakura Finetek) was used to stain the samples. An upright microscope (Olympus, Tokyo, Japan) was used to image the samples.
The specimens of the tendon injury model were divided into two groups for histological tests. The tissues were fixed in 4% paraformaldehyde solution, dehydrated with a series of concentration gradient ethanol, and then embedded in paraffin. Sections were stained as 5 mm-thick sections with Anti-Nr4a1 (1:1,000, ab13851, Abcam). After that, 5% bovine serum albumin was used to block nonspecific reactive sites, then incubate the slides overnight at 4°C with anti-rat targeted antibodies at 1:200 to 1:500 dilutions and then conjugated with goat anti-rabbit IgG (1:1500; Santa Cruz Biotechnology, USA) secondary antibody in the dark at RT for 1 h. Then, the sections were stained with 3,3′-diaminobenzidine and finally counterstained with hematoxylin.
pCDNA3.1 (+) was used as cloning vector of the Nr4a1 coding sequence. The primers were as follows:
TDSCs (5 × 103 cells/well) were plated into 6-well plates and transfected with either pCDNA3.1(+)-Nr4a1 or blank pCDNA3.1 (+) vector. Lipofectamine 2000 reagent (Invitrogen) was used to perform the transfection according to the manufacturer's instructions. After 6–8 h, TDSCs were washed and cultured for 24 h in complete medium.
All experiments were carried out at least three times and data were expressed as means ± standard deviation (SD). SPSS 13.0 (IBM, Armonk, NY, USA) was used to analyze statistics. Differences among more than two experimental groups were evaluated by one-way ANOVA. P ≤ 0.05 was considered statistically significant. Image J and GraphPad Prism software were used for plotting calculations.