2.1 Cell culture and treatment
The protocols for obtaining DPSCs were authorized by the Ethics Committee of Xuanwu Hospital, Capital Medical University (Case code 2021/004) and all experiments were conducted in line with the related regulations. DPSCs were isolated and cultured as our previous protocols [15, 16].Briefly the dental pulp tissues were isolated from extracted third molars. First, the tissues above were cleaned and then minced. Furthermore, they were digested and then the cell pellet was resuspended after centrifugation. The cells were cultured with Alpha modified Minimum Essential Medium (α-MEM, Hyclone, USA) containing 10 % fetal bovine serum (FBS, Every Green, China) at 37 ℃ cell incubator containing 5% CO2. Cells from passage 3-5 were used in this experiment to achieve stable results.
DPSCs were exposed to H2O2 with different concentrations (0, 100, 300, 500 uM) for 4 h to observe the survival rate. The cell survival rate (%) = (OD H2O2-OD blank) / (OD control-OD blank) × 100% [17]. In the following experiments in vitro, H2O2-treated DPSCs were established with 300 μM H2O2 to induce apoptosis.
2.2 PBMT irradiation
The emission spectra of light sources (810 nm LED), irradiation parameters and irradiation conditions used in this study were selected according to our previous studies [12]. The cell in irradiated group were received 3.75 J/cm2 daily in the same environment for all in vitro experiments. In vivo studies, the first irradiation was performed after DPSCs transplantation by the transcutaneous punctual contact technique repeated daily for consecutive 14 days. The hand-held delivery probe was placed light on the skin surface above the injured lesion at both ends. The irradiation parameters in vitro and in vivo were shown in Table 1.
2.3 CCK-8 assay
After specific treatment, cells in each group were submitted cell counting kit (CCK-8, Beyotime, China) assay according to manufacturer's instructions. The culture medium was discarded and rinsed thrice with PBS before assay. Then, the CCK-8 regents were added and cultured at 37 °C for 4 h. the OD of each well at 450 nm was detected by a microplate reader (Thermo, USA) at 450 nm.
2.4 TUNEL assay
Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay kit (Beyotime, China) was used to determine apoptosis according to the manufacturer’s protocol. Samples were fixed with 4% formaldehyde for 30 min, then permeabilized with 0.2 % Triton X-100 for another 10 min under room temperature. After washing with PBS twice, samples were incubated with a fluorometric terminal deoxytransferase (TdT) mixture at 37 ℃ for 1 h and then washed twice with PBS. Nuclei were counterstained with 40,6-diamidino-2-phenylindole (DAPI). Fluorescent images were captured by a Nikon Microscope.
2.5 FCM analysis
Flow cytometry (FCM) analysis Kit (Elabscience, China) was used to determine the alteration of apoptosis. Cells in different groups were collected and rinsed. After resuspending with staining buffer, 5 μL Annexin-V FITC wih 5 μL PI solution was added to the tube. Cubes were gently shaken and kept in the dark at room temperature in darkness for 10 min. Finally, 400 μL PBS were added to the tube and samples were detected by flow cytometer (BD, USA).
2.6 SOD and GSH assay
Superoxide dismutase (SOD) and malondialdehyde (MDA) contents were measured using commercial kits (Nanjing Jiancheng, China). SOD activity was investigated by quantifying the levels of formazan dye, a substance that is eliminated by SOD and produced by the reaction between xanthine oxidase (XO) and wst-8. OD values were measured at 450 nm after 30 min of incubation at 37 °C. MDA levels were quantified using the thiobarbituric acid (TBA) method following instructions of the manufacture. The samples and standards were prepared in cuvettes, and the OD value measured at 530 nm. Protein concentrations were determined using BCA protein quantification kit (Beyotime, Chian).
2.7 Western blot
Following the different treatments, we detected the alternation of proteins related to apoptosis through western blot. The cells were lysed with RIPA (Beyotime, China) supplemented with PMSF. After centrifuging, the supernatants were collected and and denatured with SDS-PAGE sample-loading buffer (Beyotime, China). The lysate proteins were separated by SDS-PAGE (Servicebio, China) and then transferred to PVDF membranes (Millipore, USA) following the routine procedures. The membranes were incubated with primary antibodies against Bcl-2, Bax, and Cleaved Caspase 3 (CST, USA). β-actin (Abcam, USA) was used as the internal reference. After incubating overnight, the membranes were labeled with conjugated secondary antibodies (Beyotime, China) for 1 h. The bands were visualized by chemiluminescence detection system (BG-gdsAUTO, China).
2.8 TEM
The cellular microstructures were observed by Transmission Electron Microscope (TEM). Cells in different groups were harvested and fixed with 2.5% glutaraldehyde at room temperature for 30 min and then kept at 4 °C overnight. Subsequently, samples were dehydrated through a series of ethanol and embedded. Ultrathin sections (50-80 μm) were cut and examined with transmission electron microscope (HITACHI, Japan).
2.9 SCI mouse model and Experimental Design
The care and use of animals were approved by Animal Care and Use Committee of Xuanwu hospital, Capital
Medical University (No. XW-20210615-1). The SCI mouse model of adult male C57BL/6 were established according to our previous studies [18]. The spinal cord was exposed at T9-10 and the lamina was removed. The injury was contused in the T9 position. A10-g impact was elevated to a 5-mm height and released freely on the spinal cord and the Dwell time was 0-s. The mice were assigned into three groups: SCI group (established SCI model and received PBS injection in the same way), DPSCs group (established SCI model and received transplantation of DPSCs) and DPSCs+irradiated group (established SCI model, then receiving transplantation of DPSCs and PBMT irradiation).
2.10 DPSCs transfection and transplantation
To track the transplanted DPSCS in vivo, DPSCs were nucleofected with lentivirus vector expressing both luciferase and GFP genes (Hanbio, China) according to manufacturer’s protocols. After infection for 24h, cells were returned to culture in standard medium at 37˚C/5% CO2. The luciferase and GFP genes transfected DPSCs (Luc-GFP-DPSCs) were transplanted into SCI mice in DPSCs and DPSCs+irradiated groups. DPSCs (1×106) were intramedullary transplanted into both sides of injured spinal cord with 5 μl Hamilton syring (Hamilton, Switzerland). Then, the lesion was closed layer by laser.
2.11 BLI analysis
To assess the luciferase activity of transfected DPSCs in vitro, different numbers of Luc-GFP-DPSCs (1, 2, 4, 6, 8, 10 × 104/well) were seeded into 96-well and 0.15 mg/mL D-luciferin solution (YeasenBio, China) was added at room temperature. After 10 min of incubation, the cells were imaged using in vivo imaging system (PerkinElmer, IVISSPE, USA). For real-time tracking of engrafted cells in vivo, mice were anesthetized with pentobarbital sodium (40 mg/kg) and injected intraperitoneally with D-luciferin (150 mg/kg). After 10 min, the mice were placed into the chamber and pictured with bioluminescence imaging (BLI).
2.12 Behavioral and footprint detection
The Basso Mouse Scale (BMS) locomotor rating scores and footprints were used to evaluate the locomotor recovery after SCI. The BMS scores were evaluated by two blinders according to scoring sheet reported in previous report [19]. Mice were tested by two examiners blinded to the treatment groups. Behavioral tests were performed before SCI surgery and 1, 3, 7, 14, 21days after surgery. On the 21th day after SCI injury, footprint detection was observed according to established article [20]. The mice were allowed to move freely on a piece of white paper and their hind legs were marked with black dye. Footprint traces were printed on the paper and digitized by the camera.
2.13 HE and Nissl staining
Tissues of spinal cord in each group were harvest at 14th day after SCI injury. According to our previous study [9], the heart was infused with 30 ml 0.9% normal saline, and then 20 ml 4% PFA, then the spinal cord of T9-T10 was carefully separated. The tissues were embedded with Tissue-Tek O.C.T. compound (Sakura, Tokyo) and sliced with freezing microtome ((Leica, Germany) at −20 ℃. The samples were then stained with Hematoxylin and eosin (H&E, WeiaoBio, China) and Nissl (G.FanBio, China) staining. Images were observed and photographed using microscopy (KEYENE, Japan).
2.14 Immunofluorescence staining
Spinal cord slides were blocked with 5% BSA at room temperature for 30 min, and then incubated with indicated primary antibodies at 4 ℃ overnight, a corresponding fluorescently secondary antibody for 1 h. DAPI was employed to stain the cell nuclei. Photographs were captured using fluorescent microscopy (KEYENE, Japan). Image J software was used to count the number of cells.
2.15 Statistical analysis
All the data were shown as mean±standard deviation (SD). The experimental results were measured via t-test or one-way analysis of variance (ANOVA). All data statistics charts making were obtained by using GraphPad Prism 7.0. P-value < 0.05 was considered statistically significant.