Intervertebral discs specimens
Intervertebral discs (≥ 5 mm) were obtained from 12 patients, including 7 males and 5 females with a combined average age of 52.8 ± 12.8 years (age range of 30-74 years) and diagnosed with lumbar disc herniation (LDH) or degenerative lumbar spondylolisthesis (DLS) (Figure 1A – C; Table 1). The NP were sampled from L4/5 and subjected to histological and immunohistochemical analyses. Intervertebral disc degeneration was evaluated using the Pfirrmann grading system.
Isolation and cell culture of BMSCs
6-week-old male Sprague-Dawley rats (Shanghai Lab, Animal Research Center Co. Ltd, Shanghai, China) were killed by cervical dislocation and soaked in the 75% ethanol for 10 min. Extraction and isolation of BMSCs from two lower legs of SD rats with sterile operation, BMSCs were cultured in Minimum Essential Medium α (MEMα) supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).
Culture of NP and AF cell-lines
The Rat’s NP and AF cell are immortalized cell lines[21] which were kindly gifted by Dr. Chen Di at the Department of Orthopedic Surgery, Rush University Medical Center (Chicago, IL, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).
RNA extraction and real-time quantitative PCR (qPCR) analyses
Total RNA were isolated from tissues and cells using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per manufacturer’s protocol. First strand complementary DNAs (cDNAs) were reversed transcribed from extracted RNAs using the cDNA Synthesis Kit (Takara Bio, Otsu, Japan). Relative mRNA expression was determined by RT-PCR using the GoTaq 1-step RT-qPCR System (Promega, Madison, WI, USA) followed by agarose gel electrophoresis (Bio-Rad Laboratories, Hercules, CA, U.S.A). Real-time qPCR was conducted using the TB Green Premix Ex Taq Kit (Takara Bio) on an Applied Biosystems QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific). Specific primer pairs were designed using NCBI BLAST and sequences provided in Table 2. The gene expression of GAPDH or β-actin was used as internal control. Target gene expression levels were determined using the 2-ΔΔCT method. The mean CT value of target genes in the experimental groups were normalized to the CT value of GAPDH or β-actin to give a ΔCT value. This was then further normalized to control samples to obtain ΔΔCT.
Cell viability analysis
Cell viability following Bleomycin treatment was evaluated using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories Co., Ltd, Kumamoto, Japan). Cells seeded onto 96-well plates at a density of 8×103 cells/well the day before were treated with increasing concentrations of Bleomycin sulfate (1, 5 and 10 μg/ml, dissolved in PBS; Selleck Chemicals, Houston, TX, USA) for 24, 48, 72, and 96 hours. NP, AF and BMSC cells were cultured in DMEM, DMEM/F12 or MEMα respectively, all supplemented with 10% FBS and 1% penicillin/streptomycin (complete DMEM or complete DMEM/F12). Cell media containing Bleomycin were changed every 2 days. At the end of the experimental periods, cells were incubated with fresh complete media containing 10 μl of CCK-8 reagent for 1 hour at 37°C. Complete media containing CCK-8 reagent but no cells and untreated cells were used as a blank and mock controls respectively. The absorbances (measured as optical density; OD) at 450 nm were measured on an Infinite M200 Pro multimode microplate reader (Tecan Life Sciences, Männedorf, Switzerland). ODs of the Bleomycin treated groups were normalized to corresponding blank ODs to account for background interference.
TGFβR1 siRNA knockdown
AF cells seeded onto 6-well plates at a density of 1×105 cells/well the day before were tranfected with small interfering RNA (siRNA) against TGFβR1 (siTGFβR1: Sense 5ʹ- GGAGAUUGUUGGUACCCAAGG-3ʹ; and Anti-sense 5ʹ-UUGGGUACCAACAAUCUCCAU-3ʹ) or with a scrambled siRNA control (NC: Sense 5ʹ-UUCUCCGAACGUGUCACGUTT-3ʹ; and Anti-sense 5ʹ-ACGUGACACGUUCGGAGAATT-3ʹ) (IBSBIO, Shanghai, China) using Lipofectamine 3000 transfection reagent in accordance with manufacturer’s protocol. After 6 hours, media containing transfection reagent and siRNAs were removed and replaced with fresh complete media. Twenty-four hours post-transfection cells were harvested for total protein or total RNA extraction.
Comet analysis (single cell gel electrophoresis)
The method for comet analysis or single cell gel electrophoresis (SCGE) was performed as previously described[22, 23] Briefly, cultured AF cells, NP cells and BMSCs were trypsinized, centrifuged and resuspended in 1×PBS (Ca2+ and Mg2+ free; Gibco, Thermo Fisher Scientific) to final cell density of 1×105 cells/ml. Next, 50 μl of cell suspensions were then mixed with 500 μl low melting point (LMP) agarose (1% at 37°C, Comet SCGE Assay Kit; Enzo Life Sciences, Farmingdale, NY, USA) and then 75 μl of cell-agarose mixture were spread onto glass slides precoated with 1% normal melting point (NMP) agarose and allowed to settle for 10 mins at 4°C in the dark. Slides were then immersed in pre-chilled Lysis Solution (Comet SCGE Assay Kit; Enzo Life Sciences) and incubated on ice for 1 hour. After lysis, the slides were placed on a horizontal gel electrophoresis unit containing electrophoretic Alkaline Solution (Comet SCGE Assay Kit; Enzo Life Sciences) for 60 mins at room temperature in the dark to allow the DNA to unwind. Electrophoresis was then carried out for 10 mins at 25 V and 300 mA (0.73 V/cm). After electrophoresis, slides were rinsed in distilled water, placed in neutralization solution (pH 7.5) to remove alkali and detergent, then dehydrated with 70% ethanol for 5 minutes and air-dried. Immediately before examination, the slides were stained with 100 μl of ethidium bromide (10 μg/ml) for 30 mins at room temperature in the dark. Comets were visualized under 400× magnification using epifluorescence microscopy (Leica DM4000 B; Leica Microsystems, Wetzlar, Germany)
Scratch-wound healing assay
The scratch-wound healing assay was performed to examine the effects of Bleomycin on collective migration and recolonization in a 2-dimensional environment. AF cells with or without TGFβR1 siRNA knockdown and BMSCs were cultured to 100% confluence. Under sterile conditions, a linear scratch line was made straight down the centre of the cell monolayer using the tip of a sterilized 200 μl micropipette tip. Cell media were carefully aspirated to remove cellular debri and floating cells and then replaced with fresh serum-free DMEM/F12 or MEMα without or with Bleomycin (5 or 10 μg/ml) ± TGFβR1 inhibitor (LY364947, 10μM; Selleck, USA). Phase contrast images were captured of the initial scratch wound for reference and designated time 0. Further images were captured at 24 and 48 hours, and the distance between the leading cell edges at each time point was measured using Imagej software (National Institutes of Health, USA).
Millicell transwell migration assay
The effects of Bleomycin on single cell migration through three-dimensional environment were examined using the transwell migration assay (Millicell Standing Cell Culture Inserts, 8 μm pore size; Merck-Millipore, Burlington, MA, USA). Briefly, AF cells with or without TGFβR1 siRNA knockdown and BMSCs were seeded into the upper chamber of the Millicell Standing Cell Culture inserts at a density of 8×104 cells/well in serum-free DMEM/F12 or MEMα media without or with Bleomycin (5 or 10 μg/ml) ± TGFβR1 inhibitor (LY364947, 10μM; Selleck, USA ). The inserts were then placed into 24-well plates filled with complete media (FBS as chemoattractant source) and cultured for 24 hours. At the end of the experiment, media in the inserts were discarded and adherent cells were fixed in 4% paraformaldehyde (PFA) for 30 mins and then stained with 0.2% crystal violet for 5 mins. Cells adhering to the membrane inside the inserts (i.e. cells that have not migrated) were gently removed using a cotton-tipped applicator. Migrated cells on the other side of the inserts were imaged under a light microscope (Leica DM4000 B; Leica Microsystems) and staining intensity analyzed with Image Pro Plus 6.0 software to evaluate the ratio of integrated optical density (expressed as the IOD/area for each sample).
Assessment of apoptosis and cell cycles by flow cytometry
The effects of Bleomycin ware evaluated using flow cytometry following staining with APC-Annexin V and propidium iodide (PI) for cell apoptosis or just PI for cell cycles based on apoptosis staining kit (Thermo Fisher Scientific) and PI/RNase Staining Buffer (BD pharmagen) according to manufacturer’s protocol. Cell suspensions were subjected to flow cytometry on a FACSCalibur Flow Cytometer (BD Biosciences) counting at least 10000 events. The apoptotic rate was quantified based on the percentage of cells in the right upper (Q2; positive staining for APC-Annexin V and PI) and right lower (Q3; positive staining for APC-Annexin V and negative for PI) quadrant of the flow cytometric scatterplot.
Senescence Assays
NP cells and BMSCs were identified using the Senescence β-Galactosidase Staining Kit (Beyotime Biotechnology) according to manufacturer’s protocol. NP cells and BMSCs were seeded onto a 12-well plate at a density of 3*106 cells/well, following with 5 μg/ml bleomycin once or three times, every treat period lasts for three days. Then cells were fixed with Fixative Solution for 15 mins at RT and then incubated with β-Galactosidase Staining buffer at 37°C overnight in a dry incubator without CO2. Then the cell percentage of positive cells were calculated.
Western blot analysis
Total cellular proteins were extracted from cultured cells using RIPA lysis buffer supplemented with phosphatase and protease inhibitors (Roche, Basel, Switzerland). Equal quantities of extracted proteins (20-30 μg) were resolved on 10% or 12.5% SDS-PAGE gel and separated proteins electroblotted onto 0.22 μm PVDF membranes (Merck-Millipore). Membranes were blocked with 5% BSA-PBS at room temperature for 1 hour and then incubated with primary antibodies (diluted 1:1000 in 5% BSA-PBS) overnight (at least 16 hours) at 4°C. Primary antibodies against SMAD2 (Ser308, D43B4; rabbit mAb), phospho-SMAD2 (Ser465/467, 138D4; rabbit mAb), SMAD3 (C67H9; rabbit mAb), phospho-SMAD3 (Ser423/425, C25A9; rabbit mAb), SMAD2/3 (D7G7; rabbit mAb), phosphor-SMAD2/3 (Ser465/467; Ser423/425; rabbit mAb), SMAD4 (D3M6U; rabbit mAb),PARP (#9542; rabbit mAb), Cleaved PARP (D64E10; rabbit mAb) and β-actin (D6A8; rabbit mAb) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against FSP1(S100A4; rabbit mAb), TGFβ Receptor I (ab31013; rabbit mAb), type I collagen (ab6308; rabbit mAb), TGFβ1 (ab64715; rabbit mAb), P21 (ab109199; rabbit mAb) and P53 (ab26; mouse pAb) were obtained from Abcam (Cambridge, UK). Membranes were then washed extensively in Tris-buffered saline-Tween20 (TBST) and subsequently incubated with anti-rabbit IgG (H+L) (DyLight™ 800 4× PEG Conjugate; Cell Signaling Technology) secondary antibody (1:5000 dilution) for 1 hour at room temperature in the dark. Membranes were again extensively washed in TBST and protein immunoreactivity were detected on a LI-COR Odyssey Fluorescence Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Semi-quantitative analysis of protein immunoreactive band intensity was measured using Image-Pro Plus 6.0 software and normalized to the internal loading control β-actin.
Animals and surgical procedures
All animal experimentation was approved by the Institutional Animal Care and Ethics Committee of Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine (Shanghai, China) and performed in accordance with the principles and procedures of the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and the Guidelines for Animal Treatment of Shanghai Jiaotong University. Six 8-week-old male Sprague-Dawley rats (Shanghai Lab, Animal Research Center Co. Ltd, Shanghai, China) were housed under pathogen-free conditions at 26-28°C and 50-65% humidity with 12-hour day/night cycle. Animals were fed standard rodent chow and had access to fresh water ad libitum. Before surgical procedures, rats were anesthetized by intraperitoneal injections of pentobarbital sodium (5 mg/100 g of body weight). The tails were sterilized with iodinated polyvinylpyrrolidone and then a ventral longitudinal skin incision was made over the tail to reveal the intervertebral disc at coccyx vertebrae 6-10. The intervertebral discs at Co6/7 were used as Sham controls and the intervertebral discs at Co7/8, Co8/9 and Co9/10 were used as experimental groups. Intervertebral discs were punctured with a 20-gauge sterile needle oriented perpendicular to the skin to make ensure insertion at the center of the disc level through the AF into the NP. The incision was then sutured and rats were allowed post-operative recovery for two weeks. A group of mice (n = 3) were sacrificed and the tails extracted, cleaned of soft tissues and the vertebral column fixed in 4% PFA. To the remaining rats (n = 3), surgical exposure of the intervertebral discs at Co8/9 and Co9/10 was again carried out, and 5μl of Bleomycin at concentrations of 10 and 5 μg/ml was injected respectively into each disc. Incision was sutured and rats were allowed 2 and 4 weeks of post-treatment recovery. At the end of the experimental period, all remaining rats were sacrificed and the tails extracted, cleaned of soft tissues and the vertebral column fixed in 4% PFA.
Histology and immunofluorescence staining
Fixed intervertebral disc tissue samples were embedded into paraffin blocks then subjected to histological sectioning (5 μm thickness). For histological assessment, paraffin tissue sections were processed for Safranin O-Fast Green and Sirius Red staining in accordance with standard laboratory protocols. For immunofluorescence assessment, BMSCs were cultured in a slide with a confluence of 10% and fixed with 4%PFA, then these cell slides are with tissue sections to be de-paraffinized in graded xylene, rehydrated in graded alcohol solutions and then incubated in antigen retrieval buffer (Roche) at 37°C for 30 mins. After cooling to room temperature, slides were immersed in PBS (pH 7.4) and washed 3 times for 5 mins each. Auto-fluorescence quencher was added to the sections for 5 mins, and then blocked with blocking buffer for 30 mins at room temperature. Sections were subsquently incubated with primary antibodies in a wet box at 4°C overnight. Primary antibodies were used at 1:100 dilution and included anti-Col1a1, anti-Col2a1, anti-FSP1, anti-TGFβ, anti-TGFβR1, S100A9 (all purchased from Cell Signaling Technology) and anti-Keratin 18(abs130128, absin). The next day, sections were washed with PBS and then incubated with Alexa Fluor 594 Conjugate secondary antibody (anti-rabbit, 1:500; Cell Signaling Technology) for 50 mins at room temperature in the dark. Sections were washed with PBS and then incubated with DAPI solution (Sigma-Aldrich, St Louis, MO, USA) for 10 mins in the dark to stain cell nuclei. Sections were subjected to final PBS washes, air-dried and then sealed with anti-fluorescence quenching tablets. Digital fluorescence images were captured under a Leica DM4000 B epifluorescence microscope (Leica Microsystems) and IOD measurements carried out using Image Pro Plus 6.0 software.
Radiographic and magnetic resonance imaging (MRI) analysis
Digital X-ray imaging of the punctured intervertebral discs were conducted in the anteroposterior axis with a 21 lp/mm detector that provides up to 5× geometric magnification (Faxitron VersaVision; Faxitron Bioptics LLC, Tucson, AZ. USA). MRI imaging of the same punctured intervertebral discs were carried out on a Siemens Magnetom Prisma E11 (Siemens Healthineers, Erlangen, Germany) with the following parameters: TR 3000 ms, TE 80 ms, 1.1 mm thickness, 0.22 mm interval, FOV 160×65 mm, and voxel size 0.25×0.25×1.1 mm.
Atomic Force Microscopy (AFM)
For AFM, the extracted punctured vertebrae were dissected to make paraffin section and nanoindentation was performed on a Park NX20 (Park Systems, South Korea) equipped with microspherical colloidal tips (R < 10 nm, nominal k ≈ 0.2 N/m, Tip:Si/Tipless/Top, cantilever Si/AI/Top; Park Systems). For a wide range of undulating surfaces, the scanning rate of 13 Hz was used. Large scanning rate can reduce drift, but it is generally only used for scanning small flat surfaces. Indentation was applied at a z-piezo displacement rate of 10 μm/s to a maximum load of ~120 nN using a Scan Asyst-Air probe, a curvature radius of 5 nm, and a force constant of 0.4 N/m. Young's modulus, adhesion force, deformation parameters were evaluated.
Statistical analysis
Three independent experiments or repeated measurements were conducted for all data. Data are presented as the mean ± standard deviation (S.D.). Significance differences between study groups were obtained by Student’s t-test or one-way analysis of variance (ANOVA) using SPSS 19.0 software (IBM Corporation, Armonk, NY, USA). Statistical significance was set at a If the p-value < 0.05 unless otherwise indicated.