Collagenase-induced tendon injury model
This study was approved by the Animal Research Ethics Committee of the authors’ institution (Ref no: 10/010/GRF). Twelve male Sprague Dawley rats, (6 weeks, weight 150-220 grams) were used. The procedures have been well-established and the histopathological changes were highly reproducible [
19]. After anesthesia with 2.5% pentobarbital (4.5 mg/kg body weight), hairs over the lower limb were shaved. The patellar tendon was located by positioning the knee at 90°. Twenty microliters (0.015 mg/μl in 0.9% saline, i.e. 0.3 mg) of bacterial collagenase I (Sigma-Aldrich, St Louis, MO, USA) (CI group) (n = 6) or saline (HT group) (n = 6) were injected into both patellar tendons (i.e. both tendons were injected with collagenase or both tendons were injected with saline) of each rat intratendinously with a 30 G needle. Free cage activity was allowed after injection. At week 2 after injection, rats were sacrificed and both patellar tendons of each rat were harvested and pooled together for the isolation of TDSCs (HT) or TDSCs (CI) [
19]. Week 2 was chosen for the isolation of TDSCs (CI) when the direct effect of collagenase subsided and tendon healing with increase in cell proliferation occurred while no chondrocyte-like cells were observed at this time point [
19].
Isolation and culture of rat TDSCs
The procedures for the isolation of TDSCs from the mid-substance of collagenase-injured and healthy patellar tendons have been established [
20]. Briefly, after euthanasia, the mid-substances of both patellar tendons were excised. Care was taken that only the mid-substance of patellar tendon tissue, but not the tissue at the tendon-bone junction, was collected. The peritenon was carefully removed and the tissue was stored in sterile phosphate-buffered saline (PBS). The tissue was minced, digested with type I collagenase (3 mg/ml; Sigma-Aldrich, St Louis, MO, USA) and passed through a 70 μm cell strainer (Becton Dickinson, Franklin Lakes, USA) to yield a single-cell suspension. The released cells were washed in PBS and resuspended in low glucose Dulbecco’s Modified Eagle Medium (LG-DMEM) (Gibco BRL; Life Technologies, Invitrogen, Carlsbad, CA, USA), 10% fetal bovine serum (FBS), 50 μg/ml penicillin, 50 μg/ml streptomycin 100 μg/ml neomycin (complete culture medium) (all from Invitrogen corporation, Carlsbad, USA). The isolated nucleated cells were plated at an optimal low density (50 cells/cm
2) for the isolation of TDSCs from rat patellar tendon and cultured at 37°C, 5% CO
2 to form colonies. At day 2 after initial plating, the cells were washed twice with PBS to remove the non-adherent cells. At day 7-10, they were trypsinized and mixed together as passage 0 (P0). TDSCs were subcultured when they reached 80-90% confluence. The stem cell-related surface marker expression (including CD44, CD90 and CD73), clonogenicity and multi-lineage differentiation potential of the isolated nucleated cells from the CI animal model and healthy animals were confirmed as described previously before being used for the experiments in this study [
18]. TDSCs (CI) and TDSCs (HT) at passage 5 were used for all the experiments.
Study design
TDSCs isolated from both sources were plated at 4000 cell/cm
2 in 100-mm tissue culture dish and cultured in low glucose Dulbecco’s Modified Eagle Medium (LG-DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 50 μg/ml penicillin, 50 μg/ml streptomycin and 100 μg/ml neomycin (complete culture medium) (all from Invitrogen corporation, Carlsbad, USA) at 37°C, 5% CO
2 until confluence. The cells were then subjected to mRNA and protein analysis of expression of BMPs (BMP-2, BMP-4, BMP-7) and BMP receptors (BMPR-IA, BMPR-IB, BMPR-II) using qRT-PCR and Western blotting (WB), respectively. To investigate the response of both cell types to BMP-2 stimulation, TDSCs (CI) and TDSCs (HT) isolated from each of three rats were plated at 4000 cell/cm
2 in 6-wells plate and cultured in complete medium until the cells reached 80% confluence for immunocytochemical staining (ICC) and confluence for WB. They were then treated with rhBMP-2 (100 ng/ml) (Wyeth, Cambridge, MA, USA) in complete medium for 0, 15, 30 and 60 minutes at 37°C, 5% CO
2. Time series data of TDSC (CI) and TDSC (HI) of each of 4 rats / group was obtained. The nuclear translocation of the phosphorylated form of Smad 1/5/8 (pSmad 1/5/8) was examined by ICC while the expression of the pSmad 1/5/8 and total Smad 1/5/8 was examined by WB. BMP-2 at 100 ng/ml was used in this study based on our previous study testing the effect of different concentrations of BMP-2 (0, 50, 100, 250, 500 and 1000 ng/ml) and 100 ng/ml was the lowest dose that induced the osteogenic differentiation of TDSCs (unpublished results). BMP-2 at 100 ng/ml promoted non-tenogenic (osteo-, chondro- and adiop- genic) differentiation, increased proteoglycan production but inhibited tendon-related marker expression in TDSCs [
13]. This same dose (100 ng/ml) was also used in a previous study investigating the effect of BMP-2 on the osteogenic response as well as the expression and translocation of pSmad 1/5/8 in tendon stem / progenitor cells [
21].
Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR)
Cells were harvested and homogenized for RNA extraction with Rneasy mini kit (Qiagen, Germany). The mRNA was reverse-transcribed to cDNA by the First Strand cDNA kit (Promega, Madison, WI, USA). 5 μl of total cDNA of each sample was amplified in final volume of 25 μl of reaction mixture containing Platinum® SYBR® Green qPCR SuperMix-UDG ready-to-use reaction cocktail and specific primers for
Bmp2,
Bmp4,
Bmp7,
Bmpr1a,
Bmpr1b,
Bmpr2 or
β-actin using the ABI StepOne Plus system (all from Applied Biosystems, CA, USA) (Table
1). Cycling conditions were: denaturation at 95°C for 10 minutes, 45 cycles at 95°C for 20 seconds, optimal annealing temperature (Table
1) for 20 seconds, 72°C for 30 seconds and finally at 60-95°C with a heating rate of 0.1°C/second. The expression of target gene was normalized to that of
β-actin gene. Relative gene expression was calculated with the 2
-△CT formula. The mRNA expression of BMPs and BMP receptors were the results of 6 rats from each group.
Table 1
The primer sequence, product size and annealing temperature of target genes for qRT-PCR
β-actin
| 5′-ATCGTGGGCCGCCCTAGGCA-3′ (forward) | 243 | 52 | NM_031144 |
5′-TGGCCTTAGGGTTCAGAGGGG-3′ (reverse) |
Bmp2
| 5′-TAGTGACTTTTGGCCACGACG-3′ (forward) | 81 | 58 | NM_017178 |
5′-GCTTCCGCTGTTTGTGTTTG-3′ (reverse) |
Bmp4
| 5′-CATGGCTCGCGCCTCCTAGC-3′ (forward) | 184 | 58 | NM_012827 |
5′- ATTCCGAGCGACGCACTGCC-3′ (reverse) |
Bmp7
| 5′- CAACCTAGTGGAGCACGACAAGGA-3′ (reverse) | 213 | 60 | NM_001191856 |
5′- AGGTCGGACTCCCTGCCTGAGT-3′ (reverse) |
Bmpr1a
| 5′-GCCACCCTGGACACCAGAGC-3′ (forward) | 101 | 60 | NM_030849 |
5′-GCAGGCTTGCCTTGCGTG-3′ (reverse) |
Bmpr1b
| 5′-CACCACGGAGGAAGCCAGC-3′ (forward) | 239 | 60 | NM_001024259 |
5′-GGCACAGGCCGCTGACAGAC-3′ (reverse) |
Bmpr2
| 5′-GGTGCTGGTCTCACATTG-3′ (forward) | 180 | 58 | NM_080407 |
5′-GAGGCGGACTGAGTGGTG-3′ (reverse) |
Western blotting (WB)
The cells were lysed and the concentration of total soluble protein was measured by BCA protein assay (Thermo Scientific, Rockford, IL). 50 μg (for BMPs and BMP receptors) or 20 μg (for pSmad 1/5/8 and total Smad 1/5/8) of protein was denatured, fractionated by electrophoresis on 12% (w/v) sodium dodecyl sulfate (SDS)-polyacrylamide gel and then electrophoretically transferred onto nitrocellulose membrane (Pall, Ann Arbor, MI). The membrane was blocked with 5% (w/v) non-fat dry milk in TBST solution (25 mM Trizma base (3.025 g), 125 mM NaCl (7.3 g), and 1 mL Tween-20, pH 7.6) and then incubated with primary antibody against BMP-2 (1:1000), BMP-4 (1:1000), BMP-7 (1:1000) (all from Abcam, Cambridge, UK), BMPR-IA (1: 500), BMPR-IB (1:200) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), BMPR-II (1: 500; BD BioSciences, San Jose, California, USA), total Smad 1/5/8 (1:1000; Abcam, Cambridge, UK), pSmad 1/5/8 (1:1000; Cell Signaling, Danvers, MA, USA) or β-actin antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-goat secondary antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), HRP-conjugated anti-mouse secondary antibody (1:1000) or HRP-conjugated anti-rabbit secondary antibody (1:1000) (both from Merck Millipore, Darmstadt, Germany). Immunoreactive bands were detected by ECL reagents (Amersham Bioscience, Little Chalfont, UK). The same batch of samples was used for studying the expression of BMPs and BMPRs. Except for BMP-2, all the proteins used for the detection of BMPs and BMP receptors were loaded and ran within one week. Semi-quantitative image analyses of the protein expression of BMPs, BMP receptors, total Smad 1/5/8 and pSmad 1/5/8 were performed using the Adobe Photoshop software (Adobe Systems Incorporated, CA, USA, version 10.0). After thresholding, the region of interest (ROI) was selected by applying a rectangular box with size that was wide enough to cover the largest band among protein samples and with minimum height possible. The mean expression level of the target protein relative to β-actin was presented. As the expression of BMP-2 was studied at a different time compared to other BMPs and BMP receptors, different β-actin bands were used for normalization. Different β-actin bands of each independent experiment were used for normalization for the expression of total Smad 1/5/8 and pSmad 1/5/8. The Western analyses were the results of 3-4 rats from each group.
Immunocytochemical staining (ICC)
ICC was performed using UltraVision Quanto Detection System (Thermo Scientific, Kalamazoo, MI, USA). Briefly, the cells were fixed in 70% ethanol, quenched with 3% H2O2 in methanol for 20 minutes, blocked with Ultra V Block and incubated with rabbit anti-human pSmad 1/5/8 cross-reacted with rat pSmad 1/5/8 (1:200, Cell Signaling, Danvers, MA, USA) overnight at 4°C. Primary antibody was replaced with blocking solution in the negative controls. After that, the cells were incubated with Primary Antibody Amplifier Quanto and then HRP Polymer Quanto for 10 minutes each at room temperature. Signal was visualized with DAB Quanto Chromogen/Substrate complex. The cells were rinsed in distilled water, counterstained in Harris hematoxylin, dehydrated through graded alcohol, and mounted with p-xylene-bis-pyridinium bromide (DPX) (Sigma Aldrich, St Louis, MO, USA). The cells were examined under light microscopy (DMRXA2, Leica Microsystems, Wetzlar GmbH, Germany). Representative results from 3 experiments were reported.
Data analysis
Quantitative and semi-quantitative data was shown in boxplots or time curve with mean and standard error (SE). For the boxplot, the lower, middle and upper boundaries of the box showed the 25th, 50th and 75th percentile of the dataset. Observation with value that was more than 3 box-length from the upper or lower edge of the box was shown as extreme value (*) if existed. Observation with value that was between 1.5 to 3 box-length from the upper or lower edge of the box was shown as outlier (o) if existed. The largest and smallest observations in the dataset that were not outliers or extreme values were shown as whiskers. If there were no outliers and extreme values, the whiskers represented the maximum and the minimum observations of the dataset. The comparison of mRNA and protein expression of BMPs and BMP receptors between the TDSCs (CI) group and the TDSCs (HT) group was done using Mann-Whitney U test. The comparison of the time series data of total Smad 1/5/8 and pSamd 1/5/8 between the TDSCs (CI) group and the TDSCs (HT) group was done using ANOVA fore repeated measures with time as the within-subjects factor and treatment group as the between-subjects factor. All the data analysis was done using SPSS (SPSS Inc, Chicago, IL, version 16.0). p < 0.050 was regarded as statistically significant.