Study design
This study used HRFs. This experiment used a control group and a study group composed of four subgroups, each treated with a different concentration of sodium alendronate (0.1, 1, 10, and 100 μM Ald) (Sigma, St. Louis, MO, USA). The concentrations were set by referring to several previously published studies which had used concentrations of alendronate [18,19,20]. Each study subgroup was exposed to its specific concentration of alendronate for 48 h, except during the measuring of the metabolism of 3-(4, 5-dimethyldiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), which was performed at varying times of 1 day to 6 days. These experimental groups were evaluated for cell viability, cell cycle and cell proliferation, type of cell death, caspase activity, and wound-healing ability. The schematic description of the study’s design, the number of samples, and the repetition times for each test are illustrated in Fig. 1.
Cell culture
The human supraspinatus tendon tissues were collected from patients undergoing arthroscopic rotator cuff repair. The harvested tendon tissues were obtained from three patients (2 males and 1 female, ages 50, 56, and 55 years) from 2010 to 2011. The rotator cuff tears that were arthroscopically repaired were one small bursal-side partial-thickness tear and two full-thickness tears of medium and large sizes. Briefly, the tissues were washed twice with PBS (Lonza, Walkersville, MD, USA), minced into small pieces with a sterile scalpel, and placed on a 6-well tissue culture plate (Corning, Corning, NY, USA) in DMEM (Lonza) supplemented with 20% FBS (Gibco, Grand Island, NY, USA) and 1% Antibiotic-Antimycotic (Gibco) in a humidified 5% CO2 atmosphere at 37 °C. After two weeks, the cells had reached 90% confluence. The cells were then trypsinized (0.02% trypsin, 0.02% EDTA in PBS) for 5 min, centrifuged at 330 g for 3 min, and expanded in a second passage. The cells were then harvested with trypsin/EDTA and cryopreserved. For this experiment, third-passage cells were used.
Cell viability analyses
Cell viability was estimated by measuring the MTT (Sigma). HRFs (2 × 104) were seeded in each well of a 24-well plate. The cells were maintained in an incubator at 5% CO2, 37 °C for 24 h. The cells were treated with alendronate (0 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM) for 1, 2, 3, 4, 5, and 6 days. A 500 μL MTT solution (0.5 mg/mL in free media) was briefly added to each well of the 24-well plate. Then, the plate was incubated for 2 h. Afterwards, the cell supernatant was removed and the solution of 200 μL DMSO (Merck, Darmstadt, Germany) was added to each well of the plate. Absorbance of the plate was measured at 570 nm, using a microplate reader. Cell viability was expressed as a percentage of live cells, compared with the control, which was set at 100%.
Cell viability analysis using the crystal violet assay was performed as follows. HRFs (1 × 105) were seeded in each well of a 6-well plate. The cells were maintained in an incubator at 5% CO2, 37 °C for 24 h. After that, the cells were treated with alendronate (0 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM) for 48 h. The cells were stained using 0.1% crystal violet solution (Sigma) for 2 h at room temperature, and then were washed with PBS. Then, the stained cells in the 6-well plate were analyzed using a scanner (PowerLook 2100XL, Umax, Dallas, TX, USA).
Cell viability was also assessed using the LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen, Carlsbad, CA, USA). HRFs (1 × 105) were seeded in a 35 mm confocal dish. The cells were maintained in an incubator at 5% CO2, 37 °C for 24 h. The cells were treated with alendronate (0 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM) for 48 h. Briefly, a Live/Dead kit solution (5X dye) was added to the dish. After the dish was incubated for 10 min at room temperature, the cells were evaluated using a laser-scanning confocal imaging system (IX70, Olympus, Tokyo, Japan); digital photographs were taken at 100 magnifications.
Analyses for cell cycle and cell proliferation
Cell-cycle analyses using the PI (propidium iodide, Sigma) reagent were performed as follows. HRFs (1 × 105) were seeded in each well of a 6-well plate. After 24 h of incubation, the experimental groups were exposed to alendronate (0 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM) for 48 h. The cultured HRFs were harvested after trypsinization, and then collected after centrifugation. Those cells were washed with PBS, then fixed with 70% ethanol. They were then stained, using PBS containing 0.05 mg/mL PI and 1 μg/mL RNase, and 1 μg/mL Triton X-100. Flow cytometry (Cytomics FC500, Beckman Coulter, Fullerton, CA, USA) was used to measure the fluorescence intensity of each of the cells. Then, the subG1 population (cells with DNA fragmentation) was measured from the PI histogram.
Cell proliferation was evaluated with the Ki-67 staining method. HRFs (1 × 104) were seeded on each well of a 24-well cell culture plate. Following treatment with alendronate, the cells were incubated for 48 h. The culture medium was removed from each well, and the cells were washed with PBS. A fixative solution of 4% paraformaldehyde was added to each well, which was then incubated for 20 min at 4 °C. The wells were then washed twice with PBS. The cells were permeabilized with 0.3% Triton X-100 added to each well, which was then incubated for 20 min at room temperature. The cells were then incubated in 5% bovine serum albumin (Amresco, Solon, OH, USA) in PBS for 1 h at room temperature. After that, the 1:300 diluted anti-Ki67 primary antibody (Ki-67 ab15580, Abcam, Cambridge, MA, USA) was added, and the cells were incubated for 2 h at room temperature. The wells were then washed twice with PBS. The secondary antibody (red) (goat anti-rabbit IgG, DyLight®550, A120-101D3, Bethyl, Montgomery, TX, USA) was used at the 1:500 dilutions for 1 h at room temperature and cells were counterstained with 1 μg/ml of DAPI (4′,6-diamidino-2-phenylindole, Sigma). The wells were washed again with PBS. The cells were then evaluated through a fluorescence microscope (ECLIPSE Ti-S, Nikon, Tokyo, Japan).
Analyses for type of cell death
HRFs (1 × 105) were seeded in each well of a 6-well plate. After 24 h of incubation, the experimental groups were exposed to alendronate (0, 0.1, 1, 10, and 100 μM) for 48 h. The cultured HRFs were harvested after trypsinization, and then collected after centrifugation. Those cells were washed with PBS, then stained using a fluorescein isothiocyanate (FITC) Annexin V-PI kit (BD Biosciences, San Diego, CA, USA), according to the manufacturer’s instructions. Using flow cytometry (Cytomics FC500, Beckman Coulter), cell viability was determined as follows: live cells were labeled with neither stain; apoptotic cells were labeled only with Annexin V; and necrotic cells were labeled with both Annexin V and PI.
DAPI (4′, 6-diamidino-2-phenylindole) staining for evaluation of DNA fragmentation was performed as follows. HRFs (1 × 105) were seeded in each well of a 6-well plate. After 24 h of incubation, the experimental groups were exposed to alendronate (0, 0.1, 1, 10, and 100 μM) for 48 h. After washing with PBS, the cells were fixed with methanol for 5 min at − 20 °C and were then washed with cold PBS. The cells were kept in 1% triton X-100 in PBS solution for 10 min at room temperature and were then washed with PBS. The cells were stained with 1 μg/mL DAPI staining solution (Sigma) for 5 min at 37 °C and were then washed with PBS. The cells were evaluated using a fluorescence microscope (Nikon).
Analyses of Caspases activity
HRFs (3 × 103) were seeded in each well of a 96-well cell culture white plate; then the HRFs were incubated for 24 h. Then, after treatment with alendronate (0, 0.1, 1, 10, and 100 μM), the cells were incubated for 48 h. Then, caspase-3/7 activity was measured, using the Caspase-Glo® 3/7 Assay Kit, according to the manufacturer’s guide (Promega, Madison, WI, USA).
Cleaved caspase-3 activity was also evaluated morphologically, using immunocytochemistry. HRFs (1 × 104) were seeded on the cover glass of each well of a 24-well cell culture plate. Then, after treatment with alendronate (0, 0.1, 1, 10, and 100 μM), the cells were incubated for 48 h. The culture media was removed from each well, and the cells were washed with PBS. A fixative solution of 4% paraformaldehyde was added to each well, and then washed twice with PBS. A 0.3% Triton X-100 in PBS solution was added to each well, which was then incubated for 20 min at room temperature. The wells were then washed twice with PBS, and the cells were incubated in 5% bovine serum albumin (Amresco) in PBS for 1 h at room temperature. Then, the 1:200 diluted primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling Technology, Beverly, MA, USA) were added, and the cells were incubated overnight at 4 °C. The cells were then washed twice with PBS. The cells were stained with a goat anti-rabbit IgG secondary antibody (A120-101D3, Bethyl) and counterstained with DAPI. The cells were then evaluated, using a fluorescence microscope (Nikon) under 200 magnifications. To identify the apoptosis pathways, we evaluated the activities of caspase-8 representing the extrinsic pathway and the activities of caspase-9 representing the intrinsic pathway, using the same methodological sequence for each primary antibody of caspase-8 (ab25901, Abcam) and caspase-9 (ab47537, Abcam).
Wound-healing analyses
Wound-healing was examined with a scratch assay, using a Culture-Insert (ibidi, Munich, Germany). HRFs (7 × 104) were seeded in each Culture-Insert. The cells were maintained in an incubator at 5% CO2 and 37 °C for 24 h. The study groups were treated with alendronate (0, 0.1, 1, 10, and 100 μM) for 48 h prior to wounding. After removal of the Culture-Insert, the wounds were created according to a standardized protocol, using a trimmed comb with a cell-free gap of 500 μm. Immediately after wounding, a brief wash was performed, and the culture media was added to the entire experimental group. With a phase contrast microscope, migration of cells into wounded areas of the plate was observed at 0 h, 12 h, 24 h, and 48 h. Nine fields of the wounded area were photographed at 40 magnifications. For each experimental group, the areas not covered by cells at the exposure times of 0 h, 12 h, 24 h, and 48 h were determined by analysis with the Stream image (Olympus). The migration area’s percentage in each subgroup was calculated by determining the difference between the 0-h and 48-h areas, and then dividing that by the difference in the control during the two time points, × 100.
Statistical analyses
Each experiment was performed at least three times; the results were presented as the mean of the total number of trials performed, in order to obtain more objective data. All values were expressed as mean ± standard deviation (SD). All statistical analyses were performed via one-way ANOVA, followed by Tukey’s post hoc test. Differences with a probability of less than 0.05 were considered statistically significant. All statistical analyses were done by SPSS 20.0 for Windows (SPSS, Chicago, IL, USA).