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In vitro effects of alendronate on fibroblasts of the human rotator cuff tendon

In vitro effects of alendronate on fibroblasts of the human rotator cuff tendon Background: Bone mineral density of the humeral head is an independent determining factor for postoperative rotator cuff tendon healing. Bisphosphonates, which are commonly used to treat osteoporosis, have raised concerns regarding their relationships to osteonecrosis of the jaw and to atypical fracture of the femur. In view of the prevalence of rotator cuff tear in osteoporotic elderly people, it is important to determine whether bisphosphonates affect rotator cuff tendon healing. However, no studies have investigated bisphosphonates’ cytotoxicity to human rotator cuff tendon fibroblasts (HRFs) or bisphosphonates’ effects on rotator cuff tendon healing. The purpose of this study was to evaluate the cytotoxicity of alendronate (Ald), a bisphosphonate, and its effects on HRF wound healing. Methods: HRFs were obtained from human supraspinatus tendons, using primary cell cultures. The experimental groups were control, 0.1 μM Ald, 1 μM Ald, 10 μM Ald, and 100 μM Ald. Alendronate exposure was for 48 h, except during a cell viability analysis with durations from 1 day to 6 days. The experimental groups were evaluated for cell viability, cell cycle and cell proliferation, type of cell death, caspase activity, and wound-healing ability. Results: The following findings regarding the 100 μM Ald group contrasted with those for all the other experimental groups: a significantly lower rate of live cells (p < 0.01), a higher rate of subG1 population, a lower rate of Ki-67 positive cells, higher rates of apoptosis and necrosis, a higher number of cells with DNA fragmentation, higher caspase-3/7 activity (p < 0.001), and a higher number of caspase-3 positive staining cells. In scratch-wound healing analyses of all the experimental groups, all the wounds healed within 48 h, except in the 100 μM Ald group (p <0.001). Conclusions: Low concentrations of alendronate appear to have little effect on HRF viability, proliferation, migration, and wound healing. However, high concentrations are significantly cytotoxic, impairing cellular proliferation, cellular migration, and wound healing in vitro. Keywords: Human rotator cuff tendon fibroblasts, Alendronate, Wound healing Background technique is currently popular for arthroscopic rotator Rotator cuff tendon tear, a cause of shoulder pain and cuff repair [6]. Several biomechanical studies have re- dysfunction, is the most common shoulder disease that ported that inadequate bone mineral density (BMD) in is closely related to the aging process [1–3]. Elderly pa- the tuberosities of the proximal humerus negatively in- tients, who are commonly candidates for rotator cuff re- fluences the critically important pullout strength of su- pair to reduce their shoulder pain and improve their ture anchors, which determines the final fixation shoulder function, often additionally have osteoporosis strength of inserted implants. BMD has been reported as of the proximal humerus [4, 5]. The suture anchor an independent determining factor affecting postopera- tive rotator cuff healing [7]. Previous studies additionally * Correspondence: hbinpark@gnu.ac.kr suggest that reduced BMD is caused by excessive osteo- Department of Orthopaedic Surgery, Gyeongsang National University clastic activity, which could impair bone ingrowth at the School of Medicine and Gyeongsang National University Changwon Hospital, tendon-to-bone attachment sites [8, 9]. Therefore, the Changwon, Republic of Korea 51472 Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 2 of 11 assessment of BMD and, if necessary, its improvement, DMEM (Lonza) supplemented with 20% FBS (Gibco, and the suppression of osteoclastic activity are essential Grand Island, NY, USA) and 1% Antibiotic- for rotator cuff repair in the elderly. Antimycotic (Gibco) in a humidified 5% CO atmos- Bisphosphonates, despite their long-established use as phere at 37 °C. After two weeks, the cells had reached the first line of treatment for osteoporosis, have con- 90% confluence. The cells were then trypsinized cerning relationships to osteonecrosis of the jaw and (0.02% trypsin, 0.02% EDTA in PBS) for 5 min, centri- atypical fracture of the femur [10–13]. Although bispho- fuged at 330 g for 3 min, and expanded in a second sphonates are known to suppress bone turnover, the ef- passage. The cells were then harvested with trypsin/ fect of this action on soft tissues has not been EDTA and cryopreserved. For this experiment, third- completely evaluated. Some studies have reported that passage cells were used. alendronate, a commonly used bisphosphonate, is cyto- toxic to some cells, including oral keratinocytes [14], Cell viability analyses gingival fibroblasts [15], periodontal ligament fibro- Cell viability was estimated by measuring the MTT blasts [16], and endothelial cells [17]. However, no (Sigma). HRFs (2 × 10 ) were seeded in each well of a study has addressed the cytotoxicity of bisphospho- 24-well plate. The cells were maintained in an incu- nates on human rotator cuff tendon fibroblasts bator at 5% CO , 37 °C for 24 h. The cells were (HRFs). The purpose of this study was to evaluate the treated with alendronate (0 μM, 0.1 μM, 1 μM, 10 μM, cytotoxicity of alendronate (Ald), a bisphosphonate, and 100 μM) for1, 2, 3,4,5,and6days.A500 μL and its effects on HRF wound healing. MTT solution (0.5 mg/mL in free media) was briefly added to each well of the 24-well plate. Then, the Methods plate was incubated for 2 h. Afterwards, the cell Study design supernatant was removed and the solution of 200 μL This study used HRFs. This experiment used a con- DMSO (Merck, Darmstadt, Germany) was added to trol group and a study group composed of four sub- each well of the plate. Absorbance of the plate was groups, each treated with a different concentration of measured at 570 nm, using a microplate reader. Cell sodium alendronate (0.1, 1, 10, and 100 μM Ald) viability was expressed as a percentage of live cells, (Sigma, St. Louis, MO, USA). The concentrations compared with the control, which was set at 100%. were set by referring to several previously published Cell viability analysis using the crystal violet assay studies which had used concentrations of alendronate was performed as follows. HRFs (1 × 10 ) were seeded [18–20]. Each study subgroup was exposed to its spe- in each well of a 6-well plate. The cells were main- cific concentration of alendronate for 48 h, except tained in an incubator at 5% CO ,37°Cfor 24h. during the measuring of the metabolism of 3-(4, 5- After that, the cells were treated with alendronate dimethyldiazol-2-yl)-2, 5-diphenyltetrazolium bromide (0 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM) for 48 h. (MTT), which was performed at varying times of 1 The cells were stained using 0.1% crystal violet solu- day to 6 days. These experimental groups were evalu- tion (Sigma) for 2 h at room temperature, and then ated for cell viability, cell cycle and cell proliferation, were washed with PBS. Then, the stained cells in the type of cell death, caspase activity, and wound-healing 6-well plate were analyzed using a scanner (Power- ability. The schematic description of the study’sde- Look 2100XL, Umax, Dallas, TX, USA). sign, the number of samples, and the repetition times Cell viability was also assessed using the LIVE/ for each test are illustrated in Fig. 1. DEAD Viability/Cytotoxicity Kit (Invitrogen, Carlsbad, CA, USA). HRFs (1 × 10 )wereseeded in a 35mm Cell culture confocal dish. The cells were maintained in an incu- The human supraspinatus tendon tissues were col- bator at 5% CO , 37 °C for 24 h. The cells were lected from patients undergoing arthroscopic rotator treated with alendronate (0 μM, 0.1 μM, 1 μM, 10 μM, cuff repair. The harvested tendon tissues were ob- and 100 μM) for 48 h. Briefly, a Live/Dead kit solution tained from three patients (2 males and 1 female, (5X dye) was added to the dish. After the dish was ages 50, 56, and 55 years) from 2010 to 2011. The ro- incubated for 10 min at room temperature, the cells tator cuff tears that were arthroscopically repaired were evaluated using a laser-scanning confocal im- were one small bursal-side partial-thickness tear and aging system (IX70, Olympus, Tokyo, Japan); digital two full-thickness tears of medium and large sizes. photographs were taken at 100 magnifications. Briefly, the tissues were washed twice with PBS (Lonza, Walkersville, MD, USA), minced into small Analyses for cell cycle and cell proliferation pieces with a sterile scalpel, and placed on a 6-well Cell-cycle analyses using the PI (propidium iodide, Sigma) tissue culture plate (Corning, Corning, NY, USA) in reagent were performed as follows. HRFs (1 × 10 )were Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 3 of 11 Fig. 1 A schematic drawing of the experimental design, number of culture cell donors, and the repetition times of each test seeded in each well of a 6-well plate. After 24 h of incuba- Cell proliferation was evaluated with the Ki-67 staining tion, the experimental groups were exposed to alen- method. HRFs (1 × 10 ) were seeded on each well of a dronate (0 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM) for 24-well cell culture plate. Following treatment with alen- 48 h. The cultured HRFs were harvested after trypsi- dronate, the cells were incubated for 48 h. The culture nization, and then collected after centrifugation. Those medium was removed from each well, and the cells were cells were washed with PBS, then fixed with 70% ethanol. washed with PBS. A fixative solution of 4% paraformal- They were then stained, using PBS containing 0.05 dehyde was added to each well, which was then incu- mg/mL PI and 1 μg/mL RNase, and 1 μg/mL Triton bated for 20 min at 4 °C. The wells were then washed X-100. Flow cytometry (Cytomics FC500, Beckman twice with PBS. The cells were permeabilized with 0.3% Coulter, Fullerton, CA, USA) was used to measure Triton X-100 added to each well, which was then incu- the fluorescence intensity of each of the cells. Then, bated for 20 min at room temperature. The cells were the subG1 population (cells with DNA fragmentation) then incubated in 5% bovine serum albumin (Amresco, was measured from the PI histogram. Solon, OH, USA) in PBS for 1 h at room temperature. Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 4 of 11 After that, the 1:300 diluted anti-Ki67 primary antibody Analyses for type of cell death (Ki-67 ab15580, Abcam, Cambridge, MA, USA) was HRFs (1 × 10 ) were seeded in each well of a 6-well plate. added, and the cells were incubated for 2 h at room After 24 h of incubation, the experimental groups were ex- temperature. The wells were then washed twice with posed to alendronate (0, 0.1, 1, 10, and 100 μM) for 48 h. PBS. The secondary antibody (red) (goat anti-rabbit IgG, The cultured HRFs were harvested after trypsinization, DyLight®550, A120-101D3, Bethyl, Montgomery, TX, and then collected after centrifugation. Those cells were USA) was used at the 1:500 dilutions for 1 h at room washed with PBS, then stained using a fluorescein isothio- temperature and cells were counterstained with 1 μg/ml cyanate (FITC) Annexin V-PI kit (BD Biosciences, San of DAPI (4′,6-diamidino-2-phenylindole, Sigma). The Diego, CA, USA), according to the manufacturer’s instruc- wells were washed again with PBS. The cells were then tions. Using flow cytometry (Cytomics FC500, Beckman evaluated through a fluorescence microscope (ECLIPSE Coulter), cell viability was determined as follows: live cells Ti-S, Nikon, Tokyo, Japan). were labeled with neither stain; apoptotic cells were Fig. 2 Cell viability analyses. a Cell viability was significantly decreased in a time-dependent manner in 100 μM Ald group, the group with the highest alendronate concentration (p < 0.001). After 24 h, cell viability in 100 μM Ald was significantly lower than in the other studied groups (P < 0.001). b Contrast microscope analyses demonstrated that the cell population in 100 μM Ald group was markedly more decreased than in the other studied groups. c Crystal violet stain analyses confirmed that live cells were markedly more decreased in 100 μM Ald group than in the other studied groups. d Theliveand dead assays showed that the population of live cells (green) was more decreased and the population of dead cells (red) was more increased in 100 μMAld group than in the other studied groups Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 5 of 11 labeled only with Annexin V; and necrotic cells were la- 24 h. Then, after treatment with alendronate (0, 0.1, 1, beled with both Annexin V and PI. 10, and 100 μM), the cells were incubated for 48 h. Then, DAPI (4′, 6-diamidino-2-phenylindole) staining for caspase-3/7 activity was measured, using the Caspase- evaluation of DNA fragmentation was performed as fol- Glo® 3/7 Assay Kit, according to the manufacturer’s lows. HRFs (1 × 10 ) were seeded in each well of a 6-well guide (Promega, Madison, WI, USA). plate. After 24 h of incubation, the experimental groups Cleaved caspase-3 activity was also evaluated were exposed to alendronate (0, 0.1, 1, 10, and 100 μM) morphologically, using immunocytochemistry. HRFs for 48 h. After washing with PBS, the cells were fixed (1 × 10 ) were seeded on the cover glass of each well with methanol for 5 min at − 20 °C and were then of a 24-well cell culture plate. Then, after treatment washed with cold PBS. The cells were kept in 1% triton with alendronate (0, 0.1, 1, 10, and 100 μM), the cells X-100 in PBS solution for 10 min at room temperature were incubatedfor 48h. Theculture mediawas re- and were then washed with PBS. The cells were stained moved from each well, and the cells were washed with 1 μg/mL DAPI staining solution (Sigma) for 5 min with PBS. A fixative solution of 4% paraformaldehyde at 37 °C and were then washed with PBS. The cells were was added to each well, and then washed twice with evaluated using a fluorescence microscope (Nikon). PBS. A 0.3% Triton X-100 in PBS solution was added to each well, which was then incubated for 20 min at Analyses of Caspases activity room temperature. The wells were then washed twice HRFs (3 × 10 ) were seeded in each well of a 96-well cell with PBS, and the cells were incubated in 5% bovine culture white plate; then the HRFs were incubated for serum albumin (Amresco) in PBS for 1 h at room Fig. 3 The comparisons of cell cycle and proliferation rates in various study groups. a and b The cell population of subG1 increased significantly in 100 μM Ald group, as compared with the control and the other studied groups (p ≤ 0.013). * represents the p values that are less than 0.05. c Immunocytochemical staining demonstrates that the Ki-67 positive cells representing cellular proliferation markedly decreased in 100 μM Ald group, as compared with the control and the other studied groups Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 6 of 11 temperature. Then, the 1:200 diluted primary anti- prior to wounding. After removal of the Culture- bodies against cleaved caspase-3 (Asp175, Cell Signal- Insert, the wounds were created according to a stan- ing Technology, Beverly, MA, USA) were added, and dardized protocol, using a trimmed comb with a cell- the cells were incubated overnight at 4 °C. The cells free gap of 500 μm. Immediately after wounding, a were then washed twice with PBS. The cells were brief wash was performed, and the culture media was stained with a goat anti-rabbit IgG secondary anti- added to the entire experimental group. With a phase body (A120-101D3, Bethyl) and counterstained with contrast microscope, migration of cells into wounded DAPI. The cells were then evaluated, using a fluores- areas of the plate was observed at 0 h, 12 h, 24 h, and cence microscope (Nikon) under 200 magnifications. 48 h. Nine fields of the wounded area were photo- To identify the apoptosis pathways, we evaluated the graphed at 40 magnifications. For each experimental activities of caspase-8 representing the extrinsic path- group, the areas not covered by cells at the exposure way and the activities of caspase-9 representing the times of 0 h, 12 h, 24 h, and 48 h were determined by intrinsic pathway, using the same methodological se- analysis with the Stream image (Olympus). The mi- quence for each primary antibody of caspase-8 gration area’s percentage in each subgroup was calcu- (ab25901, Abcam) and caspase-9 (ab47537, Abcam). lated by determining the difference between the 0-h and 48-h areas, and then dividing that by the differ- Wound-healing analyses ence in the control during the two time points, × 100. Wound-healing was examined with a scratch assay, using a Culture-Insert (ibidi, Munich, Germany). Statistical analyses HRFs (7 × 10 ) were seeded in each Culture-Insert. Each experiment was performed at least three times; The cells were maintained in an incubator at 5% CO the results were presented as the mean of the total and 37 °C for 24 h. The study groups were treated number of trials performed, in order to obtain more with alendronate (0, 0.1, 1, 10, and 100 μM) for 48 h objective data. All values were expressed as mean ± Fig. 4 Analyses for type of cell death in various study groups. a and b In the Annexin V-PI double staining analyses, the rates of apoptosis and necrosis in 100 μM Ald group were significantly higher than in the control and the other studied groups (p < 0.01). ## signifies the p values of the apoptosis that are less than 0.01 and ** represents the p vlaues of the necrosis that are less than 0.01. c In the DAPI staining analyses, DNA fragmentation, a marker for apoptosis, was markedly increased in 100 μM Ald group as compared with control and the other studied groups Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 7 of 11 standard deviation (SD). All statistical analyses were did not differ markedly among control and the other performed via one-way ANOVA, followed by Tukey’s studied groups (Fig. 4c). post hoc test. Differences with a probability of less than 0.05 were considered statistically significant. All Caspases activity assay statistical analyses were done by SPSS 20.0 for Win- Caspase-3/7 activity in the 100 μM Ald group was sig- dows (SPSS, Chicago, IL, USA). nificantly higher than in control and the other studied groups (p <0.001). Caspase-3/7 activity did not differ sig- Results nificantly among the control and the other studied Cytotoxicity analyses groups (p > 0.05) (Fig. 5a). The comparatively higher Cultures treated with the highest alendronate concen- caspase-3 expression in the 100 μM Ald group was con- tration (100 μM Ald) showed significantly lower per- firmed by immunocytochemistry staining (Fig. 5b); centages of live cells than the other groups (control, Caspase-8 expression in the 100 μM Ald group was 0.1, 1 and 10 μMAld)after 24h(p < 0.001). The rate higher than in control and the other studied groups (Fig. of live cells in the 100 μM Ald group significantly de- 5c); Caspase-9 expression in the 100 μM Ald group was creased with time dependence (p < 0.001), reaching also higher than in control and the other studied groups 24.53 ± 7.71% (mean ± SD) of total cells in 5 days. The (Fig. 5d). rates of live cells in control, 0.1, 1 and 10 μM Ald did not differ significantly from one another (p > 0.05) Wound-healing analyses (Fig. 2a). In a comparison of scratch-wound healing in a 5% FBS According to the morphological analyses using media, all the wounds healed within 48 h, except in the phase contrast microscope and crystal violet staining, 100 μM Ald group. The mean values in each group were the cell population was markedly more decreased in control, 100.00 ± 1.25; 0.1 μM Ald, 101.01 ± 1.99; 1 μM the 100 μM Ald group than in the other studied Ald, 96.84 ± 2.39; 10 μM Ald, 96.56 ± 3.45; and 100 μM groups (Fig. 2b and c). According to the cell viability Ald, 9.04 ± 5.75. The wound-healing results in the analyses using a LIVE/DEAD kit, the number of dead 100 μM Ald group were significantly lower than in the cells stained with red color in the 100 μM Ald group other studied groups and the control (p < 0.001). The was markedly higher than in the control group and mean percentages of wound healing in the control, 0.1, 1 the other studied groups (0.1, 1 and 10 μMAld)(Fig. and 10 μM Ald groups were not significantly different 2d). from each other (p > 0.05) (Fig. 6a and b). Analyses of cell cycle and cellular proliferation Discussion The subG1 population increased significantly in the The notable finding of the current study was that a high 100 μM Ald group, as compared with the control and the concentration of alendronate (100 μM Ald) has a cyto- other studied groups, according to the FACS analyses toxic effect on HRF. In this study, cell viability was sig- using PI staining (p ≤ 0.013). The subG1 populations in nificantly decreased in the subgroup with the highest thecontrol,0.1,1,and 10 μM Ald groups did not differ alendronate concentration, as compared to the control significantly from each other (p > 0.05) (Fig. 3a and b). and the other studied subgroups. The decreased cell via- Cellular proliferation shown by Ki-67 positive cells bility was mainly caused by apoptosis involving the was more decreased in the 100 μM Ald group than in caspase-3 pathway. Alendronate-induced cytotoxicity the control and the other studied groups (Fig. 3c). has been reported in several studies involving oral kera- tinocytes [18], gingival fibroblasts [21], human periodon- Analysis of Annexin V-PI and DAPI staining tal ligament fibroblasts [18], endothelial cells [22], and The mean percentages of apoptotic and necrotic cells human bone-marrow-derived stem cells [23]. Those were significantly higher in the 100 μMAld group studies all found that the highest concentrations of alen- −11 than in the control and the other studied groups, ac- dronate, ranging from 10 M to 100 M, had various cording to the FACS analyses using Annexin V-PI levels of cytotoxicity to the studied cells. Those findings double staining (p < 0.01). The mean percentages of support the results of this study. apoptotic and necrotic cells did not differ signifi- This study showed that the population of subG1 cells cantly among the control and the other studied increased significantly in the 100 μM Ald group, as com- groups (p >0.05) (Fig. 4aand b). pared with the control and other studied groups. This The number of cells with DNA fragmentation was means that many cells did not actively participate in cel- higher in the 100 μM Ald group than in the control and lular proliferation due to DNA fragmentation. We also the other studied groups, according to the DAPI staining found that 100 μM Ald inhibited cellular proliferation, analyses. The numbers of cells with DNA fragmentation using immunocytochemistry for the Ki-67 antigen, Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 8 of 11 Fig. 5 Caspase-3/7 activity and immunocytochemistry for caspase-3, caspase-8, and caspase-9 activity. a Caspase-3/7 activity was significantly higher in 100 μM Ald group, as compared with control and the other studied groups (p < 0.001). *** represents the p vlaues that are less than 0.001. b Cleaved caspase-3 expression (red) was higher in 100 μM Ald group than in control and the other studied groups in the immunocytochemistry study. c Caspase-8 expression (red) in 100 μM Ald group was higher than in control and the other studied groups. d Caspase-9 expression (red) in 100 μM Ald group was higher than in control and the other studied groups Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 9 of 11 Fig. 6 Wound-healing analyses. a In a comparison of scratch wound-healing, all the wounds healed within 48 h, except in 100 μM Ald. b The percentage of wound healing in 100 μM Ald group was significantly lower than in control and the other studied groups (p < 0.001). *** represents the p vlaues that are less than 0.001. The mean percentages of wound-healing in control, 0.1, 1 and 10 μM Ald group did not differ significantly from one another (p > 0.05) which is a well-known marker for cell proliferation. The mechanism, operates in alendronate-induced cytotoxicity Ki-67 antigen is always present in proliferating cells in to HRFs [25, 26]. We also evaluated caspase-8, involving all active phases of the cell cycle (G ,S,G , and mitosis), extrinsic apoptosis pathways, and caspase-9, involving 1 2 but is absent from resting cells (G )[24]. All these find- intrinsic pathways [27, 28]. In this study, both caspases ings suggest that the highest concentration of alendro- were highly expressed in the group with the highest con- nate inhibited cellular proliferation. centration (100 μM Ald). That means that both extrinsic Although previous studies reported that, in high con- and intrinsic apoptosis pathways are involved in centrations and in a time-dependent manner, alendro- alendronate-induced apoptosis. nate has cytotoxic effects, those studies did not evaluate The wound-healing analysis indicated that the highest the types or the molecular mechanisms of alendronate- concentration of alendronate reduced wound-healing, induced cell death. We evaluated the types of not only affecting cell survival but also inhibiting cell alendronate-induced cell death and determined that proliferation and cell migration, which are processes apoptosis is the predominant type. Although we did not vital to rotator cuff healing. One study that evaluated evaluate in detail the cascade mechanisms of apoptosis alendronate’s effect on wound-healing used a rat gastric related to alendronate, we determined that caspase-3, a epithelial cell line [29]. That study reported that alendro- well-known executioner of cells in the apoptosis nate did not affect the wound-healing ability of rat Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 10 of 11 gastric epithelial cells; however, in a coculture with tissue. Therefore, the appropriateness of immediate endothelial-derived growth factor and alendronate, the alendronate treatment for patients following rotator wound-healing ability was less than with endothelial- cuff tendon repair should be further evaluated in derived growth factor alone. That study indicated that animal-model or clinical research. alendronate diminishes the endothelial-derived growth factor’s wound-healing effect [29]. The current and pre- Conclusion vious studies suggest that, above a certain concentration, Low concentrations of alendronate appear to have little alendronate has a negative effect on wound healing. In effect on HRF viability, proliferation, migration, and animal experiments, the effects of bisphosphonates for wound healing. However, high concentrations are sig- tendon-to-bone healing are controversial. Thomopoulos nificantly cytotoxic, impairing cellular proliferation, cel- et al. [30] reported that alendronate prevents bone loss lular migration, and wound healing in vitro. and improves tendon-to-bone repair. However, not only Abbreviations did alendronate not improve tendon stiffness, but sys- Ald: Alendronate; BMD: Bone mineral density; FITC: Fluorescein temic plus local treatment of alendronate was detrimen- isothiocyanate; HRFs: Human rotator cuff tendon fibroblasts; MTT: Metabolism of 3-(4, 5-dimethyldiazol-2-yl)-2, 5-diphenyltetrazolium tal to stiffness of the repair due to alendronate’s effect bromide; SD: Standard deviation on the inflammatory process. Thomopoulos et al. [30] suggested that the high local dose of alendronate may Acknowledgements have led to an enhanced inflammatory reaction in the None. bone tunnel that was detrimental to the healing process. Authors’ contributions Lui et al. [31] reported that alendronate reduced peri- Conception and study design: CMS, HBP and RJK. Data collection: JYG and tunnel bone resorption, increased mineralized tissue in- RJK. Data analysis and interpretation: HBP, YSH, and RJK. Drafting of article or revising content: HBP, CMS, RJK, and JYG. Final approval of the article: HBP, side the bone tunnel, and histologically and biomechan- CMS, RJK, YSH, and JYG. All authors read and approved the final manuscript. ically promoted graft-bone tunnel healing. That study also reported that alendronate might be used for redu- Funding cing peri-tunnel bone loss and promoting graft-bone None. tunnel healing at the early stage of anterior cruciate liga- Availability of data and materials ment (ACL) reconstruction. The Lui et al. [31] study’s The dataset generated and/or analyzed during the current study is not result differs from those of the Thomopoulos et al. [30] publicly available due to legal and ethical considerations. However, the data are available from the corresponding author, upon reasonable request. study. Conversely, Hjorthaug et al. [32] showed a nega- tive effect of a bisphosphonate, in which zoledronic acid Ethics approval and consent to participate reduced pullout strength and the stiffness of the tendon- This study was carried out in accordance with the permission of the Gyeongsang National University Institutional Review Board (GNUHIRB-2009- bone interface. That study reported no evidence sup- 022) in the Gyeongsang National University Hospital. All participants signed porting the use of zoledronic acid as adjuvant treatment an informed consent. in tendon-to-bone healing. There is still controversy as to whether bisphosphonates enhance tendon-to-bone Consent for publication Not Applicable. healing. However, those previous studies may not be dir- ectly comparable to our study due to the differences in Competing interests the study designs. The authors declare that they have no conflicts of interest with the contents of this article. This study has several limitations. We did not evaluate every bisphosphonate that is used in clinical Author details practice because most injectable bisphosphonates are Department of Orthopaedic Surgery, Gyeongsang National University College of Medicine, Jinju, South Korea. Department of Convergence used to treat metastatic cancer and are not practical Medical Science, Gyeongsang National University, Jinju, South Korea. for patients needing rotator cuff repair [33]. We fo- Institute of Health Sciences, Gyeongsang National University School of cused on alendronate, a lower-potency bisphospho- Medicine and Biomedical Research Institute, Gyeongsang National University Hospital, Jinju, South Korea. Department of Orthopaedic Surgery, nate commonly used to treat osteoporosis, giving this Gyeongsang National University School of Medicine and Gyeongsang study clinical significance for rotator cuff surgery [34, National University Changwon Hospital, Changwon, Republic of Korea 51472. 35]. 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In vitro effects of alendronate on fibroblasts of the human rotator cuff tendon

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10.1186/s12891-019-3014-1
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Abstract

Background: Bone mineral density of the humeral head is an independent determining factor for postoperative rotator cuff tendon healing. Bisphosphonates, which are commonly used to treat osteoporosis, have raised concerns regarding their relationships to osteonecrosis of the jaw and to atypical fracture of the femur. In view of the prevalence of rotator cuff tear in osteoporotic elderly people, it is important to determine whether bisphosphonates affect rotator cuff tendon healing. However, no studies have investigated bisphosphonates’ cytotoxicity to human rotator cuff tendon fibroblasts (HRFs) or bisphosphonates’ effects on rotator cuff tendon healing. The purpose of this study was to evaluate the cytotoxicity of alendronate (Ald), a bisphosphonate, and its effects on HRF wound healing. Methods: HRFs were obtained from human supraspinatus tendons, using primary cell cultures. The experimental groups were control, 0.1 μM Ald, 1 μM Ald, 10 μM Ald, and 100 μM Ald. Alendronate exposure was for 48 h, except during a cell viability analysis with durations from 1 day to 6 days. The experimental groups were evaluated for cell viability, cell cycle and cell proliferation, type of cell death, caspase activity, and wound-healing ability. Results: The following findings regarding the 100 μM Ald group contrasted with those for all the other experimental groups: a significantly lower rate of live cells (p < 0.01), a higher rate of subG1 population, a lower rate of Ki-67 positive cells, higher rates of apoptosis and necrosis, a higher number of cells with DNA fragmentation, higher caspase-3/7 activity (p < 0.001), and a higher number of caspase-3 positive staining cells. In scratch-wound healing analyses of all the experimental groups, all the wounds healed within 48 h, except in the 100 μM Ald group (p <0.001). Conclusions: Low concentrations of alendronate appear to have little effect on HRF viability, proliferation, migration, and wound healing. However, high concentrations are significantly cytotoxic, impairing cellular proliferation, cellular migration, and wound healing in vitro. Keywords: Human rotator cuff tendon fibroblasts, Alendronate, Wound healing Background technique is currently popular for arthroscopic rotator Rotator cuff tendon tear, a cause of shoulder pain and cuff repair [6]. Several biomechanical studies have re- dysfunction, is the most common shoulder disease that ported that inadequate bone mineral density (BMD) in is closely related to the aging process [1–3]. Elderly pa- the tuberosities of the proximal humerus negatively in- tients, who are commonly candidates for rotator cuff re- fluences the critically important pullout strength of su- pair to reduce their shoulder pain and improve their ture anchors, which determines the final fixation shoulder function, often additionally have osteoporosis strength of inserted implants. BMD has been reported as of the proximal humerus [4, 5]. The suture anchor an independent determining factor affecting postopera- tive rotator cuff healing [7]. Previous studies additionally * Correspondence: hbinpark@gnu.ac.kr suggest that reduced BMD is caused by excessive osteo- Department of Orthopaedic Surgery, Gyeongsang National University clastic activity, which could impair bone ingrowth at the School of Medicine and Gyeongsang National University Changwon Hospital, tendon-to-bone attachment sites [8, 9]. Therefore, the Changwon, Republic of Korea 51472 Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 2 of 11 assessment of BMD and, if necessary, its improvement, DMEM (Lonza) supplemented with 20% FBS (Gibco, and the suppression of osteoclastic activity are essential Grand Island, NY, USA) and 1% Antibiotic- for rotator cuff repair in the elderly. Antimycotic (Gibco) in a humidified 5% CO atmos- Bisphosphonates, despite their long-established use as phere at 37 °C. After two weeks, the cells had reached the first line of treatment for osteoporosis, have con- 90% confluence. The cells were then trypsinized cerning relationships to osteonecrosis of the jaw and (0.02% trypsin, 0.02% EDTA in PBS) for 5 min, centri- atypical fracture of the femur [10–13]. Although bispho- fuged at 330 g for 3 min, and expanded in a second sphonates are known to suppress bone turnover, the ef- passage. The cells were then harvested with trypsin/ fect of this action on soft tissues has not been EDTA and cryopreserved. For this experiment, third- completely evaluated. Some studies have reported that passage cells were used. alendronate, a commonly used bisphosphonate, is cyto- toxic to some cells, including oral keratinocytes [14], Cell viability analyses gingival fibroblasts [15], periodontal ligament fibro- Cell viability was estimated by measuring the MTT blasts [16], and endothelial cells [17]. However, no (Sigma). HRFs (2 × 10 ) were seeded in each well of a study has addressed the cytotoxicity of bisphospho- 24-well plate. The cells were maintained in an incu- nates on human rotator cuff tendon fibroblasts bator at 5% CO , 37 °C for 24 h. The cells were (HRFs). The purpose of this study was to evaluate the treated with alendronate (0 μM, 0.1 μM, 1 μM, 10 μM, cytotoxicity of alendronate (Ald), a bisphosphonate, and 100 μM) for1, 2, 3,4,5,and6days.A500 μL and its effects on HRF wound healing. MTT solution (0.5 mg/mL in free media) was briefly added to each well of the 24-well plate. Then, the Methods plate was incubated for 2 h. Afterwards, the cell Study design supernatant was removed and the solution of 200 μL This study used HRFs. This experiment used a con- DMSO (Merck, Darmstadt, Germany) was added to trol group and a study group composed of four sub- each well of the plate. Absorbance of the plate was groups, each treated with a different concentration of measured at 570 nm, using a microplate reader. Cell sodium alendronate (0.1, 1, 10, and 100 μM Ald) viability was expressed as a percentage of live cells, (Sigma, St. Louis, MO, USA). The concentrations compared with the control, which was set at 100%. were set by referring to several previously published Cell viability analysis using the crystal violet assay studies which had used concentrations of alendronate was performed as follows. HRFs (1 × 10 ) were seeded [18–20]. Each study subgroup was exposed to its spe- in each well of a 6-well plate. The cells were main- cific concentration of alendronate for 48 h, except tained in an incubator at 5% CO ,37°Cfor 24h. during the measuring of the metabolism of 3-(4, 5- After that, the cells were treated with alendronate dimethyldiazol-2-yl)-2, 5-diphenyltetrazolium bromide (0 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM) for 48 h. (MTT), which was performed at varying times of 1 The cells were stained using 0.1% crystal violet solu- day to 6 days. These experimental groups were evalu- tion (Sigma) for 2 h at room temperature, and then ated for cell viability, cell cycle and cell proliferation, were washed with PBS. Then, the stained cells in the type of cell death, caspase activity, and wound-healing 6-well plate were analyzed using a scanner (Power- ability. The schematic description of the study’sde- Look 2100XL, Umax, Dallas, TX, USA). sign, the number of samples, and the repetition times Cell viability was also assessed using the LIVE/ for each test are illustrated in Fig. 1. DEAD Viability/Cytotoxicity Kit (Invitrogen, Carlsbad, CA, USA). HRFs (1 × 10 )wereseeded in a 35mm Cell culture confocal dish. The cells were maintained in an incu- The human supraspinatus tendon tissues were col- bator at 5% CO , 37 °C for 24 h. The cells were lected from patients undergoing arthroscopic rotator treated with alendronate (0 μM, 0.1 μM, 1 μM, 10 μM, cuff repair. The harvested tendon tissues were ob- and 100 μM) for 48 h. Briefly, a Live/Dead kit solution tained from three patients (2 males and 1 female, (5X dye) was added to the dish. After the dish was ages 50, 56, and 55 years) from 2010 to 2011. The ro- incubated for 10 min at room temperature, the cells tator cuff tears that were arthroscopically repaired were evaluated using a laser-scanning confocal im- were one small bursal-side partial-thickness tear and aging system (IX70, Olympus, Tokyo, Japan); digital two full-thickness tears of medium and large sizes. photographs were taken at 100 magnifications. Briefly, the tissues were washed twice with PBS (Lonza, Walkersville, MD, USA), minced into small Analyses for cell cycle and cell proliferation pieces with a sterile scalpel, and placed on a 6-well Cell-cycle analyses using the PI (propidium iodide, Sigma) tissue culture plate (Corning, Corning, NY, USA) in reagent were performed as follows. HRFs (1 × 10 )were Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 3 of 11 Fig. 1 A schematic drawing of the experimental design, number of culture cell donors, and the repetition times of each test seeded in each well of a 6-well plate. After 24 h of incuba- Cell proliferation was evaluated with the Ki-67 staining tion, the experimental groups were exposed to alen- method. HRFs (1 × 10 ) were seeded on each well of a dronate (0 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM) for 24-well cell culture plate. Following treatment with alen- 48 h. The cultured HRFs were harvested after trypsi- dronate, the cells were incubated for 48 h. The culture nization, and then collected after centrifugation. Those medium was removed from each well, and the cells were cells were washed with PBS, then fixed with 70% ethanol. washed with PBS. A fixative solution of 4% paraformal- They were then stained, using PBS containing 0.05 dehyde was added to each well, which was then incu- mg/mL PI and 1 μg/mL RNase, and 1 μg/mL Triton bated for 20 min at 4 °C. The wells were then washed X-100. Flow cytometry (Cytomics FC500, Beckman twice with PBS. The cells were permeabilized with 0.3% Coulter, Fullerton, CA, USA) was used to measure Triton X-100 added to each well, which was then incu- the fluorescence intensity of each of the cells. Then, bated for 20 min at room temperature. The cells were the subG1 population (cells with DNA fragmentation) then incubated in 5% bovine serum albumin (Amresco, was measured from the PI histogram. Solon, OH, USA) in PBS for 1 h at room temperature. Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 4 of 11 After that, the 1:300 diluted anti-Ki67 primary antibody Analyses for type of cell death (Ki-67 ab15580, Abcam, Cambridge, MA, USA) was HRFs (1 × 10 ) were seeded in each well of a 6-well plate. added, and the cells were incubated for 2 h at room After 24 h of incubation, the experimental groups were ex- temperature. The wells were then washed twice with posed to alendronate (0, 0.1, 1, 10, and 100 μM) for 48 h. PBS. The secondary antibody (red) (goat anti-rabbit IgG, The cultured HRFs were harvested after trypsinization, DyLight®550, A120-101D3, Bethyl, Montgomery, TX, and then collected after centrifugation. Those cells were USA) was used at the 1:500 dilutions for 1 h at room washed with PBS, then stained using a fluorescein isothio- temperature and cells were counterstained with 1 μg/ml cyanate (FITC) Annexin V-PI kit (BD Biosciences, San of DAPI (4′,6-diamidino-2-phenylindole, Sigma). The Diego, CA, USA), according to the manufacturer’s instruc- wells were washed again with PBS. The cells were then tions. Using flow cytometry (Cytomics FC500, Beckman evaluated through a fluorescence microscope (ECLIPSE Coulter), cell viability was determined as follows: live cells Ti-S, Nikon, Tokyo, Japan). were labeled with neither stain; apoptotic cells were Fig. 2 Cell viability analyses. a Cell viability was significantly decreased in a time-dependent manner in 100 μM Ald group, the group with the highest alendronate concentration (p < 0.001). After 24 h, cell viability in 100 μM Ald was significantly lower than in the other studied groups (P < 0.001). b Contrast microscope analyses demonstrated that the cell population in 100 μM Ald group was markedly more decreased than in the other studied groups. c Crystal violet stain analyses confirmed that live cells were markedly more decreased in 100 μM Ald group than in the other studied groups. d Theliveand dead assays showed that the population of live cells (green) was more decreased and the population of dead cells (red) was more increased in 100 μMAld group than in the other studied groups Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 5 of 11 labeled only with Annexin V; and necrotic cells were la- 24 h. Then, after treatment with alendronate (0, 0.1, 1, beled with both Annexin V and PI. 10, and 100 μM), the cells were incubated for 48 h. Then, DAPI (4′, 6-diamidino-2-phenylindole) staining for caspase-3/7 activity was measured, using the Caspase- evaluation of DNA fragmentation was performed as fol- Glo® 3/7 Assay Kit, according to the manufacturer’s lows. HRFs (1 × 10 ) were seeded in each well of a 6-well guide (Promega, Madison, WI, USA). plate. After 24 h of incubation, the experimental groups Cleaved caspase-3 activity was also evaluated were exposed to alendronate (0, 0.1, 1, 10, and 100 μM) morphologically, using immunocytochemistry. HRFs for 48 h. After washing with PBS, the cells were fixed (1 × 10 ) were seeded on the cover glass of each well with methanol for 5 min at − 20 °C and were then of a 24-well cell culture plate. Then, after treatment washed with cold PBS. The cells were kept in 1% triton with alendronate (0, 0.1, 1, 10, and 100 μM), the cells X-100 in PBS solution for 10 min at room temperature were incubatedfor 48h. Theculture mediawas re- and were then washed with PBS. The cells were stained moved from each well, and the cells were washed with 1 μg/mL DAPI staining solution (Sigma) for 5 min with PBS. A fixative solution of 4% paraformaldehyde at 37 °C and were then washed with PBS. The cells were was added to each well, and then washed twice with evaluated using a fluorescence microscope (Nikon). PBS. A 0.3% Triton X-100 in PBS solution was added to each well, which was then incubated for 20 min at Analyses of Caspases activity room temperature. The wells were then washed twice HRFs (3 × 10 ) were seeded in each well of a 96-well cell with PBS, and the cells were incubated in 5% bovine culture white plate; then the HRFs were incubated for serum albumin (Amresco) in PBS for 1 h at room Fig. 3 The comparisons of cell cycle and proliferation rates in various study groups. a and b The cell population of subG1 increased significantly in 100 μM Ald group, as compared with the control and the other studied groups (p ≤ 0.013). * represents the p values that are less than 0.05. c Immunocytochemical staining demonstrates that the Ki-67 positive cells representing cellular proliferation markedly decreased in 100 μM Ald group, as compared with the control and the other studied groups Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 6 of 11 temperature. Then, the 1:200 diluted primary anti- prior to wounding. After removal of the Culture- bodies against cleaved caspase-3 (Asp175, Cell Signal- Insert, the wounds were created according to a stan- ing Technology, Beverly, MA, USA) were added, and dardized protocol, using a trimmed comb with a cell- the cells were incubated overnight at 4 °C. The cells free gap of 500 μm. Immediately after wounding, a were then washed twice with PBS. The cells were brief wash was performed, and the culture media was stained with a goat anti-rabbit IgG secondary anti- added to the entire experimental group. With a phase body (A120-101D3, Bethyl) and counterstained with contrast microscope, migration of cells into wounded DAPI. The cells were then evaluated, using a fluores- areas of the plate was observed at 0 h, 12 h, 24 h, and cence microscope (Nikon) under 200 magnifications. 48 h. Nine fields of the wounded area were photo- To identify the apoptosis pathways, we evaluated the graphed at 40 magnifications. For each experimental activities of caspase-8 representing the extrinsic path- group, the areas not covered by cells at the exposure way and the activities of caspase-9 representing the times of 0 h, 12 h, 24 h, and 48 h were determined by intrinsic pathway, using the same methodological se- analysis with the Stream image (Olympus). The mi- quence for each primary antibody of caspase-8 gration area’s percentage in each subgroup was calcu- (ab25901, Abcam) and caspase-9 (ab47537, Abcam). lated by determining the difference between the 0-h and 48-h areas, and then dividing that by the differ- Wound-healing analyses ence in the control during the two time points, × 100. Wound-healing was examined with a scratch assay, using a Culture-Insert (ibidi, Munich, Germany). Statistical analyses HRFs (7 × 10 ) were seeded in each Culture-Insert. Each experiment was performed at least three times; The cells were maintained in an incubator at 5% CO the results were presented as the mean of the total and 37 °C for 24 h. The study groups were treated number of trials performed, in order to obtain more with alendronate (0, 0.1, 1, 10, and 100 μM) for 48 h objective data. All values were expressed as mean ± Fig. 4 Analyses for type of cell death in various study groups. a and b In the Annexin V-PI double staining analyses, the rates of apoptosis and necrosis in 100 μM Ald group were significantly higher than in the control and the other studied groups (p < 0.01). ## signifies the p values of the apoptosis that are less than 0.01 and ** represents the p vlaues of the necrosis that are less than 0.01. c In the DAPI staining analyses, DNA fragmentation, a marker for apoptosis, was markedly increased in 100 μM Ald group as compared with control and the other studied groups Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 7 of 11 standard deviation (SD). All statistical analyses were did not differ markedly among control and the other performed via one-way ANOVA, followed by Tukey’s studied groups (Fig. 4c). post hoc test. Differences with a probability of less than 0.05 were considered statistically significant. All Caspases activity assay statistical analyses were done by SPSS 20.0 for Win- Caspase-3/7 activity in the 100 μM Ald group was sig- dows (SPSS, Chicago, IL, USA). nificantly higher than in control and the other studied groups (p <0.001). Caspase-3/7 activity did not differ sig- Results nificantly among the control and the other studied Cytotoxicity analyses groups (p > 0.05) (Fig. 5a). The comparatively higher Cultures treated with the highest alendronate concen- caspase-3 expression in the 100 μM Ald group was con- tration (100 μM Ald) showed significantly lower per- firmed by immunocytochemistry staining (Fig. 5b); centages of live cells than the other groups (control, Caspase-8 expression in the 100 μM Ald group was 0.1, 1 and 10 μMAld)after 24h(p < 0.001). The rate higher than in control and the other studied groups (Fig. of live cells in the 100 μM Ald group significantly de- 5c); Caspase-9 expression in the 100 μM Ald group was creased with time dependence (p < 0.001), reaching also higher than in control and the other studied groups 24.53 ± 7.71% (mean ± SD) of total cells in 5 days. The (Fig. 5d). rates of live cells in control, 0.1, 1 and 10 μM Ald did not differ significantly from one another (p > 0.05) Wound-healing analyses (Fig. 2a). In a comparison of scratch-wound healing in a 5% FBS According to the morphological analyses using media, all the wounds healed within 48 h, except in the phase contrast microscope and crystal violet staining, 100 μM Ald group. The mean values in each group were the cell population was markedly more decreased in control, 100.00 ± 1.25; 0.1 μM Ald, 101.01 ± 1.99; 1 μM the 100 μM Ald group than in the other studied Ald, 96.84 ± 2.39; 10 μM Ald, 96.56 ± 3.45; and 100 μM groups (Fig. 2b and c). According to the cell viability Ald, 9.04 ± 5.75. The wound-healing results in the analyses using a LIVE/DEAD kit, the number of dead 100 μM Ald group were significantly lower than in the cells stained with red color in the 100 μM Ald group other studied groups and the control (p < 0.001). The was markedly higher than in the control group and mean percentages of wound healing in the control, 0.1, 1 the other studied groups (0.1, 1 and 10 μMAld)(Fig. and 10 μM Ald groups were not significantly different 2d). from each other (p > 0.05) (Fig. 6a and b). Analyses of cell cycle and cellular proliferation Discussion The subG1 population increased significantly in the The notable finding of the current study was that a high 100 μM Ald group, as compared with the control and the concentration of alendronate (100 μM Ald) has a cyto- other studied groups, according to the FACS analyses toxic effect on HRF. In this study, cell viability was sig- using PI staining (p ≤ 0.013). The subG1 populations in nificantly decreased in the subgroup with the highest thecontrol,0.1,1,and 10 μM Ald groups did not differ alendronate concentration, as compared to the control significantly from each other (p > 0.05) (Fig. 3a and b). and the other studied subgroups. The decreased cell via- Cellular proliferation shown by Ki-67 positive cells bility was mainly caused by apoptosis involving the was more decreased in the 100 μM Ald group than in caspase-3 pathway. Alendronate-induced cytotoxicity the control and the other studied groups (Fig. 3c). has been reported in several studies involving oral kera- tinocytes [18], gingival fibroblasts [21], human periodon- Analysis of Annexin V-PI and DAPI staining tal ligament fibroblasts [18], endothelial cells [22], and The mean percentages of apoptotic and necrotic cells human bone-marrow-derived stem cells [23]. Those were significantly higher in the 100 μMAld group studies all found that the highest concentrations of alen- −11 than in the control and the other studied groups, ac- dronate, ranging from 10 M to 100 M, had various cording to the FACS analyses using Annexin V-PI levels of cytotoxicity to the studied cells. Those findings double staining (p < 0.01). The mean percentages of support the results of this study. apoptotic and necrotic cells did not differ signifi- This study showed that the population of subG1 cells cantly among the control and the other studied increased significantly in the 100 μM Ald group, as com- groups (p >0.05) (Fig. 4aand b). pared with the control and other studied groups. This The number of cells with DNA fragmentation was means that many cells did not actively participate in cel- higher in the 100 μM Ald group than in the control and lular proliferation due to DNA fragmentation. We also the other studied groups, according to the DAPI staining found that 100 μM Ald inhibited cellular proliferation, analyses. The numbers of cells with DNA fragmentation using immunocytochemistry for the Ki-67 antigen, Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 8 of 11 Fig. 5 Caspase-3/7 activity and immunocytochemistry for caspase-3, caspase-8, and caspase-9 activity. a Caspase-3/7 activity was significantly higher in 100 μM Ald group, as compared with control and the other studied groups (p < 0.001). *** represents the p vlaues that are less than 0.001. b Cleaved caspase-3 expression (red) was higher in 100 μM Ald group than in control and the other studied groups in the immunocytochemistry study. c Caspase-8 expression (red) in 100 μM Ald group was higher than in control and the other studied groups. d Caspase-9 expression (red) in 100 μM Ald group was higher than in control and the other studied groups Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 9 of 11 Fig. 6 Wound-healing analyses. a In a comparison of scratch wound-healing, all the wounds healed within 48 h, except in 100 μM Ald. b The percentage of wound healing in 100 μM Ald group was significantly lower than in control and the other studied groups (p < 0.001). *** represents the p vlaues that are less than 0.001. The mean percentages of wound-healing in control, 0.1, 1 and 10 μM Ald group did not differ significantly from one another (p > 0.05) which is a well-known marker for cell proliferation. The mechanism, operates in alendronate-induced cytotoxicity Ki-67 antigen is always present in proliferating cells in to HRFs [25, 26]. We also evaluated caspase-8, involving all active phases of the cell cycle (G ,S,G , and mitosis), extrinsic apoptosis pathways, and caspase-9, involving 1 2 but is absent from resting cells (G )[24]. All these find- intrinsic pathways [27, 28]. In this study, both caspases ings suggest that the highest concentration of alendro- were highly expressed in the group with the highest con- nate inhibited cellular proliferation. centration (100 μM Ald). That means that both extrinsic Although previous studies reported that, in high con- and intrinsic apoptosis pathways are involved in centrations and in a time-dependent manner, alendro- alendronate-induced apoptosis. nate has cytotoxic effects, those studies did not evaluate The wound-healing analysis indicated that the highest the types or the molecular mechanisms of alendronate- concentration of alendronate reduced wound-healing, induced cell death. We evaluated the types of not only affecting cell survival but also inhibiting cell alendronate-induced cell death and determined that proliferation and cell migration, which are processes apoptosis is the predominant type. Although we did not vital to rotator cuff healing. One study that evaluated evaluate in detail the cascade mechanisms of apoptosis alendronate’s effect on wound-healing used a rat gastric related to alendronate, we determined that caspase-3, a epithelial cell line [29]. That study reported that alendro- well-known executioner of cells in the apoptosis nate did not affect the wound-healing ability of rat Sung et al. BMC Musculoskeletal Disorders (2020) 21:19 Page 10 of 11 gastric epithelial cells; however, in a coculture with tissue. Therefore, the appropriateness of immediate endothelial-derived growth factor and alendronate, the alendronate treatment for patients following rotator wound-healing ability was less than with endothelial- cuff tendon repair should be further evaluated in derived growth factor alone. That study indicated that animal-model or clinical research. alendronate diminishes the endothelial-derived growth factor’s wound-healing effect [29]. The current and pre- Conclusion vious studies suggest that, above a certain concentration, Low concentrations of alendronate appear to have little alendronate has a negative effect on wound healing. In effect on HRF viability, proliferation, migration, and animal experiments, the effects of bisphosphonates for wound healing. However, high concentrations are sig- tendon-to-bone healing are controversial. Thomopoulos nificantly cytotoxic, impairing cellular proliferation, cel- et al. [30] reported that alendronate prevents bone loss lular migration, and wound healing in vitro. and improves tendon-to-bone repair. However, not only Abbreviations did alendronate not improve tendon stiffness, but sys- Ald: Alendronate; BMD: Bone mineral density; FITC: Fluorescein temic plus local treatment of alendronate was detrimen- isothiocyanate; HRFs: Human rotator cuff tendon fibroblasts; MTT: Metabolism of 3-(4, 5-dimethyldiazol-2-yl)-2, 5-diphenyltetrazolium tal to stiffness of the repair due to alendronate’s effect bromide; SD: Standard deviation on the inflammatory process. Thomopoulos et al. [30] suggested that the high local dose of alendronate may Acknowledgements have led to an enhanced inflammatory reaction in the None. bone tunnel that was detrimental to the healing process. Authors’ contributions Lui et al. [31] reported that alendronate reduced peri- Conception and study design: CMS, HBP and RJK. Data collection: JYG and tunnel bone resorption, increased mineralized tissue in- RJK. Data analysis and interpretation: HBP, YSH, and RJK. Drafting of article or revising content: HBP, CMS, RJK, and JYG. Final approval of the article: HBP, side the bone tunnel, and histologically and biomechan- CMS, RJK, YSH, and JYG. All authors read and approved the final manuscript. ically promoted graft-bone tunnel healing. That study also reported that alendronate might be used for redu- Funding cing peri-tunnel bone loss and promoting graft-bone None. tunnel healing at the early stage of anterior cruciate liga- Availability of data and materials ment (ACL) reconstruction. The Lui et al. [31] study’s The dataset generated and/or analyzed during the current study is not result differs from those of the Thomopoulos et al. [30] publicly available due to legal and ethical considerations. However, the data are available from the corresponding author, upon reasonable request. study. Conversely, Hjorthaug et al. [32] showed a nega- tive effect of a bisphosphonate, in which zoledronic acid Ethics approval and consent to participate reduced pullout strength and the stiffness of the tendon- This study was carried out in accordance with the permission of the Gyeongsang National University Institutional Review Board (GNUHIRB-2009- bone interface. That study reported no evidence sup- 022) in the Gyeongsang National University Hospital. All participants signed porting the use of zoledronic acid as adjuvant treatment an informed consent. in tendon-to-bone healing. There is still controversy as to whether bisphosphonates enhance tendon-to-bone Consent for publication Not Applicable. healing. However, those previous studies may not be dir- ectly comparable to our study due to the differences in Competing interests the study designs. The authors declare that they have no conflicts of interest with the contents of this article. This study has several limitations. We did not evaluate every bisphosphonate that is used in clinical Author details practice because most injectable bisphosphonates are Department of Orthopaedic Surgery, Gyeongsang National University College of Medicine, Jinju, South Korea. Department of Convergence used to treat metastatic cancer and are not practical Medical Science, Gyeongsang National University, Jinju, South Korea. for patients needing rotator cuff repair [33]. We fo- Institute of Health Sciences, Gyeongsang National University School of cused on alendronate, a lower-potency bisphospho- Medicine and Biomedical Research Institute, Gyeongsang National University Hospital, Jinju, South Korea. Department of Orthopaedic Surgery, nate commonly used to treat osteoporosis, giving this Gyeongsang National University School of Medicine and Gyeongsang study clinical significance for rotator cuff surgery [34, National University Changwon Hospital, Changwon, Republic of Korea 51472. 35]. 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Published: Jan 11, 2020

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