Nitrogen-Containing Bisphosphonates Downregulate Cathepsin K and Upregulate Annexin V in Osteoclasts Cultured In Vitro

Introduction Bisphosphonates are widely used in the treatment of osteoporosis; however, they are associated with the serious adverse event of bisphosphonate‐related osteonecrosis of the jaw (BRONJ). Aim The aim of this study is to assess the effects of nitrogen-containing bisphosphonates (N-PHs) on the synthesis of IL-1β, TNF-α, sRANKL, cathepsin K, and annexin V in bone cells cultured in vitro. Materials and Methods Osteoblasts and bone marrow-derived osteoclasts were cultured in vitro, subjected to treatment with alendronate, risedronate, or ibandronate at a concentration of 10−5 M for 0 to 96 h and then assayed for IL-1β, sRANKL, and TNF-α production by ELISA. Cathepsin K and Annexin V-FITC staining in osteoclasts were assessed by flow cytometry. Results There was significant downregulation of IL-1β, sRANKL, and TNF-α in experimental osteoblasts compared to control cells, and there was upregulation of IL-1β and downregulation of RANKL and TNF-α in experimental osteoclasts. Furthermore, in osteoclasts, cathepsin K expression was downregulated at 48–72 h with alendronate treatment, while risedronate treatment resulted in upregulated annexin V expression at 48 h compared to the control treatment. Conclusion Bisphosphonates added to bone cells inhibited osteoclastogenesis, which led to the downregulation of cathepsin K and induction of apoptosis in osteoclasts; these changes limited the capacity of bone remodelling and healing that may contribute to BRONJ induced by surgical dental procedures.


Introduction
Bisphosphonates (BPs) are widely used in the treatment of osteoporosis and cancer-related bone disease and reduce the incidence of pathological fractures [1]. Oral and intravenous administration of BPs is associated with adverse events of toxicity in the short and long term, including upper gastrointestinal adverse efects, hypocalcaemia, ocular complications, renal failure, and osteonecrosis of the jaws [2]. Moreover, BPs accumulate in human jaws at higher levels than the skeleton [3] and are potent inhibitors of bone resorption [4,5]. Te most efcient BPs have a nitrogen atom in one of the side chains; therefore, they are called nitrogen-containing bisphosphonates (N-BPs). N-BPs include ibandronate, alendronate, and risedronate, among others. Tese N-BPs decrease bone turnover and prevent bone loss by inhibiting the osteoclast mevalonate pathway [6][7][8][9][10][11]. Extended treatment with BPs causes severe suppression of bone turnover associated with signifcant side efects [12,13]. Bisphosphonate-related osteonecrosis of the jaw (BRONJ), which is characterized by refractory bone exposure, has been recognized as a severe side efect of treatment with BPs. Initially, it was argued that the appearance of BRONJ was due to the intravenous administration of BPs at high doses for the treatment of metastatic bone lesions or multiple myelomas. However, recent studies have indicated that BRONJ also occurs frequently in patients receiving therapeutic oral doses of BPs to treat osteoporosis [14][15][16].
As bioflms are present in the oral environment [23], dental professionals should maintain a healthy oral condition prior to BP therapy; nevertheless, some invasive dental procedures are expected after N-BPs have been prescribed. Although there is no specifc rule for the cessation of N-BPs in invasive dental procedures, these procedures must be ceased at least one to three months before and during the healing process [24]. If there was a surgical dental procedure, the administration of amoxicillin-clavulanate (875 mg) and chlorhexidine mouth rinses should be implemented. In cases of allergies to antibiotics, 300 mg of clindamycin should be indicated.
Terefore, the proposed null hypothesis of this study was to evaluate if there is a decrease in the expression of cytokines such as IL-1β, TNF-α, sRANKL Cathepsin K, and cell apoptosis with the addition of bisphosphonates to in vitro cultures of osteoclasts that may afect bone turnover.

Murine Osteoblasts.
Osteoblasts were prepared and characterized by a method previously described by Heath et al. [25] and García-López et al. [26]. Te calvaria was carefully dissected from one-week-old neonatal BALB/c mice by removing the adherent soft tissue, washed with a Tyrode solution free of Ca 2+ and Mg 2+ and placed in a 30mm Petri dish. Te small bones were cut into smaller pieces with a scalpel blade and sequentially digested with 1 mg/ml trypsin for 20 minutes. Te released cells were placed in a falcon tube with 500 mL of medium and centrifuged at 1000 rpm for 5 minutes. Te pelleted cells were resuspended in 1 : 1 F12 : Dulbecco's modifed Eagle's medium (DMEM) supplemented with 100 units/mL penicillin, 100 mg/mL streptomycin, and 20% foetal calf serum (Gibco, Invitrogen, Carlsbad, CA, USA). Cells were plated in 1.9-cm 2 24-well plates (Corning, MA, USA) at a density of 1 × 10 6 cells/well and cultured at 37°C in a humidifed atmosphere of 5% CO 2 /95% air ( Figure 1).

2.4.
Tartrate-Resistant Acid Phosphatase Assay. Osteoclastic cells were stained to identify tartrate-resistant acid phosphatase (TRAP) following a previous report [29] with a K-ASSAY ® TRAP staining kit (Kamiya Biomedical Company, Seattle, WA, USA). Briefy, cells were washed with 100 μL of PBS, fxed with 50 μL of 10% formalin for 5 minutes, and then washed three times with 250 μL of dH 2 O. One vial of the chromogenic substrate (50 μL) was added to each well, and the plates were incubated at 37°C for 20-60 minutes to allow the colour to develop. Finally, the wells were washed with dH 2 O and imaged with an inverted microscope (Motic AE2000, USA) with the New Motic Images Plus 2.0 software (New Motic Images Plus 2.0, BC, Canada). Positive TRAP staining was quantifed (>3 nuclei per cell) using the ImageJ software (ImageJ/Fiji 1.53a, NIH, USA) ( Figure 3).

Murine Osteoblasts and Osteoclasts Stimulated with BPs.
Osteoblasts and osteoclasts were stimulated separately with three diferent BPs: alendronate, risedronate, and ibandronate (all at 10 −5 M). Both types of cells were stimulated with a conditioned medium from 0 to 96 h. During the course of the experiments, a 500-μL sample of the supernatant was collected from both the control and experimental groups for 0 h to 96 h. Te collected supernatant was stored at −70°C in an ultrafreezer (New Air, USA) for subsequent analysis of IL-1β, TNF-α, and sRANKL using enzyme-linked immunosorbent assays (ELISAs; R and D Systems, Minneapolis, MN, USA; PeproTech, USA). Te absorbance at 450 nm was measured according to the manufacturer's instructions.

Flow Cytometry assay for Cathepsin K.
Osteoclastic culture medium was supplemented with alendronate or risedronate (10 −5 M) from 0 to 72 h, and a control group was treated with osteoclastic medium without BP stimulation in the presence of 2 brefeldins (BFAs) for four hours. Ten, the levels of cathepsin K were evaluated by fow cytometry according to a previous report with modifcations [30]. Te cellular analysis was performed on a BD FACSCalibur platform (Becton Dickinson, San José, CA), with a total of 5,000 events collected with CellQuest software (Becton Dickinson). Cells were harvested from the culture plate using 0.25% trypsin (Gibco) and centrifuged at 1,500 rpm for 5 minutes. Te pelleted cells were washed twice with 1 ml of 1x PBS containing 0.8% bovine serum albumin (BSA) and 0.02% sodium azide. Fixation and permeabilization were performed with an intracellular staining kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's specifcations. Osteoclastic cells were washed in 1 ml of 1x PBS plus BSA. A primary rabbit antihuman cathepsin k polyclonal antibody was incubated with the cells for 30-45 minutes at room temperature; we added a secondary rabbit anti-IgG antibody coupled to phycoerythrin isothiocyanate (PE) for 30-45 minutes at room temperature. A control sample was generated following the same steps for intracellular staining (Figure 4).

Flow Cytometry Assay for Annexin V-FITC Staining.
Osteoclast cultures were stimulated with alendronate or risedronate at 10 −5 M for 0 to 72 h, and a control group without BP stimulation was included. Te levels of Annexin V-fuorescein isothiocyanate (FITC) staining were assessed by fow cytometry. Cell analysis was performed on the BD FACSCalibur ™ platform (Becton Dickinson, San José, CA), with a total of 5,000 events collected using CellQuest software (Becton Dickinson). Cells were harvested from the culture plate using 0.25% trypsin (Gibco) and centrifuged at 1,500 rpm for 5 minutes. Te pelleted cells were washed twice with 1 ml of 1x PBS containing 0.8% BSA and 0.02% sodium azide. Cell staining was performed with an Annexin V-FITC I apoptosis detection kit from BD Pharmingen ™ following the manufacturer's specifcations. Osteoclastic cells were washed in 1 ml of 1x PBS containing BSA. Two microlitres of Annexin V + FITC and 2 μL of propidium iodide (PI) were added and incubated for 15-30 minutes in the dark at room temperature (24°C). Finally, 400 μL of 1x binding bufer was added for analysis by fow cytometry ( Figure 5).

Statistical
Analysis. Te data are expressed as the mean ± standard deviation (SD). Diferences between control and experimental cultures were determined by Student's t-test (two tails) using the GraphPad Prism 9 software   (GraphPad Software Inc., San Diego, CA, USA.) Te level of signifcance was set at P < 0.05.

Results
(i) Cytokine expression in osteoblasts.
(iii) TRAP expression in bone marrow cells.
(iv) Comparison of cathepsin K expression in osteoclasts cultured in vitro with BP stimulation evaluated by the fow cytometry.

Discussion
Tis study evaluated the efects of N-BPs on primary bone cell cultures at a dose of 10 −5 M, which is within the therapeutic range according to Chen et al. [26], for 0-72 hours as described in previous studies [26,28,29]. During the course of the experiments with osteoblasts in culture, constitutive production of IL-1β, TNF-a, and RANKL was observed in both types of cells. Te production of IL-1β, TNF-a, and IL-6 by osteoblasts synergistically enhances RANKL [28,29] and may also contribute to the stimulation, diferentiation, and formation of bone. Terefore, peripheral blood monocytes diferentiate into osteoclasts by signalling through their receptor RANKr, and large amounts of RANKL have to be expressed by osteoblasts to diferentiate mature osteoclasts, consolidate the osteoclastogenic process to initiate bone resorption, maintain the homeostatic activity coordinated by osteoblasts, and induce the expression of OPG, the natural decoy of RANKL that inhibits osteoclastogenesis, an efect mediated by the expression of IL-4 and IL-13 [26]. IL-17 is also expressed by bone cells to stimulate CatK to degrade bone matrix, which stimulates periostin and thereby moderates Wnt-β-catenin signalling to induce cortical bone formation [31].
Tis study showed downregulation followed by upregulation and then downregulation of IL-1β expression with alendronate or ibandronate treatment, while there was only downregulation with risedronate treatment in osteoblast cultures. Te N-BPs alendronate and ibandronate produced alterations in IL-1β at a therapeutic dose, and it has been shown that low concentrations of N-BPs (10 −3 M) may increase cell proliferation and reduce the proliferation of osteoblasts, while high doses (10 6−10 M) may induce apoptosis [32][33][34]. In osteoclasts, there was upregulation of IL-1β with treatment with any of the three N-PHs at a therapeutic dose (10 −5 M), which may induce proliferation of these cells and activate RANKL, as seen at low concentrations [35,36]. Nevertheless, there was also downregulation of TNF-a in the experimental group of osteoblast cultures with each of the three N-PHs, which is consistent with other studies [37,38]. Te downregulation and upregulation observed in the experimental groups of osteoclasts treated with alendronate or ibandronate and the downregulation observed with risedronate treatment could be due to the accumulation or consumption of N-BPs, which may enhance TNF-a production or inhibit osteoclast formation [39].
In relation to the expression of RANKL, both osteoclasts and osteoblasts showed downregulation of RANKL. Tus, taken together with the downregulation of IL-1β and TNF-α,  the study results show that N-PHs produce an inhibitory efect on the osteoclastogenic process, which may produce the side efects of inhibiting bone resorption as a result of osteoclast apoptosis. Terefore, there is no stimulation of bone remodelling or only low remodelling, which may result in fractures in the bone to contribute to BRONJ [9]. Moreover, another study showed that at high concentration of 5 × 10 −5 M, N-PHs may enhance OPG gene expression and strongly increase that of RANKL [40]. Furthermore, cathepsin K and annexin V were evaluated in only osteoclast cultures treated with alendronate or risedronate; nonetheless, these results, similar to those of another study [41], demonstrated that alendronate and risedronate at a dose of 10 −5 M inhibited osteoclast proliferation and induced apoptosis in vitro [34,42].
Cathepsin K is expressed at high levels in osteoclasts and functions as a protease, being the primary enzyme responsible for the degradation of type I collagen, which composes the organic matrix; cathepsin K is able to completely dissolve human cortical bone [43]. Nevertheless, cathepsin K was downregulated in osteoclast cultures by the N-PHs studied, which suggested a lack of matrix degradation, as cathepsin K production by osteoclasts should induce bone remodelling stimulated by periostin degradation [31,44,45].
Te evaluation of Annexin V-FITC staining in the osteoclast culture group showed signifcant upregulation in the experimental group compared to the control group, as it is known that BPs induce osteoclast apoptosis via the inhibition of farnesyl pyrophosphate synthase through the mevalonate pathway, which produces prenylation in osteoclasts that contributes to the inhibition of bone resorption by disrupting osteoclast function [8]. However, it has been shown that apoptotic bodies derived from osteoclasts possess osteogenic potency mediated by activating RANKL reverse signalling in preosteoblasts [46] to stimulate osteoblasts and other cells around the bone; therefore, it has been suggested that the administration of calcitonin in conjunction with N-PHs may prolong osteoclast survival by inhibiting apoptosis [47].
Te efects of apoptosis in endothelial cells induced by BPs and the impaired viability of fbroblasts and oral keratinocytes may induce vascular damage and produce a lack of circulation in the jawbones, which afects bone healing. Likewise, the presence of Actinomyces species in signifcant numbers led Naik and Russo [48] to suggest a critical role for these organisms, among other factors, in the development of BRONJ.
Within the limitations of our in vitro study, these fndings confrm our hypothesis, and we concluded from  a clinical standpoint that murine osteoblasts and bone marrow-derived osteoclasts stimulated with a therapeutic dose of N-PHs (10 −5 M) showed inhibition of IL-1β, TNF-α, and RANKL, although IL-1β and TNF-α contribute to the expression of RANKL, which stimulates osteoclast activity. Tis was inhibited by the N-PHs and there was also downregulation of cathepsin K. Terefore, there is no stimulation of periostin to activate cortical bone formation and the stimulation of apoptosis in osteoclasts, which does not allow the development of mature osteoclasts to induce bone resorption. Tese changes limited the capacity of bone remodelling and healing that may contribute to BRONJ induced by surgical dental procedures, among other factors.

Data Availability
Te data that support the fndings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.