Nitrogen-containing bisphosphonate for vascular calcification: animal experiments and a meta-analysis

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Methods: In our animal experiments, the SD rats were randomly divided into control group, VC group, low-dose Zoledronic acid (ZOL) group (20ug/kg) and high-dose ZOL group (100ug/kg). The calcification of aortic arch was detected by alizarin red staining. The calcium content of aortic arch was detected. In our meta-analysis, databases including PubMed, EMbase, the Cochrane Library, CNKI , Wanfang database were searched from the inception to December 20th, 2023. Eligible studies comparing N-BP versus no N-BP in the treatment of VC were included. Results: In our animal experiment, compared with the VC group, the red-stained calcification structure in the low-dose ZOL group were slightly reduced, and the red-stained calcification structure in the high-dose ZOL group were significantly reduced. The calcium content in the low-dose ZOL group was slightly lower than that in the VC group, but there was no statistical difference(P > 0.05).The calcium content in the high-dose ZOL group was significantly lower than that in the VC group(P 0.05). Our meta-analysis from animal studies showed that N-BP did not reduce arterial calcification score significantly(P > 0.05), but N-BP reduce arterial calcification area, arterial calcium and PO4 content(P < 0.05). Conclusions: Our animal experiment revealed that high-dose ZOL inhibited VC, but low-dose ZOL did not inhibit VC significantly. Our meta-analysis from humans studies revealed that N-BP was not effective for the treatment of VC, but Our meta-analysis result from animal studies was inclined to supporting the role of N-BP in inhibiting VC. Nitrogen-containing bisphosphonate Vascular calcification Animal experiments Meta-Analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Vascular calcification（VC） is defined as the deposition of phosphate-calcium crystals on the cardiovascular system, which increases the incidence and mortality of cardiovascular diseases[1,2]. At present, there is no effective treatment for VC. Therefore, it is of great significance to explore the mechanism of VC and find drug to inhibit VC. In recent years, epidemiological and clinical studies have shown that patients with low bone density have a significantly increased risk of VC [3,4]. Some studies also suggested that drugs that were effective for osteoporosis might be effective for VC[5,6]. Bisphosphonate is the first choice for the treatment of osteoporosis [7], which are divided into non-nitrogen-containing bisphosphonate（Non-N-BP） and nitrogen-containing bisphosphonate（N-BP） according to the chemical structure and molecular mechanism. N-BP,such as zoledronic acid (ZOL),is the second or newer generation of bisphosphonate, which has a high affinity for bone tissue and can inhibit the activity of farnesyl pyrophosphate synthetase, leading to apoptosis of osteoclasts [8]. At present, N-BP has been studied for the treatment of VC in humans and animals. However,it is uncertain about the efficacy of N-BP in the treatment of VC.Therefore, we explored the effect of N-BP on VC by animal experiments. At the same time, we conducted a meta-analysis based on the studies of humans and animals to evaluate efficacy of N-BPs in the treatment of VC. Methods Animal experiments Experimental protocol Twenty-four male SD rats with weight 250-300 g, 8 weeks old, were purchased from Huachuang Xinuo Pharmaceutical Technology（China）.All rats were randomly divided into four groups with six rats in each group,which were control group, VC group, low-dose ZOL group and high-dose ZOL group. The VC model was established in VC group, low-dose ZOL group and high-dose ZOL group. The preparation method of the VC model was as follows [9,10] . 450 mg/kg/day of adenine was given by gavage in the first week, 300 mg/kg/day of adenine was given by gavage in the second to fourth week, and high phosphorus feed(1.8% phosphorus,1% calcium ) was given at the same time. The low-dose and high-dose ZOL groups were given intraperitoneal injection of 20 and 100 ug/kg ZOL once a week for 4 weeks. ZOL was given on the first day of VC modeling. The VC group was given intraperitoneal injection of normal saline at the same dose. The control group was given ordinary feed(0.6% phosphorus,1% calcium ) and intraperitoneal injection of normal saline with equal dose. After 4 weeks, the rats were euthanized by intraperitoneal injection of 150mg/kg phenobarbital, and the aorta tissues of the rats were extracted.The animal experiments were approved by the Institutional Animal Care Committee at Zhujiang Hospital, Jiangsu University, China (UJS-IACUC-AP-2023030310) . Alizarin red staining Alizarin red staining was performed to detect calcium deposition. The aortic arch tissues of rats in each group were paraffin embedded, sliced,roasted, dewaxed and dehydrated. The tissue sections were incubated with 1% Alizarin red S solution (Solarbio, China) at room temperature for 1 hour, and washed with double steaming water. Then, the tissue sections were sealed and the images were collected by microscopy. Determination of calcium content The calcium contents were determined by Calcium Assay Kit (Beyotime, China). The aortic arch tissues taken from each group were put into a centrifugal tube, added with the sample lysate, homogenized by the grinder, centrifuged at 12000 rpm at 4℃ for 5 minutes, and taken the supernatant . Then, the samples were added to the detection buffer and color developing solution, and incubated at room temperature for 10 min. Finally, the samples were measured at the 575nm absorbance by microplate reader, and the calcium content of the samples were calculated by a standard curve. Statistical analysis All the data were presented as mean ± SD. Differences among the groups were compared using one-way ANOVA. All statistical analyses were performed by SPSS 20.0 software. Graphs were plotted by GraphPad Prism 8.0 software. P < 0.05 was considered as statistically significant. Meta-analysis Search strategy Our meta-analysis was reported in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . We searched PubMed, EMbase,the Cochrane Library, CNKI(China National Knowledge Infrastructure) ,Wanfang database from the inception to December 20th, 2023. The combined text and MeSH terms included (“nitrogen-containing bisphosphonate” or “bisphosphonate” or “diphosphonate” or “minodronicacid” or “alendronate” or “risedronate” or “ibandronate” or “zoledronate” or “pamidronate”) and (“Vascular Calcification” . In addition, the relevant references and cited papers were searched manually to identify additional studies meeting the inclusion criteria. There was no language restrictions. Inclusion and exclusion criteria The inclusion criteria were (1) human randomized controlled trials (RCTs), human observational studies or animal experiments that compared N-BP versus no N-BP treatment, (2) outcomes included at least one of the indicators, such as arterial calcification score, arterial calcification area, arterial calcium or PO 4 content determination. Exclusion criteria were (1) case series, comments, reviews, (2) no control group, and (3) lack of relevant outcomes data. Data extraction and quality assessment Data were extracted independently by two investigators using standard data extraction forms. In the case of disagreement, a third investigator was consulted. We extracted characteristics including first author, year of publication, location, study design, population, specific methods of experiment and control group, follow-up period, sample size, mean age, sex, weight, treatment outcomes. The Cochrane assessment tool was used to assess the quality of human RCTs[11], whereas the Newcastle–Ottawa scale (NOS) was used to assess human non-randomized studies[12]. The Systematic Review Centre for Laboratory animal experimentsation (SYRCLE) tool was used to assess the quality of animal studies[13]. Statistical analysis This meta-analysis was performed using Review Manager Version 5.3 (Cochrane Collaboration).We summarized treatment outcomes as weighted mean differences for continuous variables with 95% confidence intervals (CIs). P 50% and P < 0.10 to indicate significant heterogeneity. Meta-analysis with insignificant heterogeneity was performed using the fixed-effects model. For meta-analyses with significant heterogeneity, the random-effects model was used. Publication bias was assessed using subgroup analysis or sensitivity analyses. Results The results of animal experiments Results of alizarin red staining in rats with VC treated with ZOL As shown in Figure 1 , the red-stained structure represented calcification structure in the aortic arch.The control group had no obvious red calcification structure. The VC group had obvious red calcification structure. Compared with the VC group, the red calcification structure in the low-dose ZOL group were slightly reduced, and the red calcification structure in the high-dose ZOL group were significantly reduced. Results of calcium content determination in rats with VC treated with ZOL The results of calcium content determination in the aortic arch were shown in Figure 2 . The calcium content in the VC group was significantly higher than that in control group (P 0.05).The calcium content in the high-dose ZOL group was significantly lower than that in the VC group(P < 0.05). The results of meta-analysis Study selection and characteristics A flow diagram of the selection process is shown in Figure 3 . Finally, a total of eleven studies were included in this meta-analysis[14-24]. Of the eleven studies, three were human studies and eight were animal studies. The risk of bias in the included human RCTs were moderate. The human non-randomized studies achieved scores of ≥6 points, which were considered to be of high quality. The risk of bias in the included animal studies was moderate. The baseline characteristics of human studies are listed in Table 1 and the baseline characteristics of animal studies are listed in Table 2 . The Cochrane assessments are listed in Table 3 , the NOS assessments are listed in Table 4 , and SYRCLE assessments are listed in Table 5 . The result of arterial calcification score in human and animal studies The data about the arterial calcification score after the N-BP or no N-BP treatment were reported in three human and two animal studies. A subgroup analysis was performed according to human and animal study. In the human subgroup, there was no significant difference between the N-BP and no N-BP treatment group concerning the arterial calcification score (SMD -0.11, 95% CI -0.40—0.18, P=0.46). In the animal subgroup, there was no significant difference between the N-BP and no N-BP treatment group concerning the arterial calcification score (SMD -1.45, 95% CI -3.10—0.20, P=0.09). When the results of all subgroups were summarized, there was also no statistical difference between the N-BP and no N-BP treatment concerning the arterial calcification score (SMD -0.59, 95% CI -1.27—0.10, P=0.09) （ Figure 4 ） . The result of arterial calcification area in animal studies The data about the arterial calcification area after the N-BP or no N-BP treatment were only reported in two animal studies.The arterial calcification area in the N-BP treatment group were significantly lower than that in the no N-BP treatment group (SMD -2.74, 95% CI -3.48—-2.00, P< 0.00001) （ Figure 5 ） . The result of the arterial calcium content in animal studies The data about the arterial calcium content after the N-BP or no N-BP treatment were only reported in four animal studies.The arterial calcium content in the N-BP treatment group was significantly lower than that in the no N-BP treatment group (SMD -4.29, 95% CI -7.51—-1.07, P= 0.009) （ Figure 6 ） . The result of the arterial PO 4 content in animal studies The data about the arterial PO 4 content after the N-BP or no N-BP treatment were only reported in two animal studies. In the study of Price(2001), the groups of alendronate treatment were divided into two groups according to the dose of alendronate, which were at a dose of 0.025 and 0.25 mg/kg/day. The arterial PO 4 content in the N-BP treatment group was significantly lower than that in the no N-BP treatment group (SMD -2.41, 95% CI -3.65—-1.18, P= 0.0001) （ Figure 7 ） . Sensitivity analyses The sensitivity analyses for all results after the N-BP or no N-BP treatment was used to judge the dependability of the results. In the animal subgroup concerning the arterial calcification score, when we deleted the study of Synetos（2018）,the arterial calcification score in the N-BP treatment group was lower than that in the no N-BP treatment group(P < 0.05). Other results remained unchanged when we deleted one study at a time. Discussion The purpose of our study was to explore the effect of N-BP on VC by animal experiments and a meta-analysis. ZOL is a type of N-BP. In our animal experiments, high-dose ZOL inhibited VC, but low-dose ZOL did not inhibit VC significantly. Our meta-analysis from human studies showed that N-BP did not inhibit VC. In our meta-analysis from animal studies, the results regarding the effect of N-BP on VC were inconsistent. Our meta-analysis from animal studies showed that N-BP did not reduce arterial calcification score significantly, but N-BP reduced arterial calcification area, arterial calcium and PO 4 content. Notably, when deleting the animal study of Synetos(2018), the overall results of animal study supported the role of N-BP in inhibiting VC. Recent studies showed that drugs that were effective for osteoporosis might be effective for VC[ 5 , 6 ]. N-BP is the frequently-used treatment for osteoporosis. According to our meta-analysis, N-BP was not an effective treatment for VC in humans, but the results of animal studies was inclined to supporting the role of N-BP in inhibiting VC. In the previous cell studies, N-BP also inhibited osteogenic differentiation and mineralization of vascular smooth muscle cells that was the drive step of VC[ 25 , 26 ]. The reason why N-BP were not effective on VC in humans was not clear, which might be related to the dosage, type, potency and administration route of N-BP [ 27 , 28 ]. We also conducted animal studies to explore the role of N-BP in inhibiting VC. In our animal experiments, high-dose ZOL inhibited VC, but low-dose ZOL did not inhibit VC significantly. The low and high doses of ZOL given to rats were 20 and 100ug/kg. The selection of the ZOL dose in our animal experiments was based on the ZOL dose for the treatment of osteoporosis in humans. The peak serum concentration of ZOL in human body following a 4 mg dose administration is from 1 to 5 µM[ 29 ]. However,low-dose ZOL did not inhibit VC significantly,which might be due to the fact that the serum concentration of ZOL after low-dose ZOL administration could not be sustained for the effective treatment of VC, or because of the different affinity of ZOL to bone and vascular tissues[ 27 , 28 , 30 ]. There were some limitations in our study. Firstly, there were differences in the dose, type, potency, administration route of N-BP among the studies included in our meta-analysis. Secondly, The number of studies included in our meta-analysis were still too small. Conclusions Our animal experiments revealed that high-dose ZOL inhibited VC, but low-dose ZOL did not inhibit VC significantly. Our meta-analysis from humans studies revealed that N-BP was not effective for the treatment of VC, but Our meta-analysis result from animal studies was inclined to supporting the role of N-BP in inhibiting VC. To further confirm the conclusion, more large human RCTs and animal experiments are necessary. Abbreviations VC:Vascular calcification; Non-N-BP:Non-nitrogen-containing bisphosphonate; N-BP: Nitrogen-containing bisphosphonate; ZOL:Zoledronic acid; PRISMA:Preferred Reporting Items for Systematic Reviews and Meta-Analyses; CNKI:China National Knowledge Infrastructure; RCTs:Randomized controlled trials;NOS:Newcastle–Ottawa scale;SYRCLE:Systematic Review Centre for Laboratory animal experimentsation; CIs:Confidence intervals Declarations Ethics approval and consent to participate The animal experiments were approved by the Institutional Animal Care Committee at Zhujiang Hospital, Jiangsu University, China (UJS-IACUC-AP-2023030310) . This study is reported in accordance with ARRIVE guidelines. Consent for publication Not applicable. Availability of data and materials Data is provided within the manuscript or supplementary information files. Competing interests The authors declare that they have no competing interests. Funding The research was supported by the Open Research Project of Key Laboratories in Jiangsu Province Universities (No.XZSYSKF2023022), the Young Talent Development Plan of Changzhou Health Commission (No.CZQM2021026) . Authors ’ contributions Wei Xu and Guoyuan Lu contributed to the conceptualization. Wei Xu, Guoyuan Lu and Lifeng Gong contributed to the animal experiments.Wei Xu,Weigang Tang and Wei Jiang contributed to the meta-analysis. Wei Xu contributed to the analysis of the data and production of figures and tables. Wei Xu and Guoyuan Lu contributed to the writing. All authors approved final manuscript. Acknowledgements The research was supported by Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy. References Villa-Bellosta R. Vascular Calcification: Key Roles of Phosphate and Pyrophosphate. Int J Mol Sci. 2021;22(24):13536. Yuan C, Ni L, Zhang C, Hu X, et al.Vascular calcification: New insights into endothelial cells. Microvasc Res. 2021;134:104105. 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Diwan V, Brown L, Gobe GC. Adenine-induced chronic kidney disease in rats. Nephrology (Carlton). 2018;23(1):5-11. Furlan AD.Malmivaara A, Chou R, et al. 2015 Updated method guideline for systematic reviews in the Cochrane back and neck group.Spine.2015;40:1660-1673. Stang A. Critical evaluation of the Newcastle-Ottawa scale for the assessment of the quality of nonrandomized studies in meta-analyses. Eur J Epidemiol .2010;25:603-605. Hooijmans CR, Rovers MM, de Vries RB, et al. SYRCLE's risk of bias tool for animal studies. BMC Med Res Methodol. 2014;14:43. Price PA, Faus SA, Williamson MK. Bisphosphonates alendronate and ibandronate inhibit artery calcification at doses comparable to those that inhibit bone resorption. Arterioscler Thromb Vasc Biol. 2001;21(5):817-824. Synetos A, Toutouzas K, Drakopoulou M, et al. Inhibition of Aortic Valve Calcification by Local Delivery of Zoledronic Acid-an Experimental Study. J Cardiovasc Transl Res. 2018;11(3):192-200. 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Artery calcification in uremic rats is increased by a low protein diet and prevented by treatment with ibandronate. Kidney Int. 2006;70(9):1577-1583. Torregrosa JV, Fuster D, Gentil MA, et al. Open-label trial: effect of weekly risedronate immediately after transplantation in kidney recipients. Transplantation. 2010;89(12):1476-1481. Okamoto M, Yamanaka S, Yoshimoto W, et al.Alendronate as an effective treatment for bone loss and vascular calcification in kidney transplant recipients. J Transplant. 2014;2014:269613. Hill JA, Goldin JG, Gjertson D, et al. Progression of coronary artery calcification in patients taking alendronate for osteoporosis. Acad Radiol. 2002;9(10):1148-1152. Cutini PH, Rauschemberger MB, Sandoval MJ, et al. Vascular action of bisphosphonates: In vitro effect of alendronate on the regulation of cellular events involved in vessel pathogenesis. J Mol Cell Cardiol. 2016;100:83-92. Zhou S, Fang X, Xin H, et al.Effects of alendronate on the Notch1‑RBP‑Jκ signaling pathway in the osteogenic differentiation and mineralization of vascular smooth muscle cells. Mol Med Rep. 2013;8(1):89-94. Hildebrand S, Cunningham J. Is there a role for bisphosphonates in vascular calcification in chronic kidney disease?. Bone. 2021;142:115751. Caffarelli C, Montagnani A, Nuti R, et al. Bisphosphonates, atherosclerosis and vascular calcification: update and systematic review of clinical studies. Clin Interv Aging. 2017;12:1819-1828. Wu L, Zhu L, Shi WH, Zhang J, et al.Zoledronate inhibits the proliferation, adhesion and migration of vascular smooth muscle cells. Eur J Pharmacol. 2009;602(1):124-131. Pawade TA, Doris MK, Bing R, et al. Effect of Denosumab or Alendronic Acid on the Progression of Aortic Stenosis: A Double-Blind Randomized Controlled Trial. Circulation. 2021;143(25):2418-2427. Tables Table1. Characteristics of the included human studies. Study (year) Country Design Follow-up period Population Treatment group Comparison group Sample size (Treatment group/ Comparion group) Mean age (years) Male (n,%) Assessment of VC Hill (2002) America Matched- control study 24 months Patients with osteoporosis Alendronate sodium 10 mg po daily for a mean of 24 months No treatment 56 56 - - Coronary arterial calcification scores by CT Torregrosa （2010） Spain RCT 12 months Kidney transplant patients Risedronate 35 mg po weekly, Calcium and vitamin D Calcium and vitamin D 52 49 47.4 ± 14.1 50.7 ± 15.5 28（53.8） 27（55.1） VC score by abdomen and hand X-ray Okamoto （2014） Japan Prospective study 24 months Kidney Transplant patients Alendronate 35 mg/week for 24 months No treatment 5 7 52.8 ± 12.6 52.9 ± 7.3 4（80） 4（57） Abdominal arterial calcification by CT Table 2. Characteristics of the included animal studies. Study (year) Country Species（Age，Sex，Weight） The disease of animal model Treatment group Comparison group Sample size (Treatment group/ Comparion group) Follow-up period Outcome evaluated Price（2006） USA Rat（13 weeks，371.4±8.4 g） Uremia model using 0.75% adenine diet for 4 weeks 2.5% protein diet， Ibandronate ic at a dose of 0.25 mg/kg/day beginning 12 days after the start of the adenine diet 2.5% Protein diet 5 6 4 weeks after the start of the adenine diet Thoracic aorta calcium and PO 4 determined colorimetrically Synetos（2018） Greece New Zealand rabbits （3 months，male，3.7±0.2 kg） Aortic valve stenosis model using vitamin D -enriched atherogenic diet for 3 weeks Local delivery of a mixture containing 500 μg/l zoledronate on the cusps of the aortic valve using a dedicated balloon catheter A placebo mixture administered with the same process 8 8 28 days after local drug delivery Progression of aortic valve calcification by PET/CT Imaging，aortic valve calcification area by von Kossa staining Synetos（2014） Greece New Zealand rabbits（3.8 ± 0.5 kg） Arterial calcification and atherosclerosis model using vitamin D -enriched atherogenic diet for 3 weeks Local delivery of a mixture containing 500 μg/l zoledronate on the vascular wall of the iliac artery, using a dedicated balloon catheter A placebo mixture administered on the contralateral iliac artery with the same process 16 28 days after local drug delivery The calcium content of iliac artery by computer-assisted histomorphometry Li Huan（2006） China SD Rat （4 weeks，male） Arterial calcification model using warfarin and vitamin D for 4 days Alendronate ic at a dose of 1 mg/kg/day starting 4 days before the first warfarin and vitamin D injection Equal amounts of saline ic 6 6 4 days after the first warfarin and vitamin D injection Aortic calcification area by von Kossa staining Price（2001） USA Rats(42 days，male) Arterial calcifica tion model using warfarin for 4 weeks Alendronate ic at a dose of 0.025 or 0.25 mg/kg/day starting 4 days before the first warfarin injection No treatment 8 8 4 weeks after the first warfarin injection Carotid PO 4 determined colorimetrically Jia peng（2012） China SD Rat （5 weeks，male，150-200g） Arterial calcification model using warfarin and vitamin D for 4 days Alendronate ic at a dose of 1 mg/kg/day starting 4 days before the first warfarin and vitamin D injection Equal amounts of saline ic 20 20 8 days after the first alendronate injection Thoracic aorta calcium determined colorimetrically Yang liyuan (2008) China SD Rat（4 weeks，male，200-250g） Arterial calcification model using warfarin and vitamin D for 4 days Alendronate ic at a dose of 1 mg/kg/day starting 4 days before the first warfarin and vitamin D injection Equal amounts of saline ic 6 6 8 days after the first alendronate injection Thoracic aorta calcium determined colorimetrically Guo liming（2008） China SD Rat（female） Arterial calcification model using subtotal nephrectomy with vitamin D ， high phosphorus and calcium diet for 4 weeks Alendronate ic at a dose of 0.25 mg/kg/day after nephrectomy No treatment 10 10 4 weeks after nephrectomy Thoracic aorta calcium by automatic biochemical measurement Table 3 . Quality assessment of randomized control trial. Study Random sequence generation Allocation concealment Blinding of participants and personnel Incomplete outcome data Selective reporting Other bias Torregrosa （2010） ? + ? + + ? The randomized control trial was evaluated using the Cochrane assessment tool.+,low risk of bias;?,unclear risk of bias; -,high risk of bias. Table 4. Quality assessment of non-randomized control trial. Studies Selection Comparability Outcome Score Hill（2002） ★★★★ ★ ★★ 7 Okamoto（2014） ★★★★ ★ ★★ 7 The Cohort studies were evaluated using the Newcastle-Ottawa scale,which are comprised of the study of selection (Representativeness of the exposed group,Representativeness of the non exposed group,Ascertainment of exposure,Demonstration that outcome of interest was not present at start of study), group comparability(Controls for the most important factor,Controls for any additional factor),outcome measures (Assessment of outcome,Was follow-up long enough for outcomes to occur,Adequacy of follow up of cohorts), a total of nine points. ★ , 1 point. Table 5. Quality assessment of animal experiment Studies ① ② ③ ④ ⑤ ⑥ ⑦ ⑧ ⑨ ⑩ Price（2006） ? ? ? ? ? ? ? + + ? Synetos（2018） + + ? + + ? + + + ? Synetos（2014） ? ? ? ? ? ? + + + ? Li Huan（2006） + ? ? ? ? ? ? + + ? Price（2001） ? ? ? ? ? ? + + + ? Jia peng（2012） + ? ? ? ? ? ? + + ? Yang liyuan(2008) + ? ? ? ? ? ? + + ? Guo liming（2008） + ? ? ? ? ? ? + + ? The animal experiment was evaluated using the SYRCLE tool.① allocation sequence generated；② baseline similar；③ allocation concealment ④ random housing animal；⑤ investigator/caregivers blinded；⑥ random outcome assessment；⑦ outcome assessor blinded； ⑧ incomplete outcome adequatedly adressed；⑨ Selective outcome reporting；⑩ other risks of bias; +,low risk of bias;?,unclear risk of bias; -,high risk of bias. Additional Declarations No competing interests reported. Supplementary Files Supplementarytable1.supplementarymaterialtofigure2..docx Cite Share Download PDF Status: Published Journal Publication published 31 Jan, 2025 Read the published version in BMC Cardiovascular Disorders → Version 1 posted Editorial decision: Revision requested 26 Nov, 2024 Reviews received at journal 26 Nov, 2024 Reviewers agreed at journal 22 Nov, 2024 Reviewers agreed at journal 19 Nov, 2024 Reviewers agreed at journal 18 Nov, 2024 Reviewers agreed at journal 17 Nov, 2024 Reviews received at journal 14 Jun, 2024 Reviewers agreed at journal 14 Jun, 2024 Reviewers invited by journal 16 May, 2024 Editor assigned by journal 16 May, 2024 Editor invited by journal 01 Mar, 2024 Submission checks completed at journal 29 Feb, 2024 First submitted to journal 20 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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0.05, n=6.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3972838/v1/eb0de822b13c1e1f79c845aa.jpg"},{"id":51966560,"identity":"206cc23b-0a63-4153-b591-c4b6dc10a238","added_by":"auto","created_at":"2024-03-04 17:46:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":259489,"visible":true,"origin":"","legend":"\u003cp\u003eFlow diagram of the literature search.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3972838/v1/7fcb6a18daf516ab9e1cb875.jpg"},{"id":51967006,"identity":"b4af680f-e8a6-47ed-8778-b330164cd764","added_by":"auto","created_at":"2024-03-04 18:02:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":547086,"visible":true,"origin":"","legend":"\u003cp\u003eForest plots comparing the arterial calcification score between N-BP and no N-BP treatment group in human and animal studies.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3972838/v1/621fef9d8afaaf330b98a88b.jpg"},{"id":51966564,"identity":"aa28a36d-3eb0-4874-9b94-fb93e5e20397","added_by":"auto","created_at":"2024-03-04 17:46:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":254609,"visible":true,"origin":"","legend":"\u003cp\u003eForest plots comparing the VC area between N-BP and no N-BP treatment group in animal studies.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3972838/v1/31348b492cdc802e4f83e6db.jpg"},{"id":51966565,"identity":"0d88caf0-7c13-430d-8c3e-de20936cbc79","added_by":"auto","created_at":"2024-03-04 17:46:45","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":322213,"visible":true,"origin":"","legend":"\u003cp\u003eForest plots comparing the arterial calcium content between N-BP and no N-BP treatment group in animal studies.\u003c/p\u003e","description":"","filename":"floatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3972838/v1/cbb94cd03514fa6b40298bc7.jpg"},{"id":51966566,"identity":"b890baa2-fb1d-4b64-886b-3b64a4b62b8d","added_by":"auto","created_at":"2024-03-04 17:46:45","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":286195,"visible":true,"origin":"","legend":"\u003cp\u003eForest plots comparing the arterial PO\u003csub\u003e4\u003c/sub\u003e content between N-BP and no N-BP treatment group in animal studies. \u0026amp; Alendronate at a dose of 0.025 mg/kg/day in Price(2001) study , # Alendronate at a dose of 0.25 mg/kg/day in Price(2001) study.\u003c/p\u003e","description":"","filename":"floatimage7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3972838/v1/48aaf78d379dff9cd07a81b5.jpg"},{"id":75351398,"identity":"ab7e4635-8bb2-4086-8805-c01ce480c206","added_by":"auto","created_at":"2025-02-03 16:10:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3187974,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3972838/v1/4d397c58-f28d-43ff-aa9e-f7b36374ba40.pdf"},{"id":51966793,"identity":"98cdb0f4-14f1-4b5f-af08-6727391255cb","added_by":"auto","created_at":"2024-03-04 17:54:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12719,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable1.supplementarymaterialtofigure2..docx","url":"https://assets-eu.researchsquare.com/files/rs-3972838/v1/ba718c9b481ee4bcdc1cd50b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nitrogen-containing bisphosphonate for vascular calcification: animal experiments and a meta-analysis","fulltext":[{"header":"Background","content":"\u003cp\u003eVascular calcification（VC）\u0026nbsp;is defined as the deposition of phosphate-calcium crystals on the cardiovascular system, which increases the \u0026nbsp;incidence \u0026nbsp; and mortality of cardiovascular \u0026nbsp; diseases[1,2]. At present, there is no effective treatment for VC. Therefore, it is of great significance to explore the mechanism of VC and find drug to inhibit VC.\u003c/p\u003e\n\u003cp\u003eIn recent years, epidemiological and clinical studies have shown that patients with low bone density have a significantly increased risk of VC [3,4]. Some studies also suggested that drugs that were effective for osteoporosis might be effective for VC[5,6]. Bisphosphonate is the first choice for the treatment of osteoporosis [7], which are divided into non-nitrogen-containing bisphosphonate（Non-N-BP） and nitrogen-containing bisphosphonate（N-BP） according to the chemical structure and molecular mechanism. N-BP,such as zoledronic acid (ZOL),is the second or newer generation of bisphosphonate, which has a high affinity for bone tissue and can inhibit the activity of farnesyl pyrophosphate synthetase, leading to apoptosis of osteoclasts [8]. At present, N-BP has been studied for the treatment of VC in humans and animals. However,it is uncertain about the efficacy of N-BP in the treatment of VC.Therefore, we explored the effect of N-BP on VC by animal experiments. At the same time, we conducted a meta-analysis based on the studies of humans and animals to evaluate efficacy of N-BPs in the treatment of VC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExperimental protocol\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty-four\u0026nbsp;male SD rats with weight 250-300 g, 8 weeks old, were purchased from Huachuang Xinuo Pharmaceutical Technology（China）.All rats were randomly divided into four groups with six rats in each group,which were control group, VC\u0026nbsp;group, low-dose ZOL group and high-dose ZOL group. The VC model was established in VC group, low-dose ZOL group and high-dose ZOL group. The preparation method of the VC model was as follows [9,10] . 450 mg/kg/day of adenine was given by gavage in the first week, 300 mg/kg/day of adenine was given by gavage in the second to fourth week, and high phosphorus feed(1.8% phosphorus,1% calcium ) was given at the same time. The low-dose and high-dose ZOL groups were given intraperitoneal injection of 20 and 100 ug/kg ZOL once a week for 4 weeks. ZOL was given on the first day of VC modeling. The VC group was given intraperitoneal injection of normal saline at the same dose. The control group was given ordinary feed(0.6% phosphorus,1% calcium ) and intraperitoneal injection of normal saline with equal dose. After 4 weeks, the rats were euthanized by intraperitoneal injection of 150mg/kg phenobarbital, and the aorta tissues of the rats were extracted.The animal experiments were approved by the Institutional Animal Care Committee at Zhujiang Hospital, Jiangsu University, China (UJS-IACUC-AP-2023030310) .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAlizarin red staining \u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlizarin red staining was performed to detect calcium deposition. The aortic arch tissues of rats in each group were paraffin embedded, sliced,roasted, dewaxed and dehydrated. The tissue sections were incubated with 1% Alizarin red S solution (Solarbio, China) at room temperature for 1 hour, and washed with double steaming water. Then, the tissue sections were sealed and the images were collected by microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermination of calcium content\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe calcium contents were determined by Calcium Assay Kit\u0026nbsp;(Beyotime, China). The aortic arch tissues taken from each group were put into a centrifugal tube, added with the sample lysate, homogenized by the grinder, centrifuged at 12000 rpm at 4℃\u0026nbsp;for 5 minutes, and taken the supernatant . Then, the samples were added to the detection buffer and color developing solution, and incubated at room temperature for 10 min. Finally, the samples were measured at the 575nm absorbance by microplate reader, and the calcium content of the samples were calculated by a standard curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data were presented as mean\u0026nbsp;\u0026plusmn;\u0026nbsp;SD. Differences among the groups were compared using one-way ANOVA. All statistical analyses were performed by SPSS 20.0 software. Graphs were plotted by GraphPad Prism 8.0 software. P \u0026lt; 0.05 was considered as statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeta-analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSearch strategy\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur meta-analysis was reported in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) .\u0026nbsp;We searched PubMed, EMbase,the Cochrane Library, CNKI(China National Knowledge Infrastructure) ,Wanfang database from the inception to December 20th, 2023. The combined text and MeSH terms included (\u0026ldquo;nitrogen-containing bisphosphonate\u0026rdquo; or \u0026ldquo;bisphosphonate\u0026rdquo; or \u0026ldquo;diphosphonate\u0026rdquo; or \u0026ldquo;minodronicacid\u0026rdquo; or \u0026ldquo;alendronate\u0026rdquo; or \u0026ldquo;risedronate\u0026rdquo; or \u0026ldquo;ibandronate\u0026rdquo; or \u0026ldquo;zoledronate\u0026rdquo; or \u0026ldquo;pamidronate\u0026rdquo;) and (\u0026ldquo;Vascular Calcification\u0026rdquo; . In addition, the relevant references and cited papers were searched manually to identify additional studies meeting the inclusion criteria. There was no language restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInclusion and exclusion criteria\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inclusion criteria were (1) human randomized controlled trials (RCTs), human observational studies or animal experiments that compared\u0026nbsp;N-BP\u0026nbsp;versus no\u0026nbsp;N-BP\u0026nbsp;treatment, (2) outcomes included at least one of the indicators, such as arterial calcification score,\u0026nbsp;arterial\u0026nbsp;calcification\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e area,\u0026nbsp;arterial\u0026nbsp;calcium or PO\u003csub\u003e4\u003c/sub\u003e content determination.\u003c/p\u003e\n\u003cp\u003eExclusion criteria were (1) case series, comments, reviews, (2) no control group, and (3) lack of relevant outcomes data.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData extraction and quality assessment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were extracted independently by two investigators using standard data extraction forms. In the case of disagreement, a third investigator was consulted. We extracted characteristics including first author, year of publication, location, study design, population, specific methods of experiment and control group, follow-up period, sample size, mean age, sex, weight, treatment outcomes. The Cochrane assessment tool was used to assess the quality of\u0026nbsp;human\u0026nbsp;RCTs[11], whereas the Newcastle\u0026ndash;Ottawa scale (NOS) was used to assess\u0026nbsp;human\u0026nbsp;non-randomized studies[12]. The Systematic Review Centre for Laboratory animal experimentsation (SYRCLE) tool was used to assess the quality of animal studies[13].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis meta-analysis was performed using Review Manager Version 5.3 (Cochrane Collaboration).We summarized treatment outcomes as weighted mean differences for continuous variables with 95% confidence intervals (CIs). P \u0026lt; 0.05 was considered statistically significant.We used the I\u003csup\u003e2\u003c/sup\u003e statistic to assess heterogeneity among studies. We considered I\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e\u0026gt; 50% and P \u0026lt; 0.10 to indicate significant heterogeneity. Meta-analysis with insignificant heterogeneity was performed using the fixed-effects model. For meta-analyses with significant heterogeneity, the random-effects model was used. Publication bias was assessed using subgroup analysis or sensitivity analyses.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe results of animal experiments \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eResults of alizarin red staining in rats with VC treated with ZOL\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in \u003cstrong\u003eFigure 1\u003c/strong\u003e, the red-stained structure represented calcification structure in the aortic arch.The control group had no obvious red calcification structure. The VC group had obvious red calcification structure. Compared with the VC group, the red calcification structure in the low-dose ZOL group were slightly reduced, and the red calcification structure in the high-dose ZOL group were significantly reduced.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eResults of calcium content determination\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;in rats with VC treated with ZOL\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of calcium content determination in the aortic arch were shown in \u003cstrong\u003eFigure 2\u003c/strong\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe calcium content in the VC group was significantly higher than that in control group (P\u0026nbsp;\u0026lt; 0.05). The calcium content in the low-dose ZOL group was slightly lower than that in the VC group, but there was no statistical difference(P\u0026nbsp;\u0026gt;\u0026nbsp;0.05).The calcium content in the high-dose ZOL group was \u0026nbsp;significantly lower than that in the VC group(P\u0026nbsp;\u0026lt; 0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe results of meta-analysis \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStudy selection and characteristics\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA flow diagram of the selection process is shown in \u003cstrong\u003eFigure 3\u003c/strong\u003e. Finally, a total of eleven studies were included in this meta-analysis[14-24]. Of the eleven studies, three were human studies and eight were animal studies. The risk of bias in the included human RCTs were moderate. The human non-randomized studies achieved scores of\u0026nbsp;\u0026ge;6 points, which were considered to be of high quality. The risk of bias in the included animal studies was moderate. The baseline characteristics of human studies are listed in \u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eand the baseline characteristics of animal \u0026nbsp;studies are listed in \u003cstrong\u003eTable 2\u003c/strong\u003e. The Cochrane assessments are listed in \u003cstrong\u003eTable 3\u003c/strong\u003e, the NOS assessments are listed in \u003cstrong\u003eTable 4\u003c/strong\u003e, and SYRCLE assessments are listed in \u003cstrong\u003eTable 5\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe result of\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003earterial\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ecalcification\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003escore\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ein\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ehuman\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eand animal studies\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data about the\u0026nbsp;arterial\u0026nbsp;calcification\u0026nbsp;score\u0026nbsp;after the\u0026nbsp;N-BP\u0026nbsp;or no\u0026nbsp;N-BP\u0026nbsp;treatment were reported in three human and two animal studies. A subgroup analysis was performed according to human and animal study. In the human subgroup, there was no significant difference between the N-BP and no N-BP treatment group concerning the\u0026nbsp;arterial\u0026nbsp;calcification\u0026nbsp;score (SMD -0.11, 95% CI -0.40\u0026mdash;0.18, P=0.46). In the animal subgroup, there was no significant difference between the N-BP and no N-BP treatment group concerning the\u0026nbsp;arterial\u0026nbsp;calcification\u0026nbsp;score\u0026nbsp;(SMD -1.45, 95% CI -3.10\u0026mdash;0.20, P=0.09).\u0026nbsp;When the results of all subgroups were summarized, there was also no statistical difference between the N-BP and no N-BP treatment concerning the\u0026nbsp;arterial\u0026nbsp;calcification\u0026nbsp;score (SMD -0.59, 95% CI -1.27\u0026mdash;0.10, P=0.09)\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eFigure 4\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe result of\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003earterial\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ecalcification\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003earea\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ein\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eanimal studies\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data about the\u0026nbsp;arterial\u0026nbsp;calcification\u0026nbsp;area\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eafter the\u0026nbsp;N-BP\u0026nbsp;or no\u0026nbsp;N-BP\u0026nbsp;treatment were only reported in two animal studies.The\u0026nbsp;arterial\u0026nbsp;calcification\u0026nbsp;area in the\u0026nbsp;N-BP\u0026nbsp;treatment group were significantly lower than that in the no N-BP treatment group (SMD -2.74, 95% CI -3.48\u0026mdash;-2.00, P\u0026lt; 0.00001)\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eFigure 5\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe result of\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ethe arterial calcium content\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e\u003cem\u003ein\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eanimal studies\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data about the arterial calcium content after the N-BP or no N-BP treatment were only reported in four animal studies.The\u0026nbsp;arterial calcium content\u0026nbsp;in the\u0026nbsp;N-BP\u0026nbsp;treatment group was significantly lower than that in the no N-BP treatment group (SMD -4.29, 95% CI -7.51\u0026mdash;-1.07, P= 0.009)\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eFigure 6\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe result of\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ethe arterial\u0026nbsp;PO\u003csub\u003e4\u003c/sub\u003e content\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e\u003cem\u003ein\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eanimal studies\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data about the arterial PO\u003csub\u003e4\u003c/sub\u003e content after the N-BP or no N-BP treatment\u0026nbsp;were only reported in two animal studies. In the study of Price(2001), the groups of alendronate treatment were \u0026nbsp;divided into two groups according to the dose of alendronate, which were at a dose of 0.025 and 0.25 mg/kg/day.\u0026nbsp;The\u0026nbsp;arterial PO\u003csub\u003e4\u003c/sub\u003e content\u0026nbsp;in the\u0026nbsp;N-BP\u0026nbsp;treatment group was significantly lower than that in the no N-BP treatment group (SMD -2.41, 95% CI -3.65\u0026mdash;-1.18, P= 0.0001)\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eFigure 7\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSensitivity analyses\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sensitivity analyses for all results after the N-BP or no N-BP treatment was used to judge the dependability of the results. In the animal subgroup concerning the arterial calcification score, when we deleted the study of Synetos（2018）,the arterial calcification score in the N-BP treatment group was lower than that in the no N-BP treatment group(P \u0026lt; 0.05). Other results remained unchanged when we deleted one study at a time.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe purpose of our study was to explore the effect of N-BP on VC by animal experiments and a meta-analysis. ZOL is a type of N-BP. In our animal experiments, high-dose ZOL inhibited VC, but low-dose ZOL did not inhibit VC significantly. Our meta-analysis from human studies showed that N-BP did not inhibit VC. In our meta-analysis from animal studies, the results regarding the effect of N-BP on VC were inconsistent. Our meta-analysis from animal studies showed that N-BP did not reduce arterial calcification score significantly, but N-BP reduced arterial calcification area, arterial calcium and PO\u003csub\u003e4\u003c/sub\u003e content. Notably, when deleting the animal study of Synetos(2018), the overall results of animal study supported the role of N-BP in inhibiting VC.\u003c/p\u003e \u003cp\u003eRecent studies showed that drugs that were effective for osteoporosis might be effective for VC[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. N-BP is the frequently-used treatment for osteoporosis. According to our meta-analysis, N-BP was not an effective treatment for VC in humans, but the results of animal studies was inclined to supporting the role of N-BP in inhibiting VC. In the previous cell studies, N-BP also inhibited osteogenic differentiation and mineralization of vascular smooth muscle cells that was the drive step of VC[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The reason why N-BP were not effective on VC in humans was not clear, which might be related to the dosage, type, potency and administration route of N-BP [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe also conducted animal studies to explore the role of N-BP in inhibiting VC. In our animal experiments, high-dose ZOL inhibited VC, but low-dose ZOL did not inhibit VC significantly. The low and high doses of ZOL given to rats were 20 and 100ug/kg. The selection of the ZOL dose in our animal experiments was based on the ZOL dose for the treatment of osteoporosis in humans. The peak serum concentration of ZOL in human body following a 4 mg dose administration is from 1 to 5 \u0026micro;M[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However,low-dose ZOL did not inhibit VC significantly,which might be due to the fact that the serum concentration of ZOL after low-dose ZOL administration could not be sustained for the effective treatment of VC, or because of the different affinity of ZOL to bone and vascular tissues[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere were some limitations in our study. Firstly, there were differences in the dose, type, potency, administration route of N-BP among the studies included in our meta-analysis. Secondly, The number of studies included in our meta-analysis were still too small.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur animal experiments revealed that high-dose ZOL inhibited VC, but low-dose ZOL did not inhibit VC significantly. Our meta-analysis from humans studies revealed that N-BP was not effective for the treatment of VC, but Our meta-analysis result from animal studies was inclined to supporting the role of N-BP in inhibiting VC. To further confirm the conclusion, more large human RCTs and animal experiments are necessary.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eVC:Vascular calcification; Non-N-BP:Non-nitrogen-containing bisphosphonate; N-BP: Nitrogen-containing bisphosphonate; ZOL:Zoledronic acid; PRISMA:Preferred Reporting Items for Systematic Reviews and Meta-Analyses; CNKI:China National Knowledge Infrastructure; RCTs:Randomized controlled trials;NOS:Newcastle\u0026ndash;Ottawa scale;SYRCLE:Systematic Review Centre for Laboratory animal experimentsation; CIs:Confidence intervals\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiments were approved by the Institutional Animal Care Committee at Zhujiang Hospital, Jiangsu University, China (UJS-IACUC-AP-2023030310) . This study is reported in accordance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by the Open Research Project of Key Laboratories in Jiangsu Province Universities (No.XZSYSKF2023022), the Young Talent Development Plan of Changzhou Health Commission (No.CZQM2021026) .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWei Xu and Guoyuan Lu contributed to the conceptualization. Wei Xu, Guoyuan Lu and Lifeng Gong contributed to the animal experiments.Wei Xu,Weigang Tang and Wei Jiang contributed to the meta-analysis. Wei Xu contributed to the analysis of the data and production of figures and tables. Wei Xu and Guoyuan Lu contributed to the writing. All authors approved final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVilla-Bellosta R. Vascular Calcification: Key Roles of Phosphate and Pyrophosphate. Int J Mol Sci. 2021;22(24):13536. \u003c/li\u003e\n\u003cli\u003eYuan C, Ni L, Zhang C, Hu X, et al.Vascular calcification: New insights into endothelial cells. Microvasc Res. 2021;134:104105.\u003c/li\u003e\n\u003cli\u003eDanilevicius CF, Lopes JB, Pereira RM. Bone metabolism and vascular calcification. Braz J Med Biol Res. 2007;40(4):435-442. \u003c/li\u003e\n\u003cli\u003eVon der Recke P, Hansen MA, Hassager C. The association between low bone mass at the menopause and cardiovascular mortality. Am J Med. 1999;106(3):273-278.\u003c/li\u003e\n\u003cli\u003eEsposito K, Capuano A, Sportiello L, et al.Should we abandon statins in the prevention of bone fractures?. Endocrine. 2013;44(2):326-333. \u003c/li\u003e\n\u003cli\u003eSantos LL, Cavalcanti TB, Bandeira FA. Vascular effects of bisphosphonates-a systematic review. Clin Med Insights Endocrinol Diabetes. 2012;5:47-54.\u003c/li\u003e\n\u003cli\u003eRaterman HG, Bultink IEM, Lems WF. Current Treatments and New Developments in the Management of Glucocorticoid-induced Osteoporosis. Drugs. 2019;79(10):1065-1087. \u003c/li\u003e\n\u003cli\u003eTella SH, Gallagher JC. Prevention and treatment of postmenopausal osteoporosis. J Steroid Biochem Mol Biol. 2014;142:155-170.\u003c/li\u003e\n\u003cli\u003eShobeiri N, Adams MA, Holden RM. Vascular calcification in animal models of CKD: A review. Am J Nephrol. 2010;31(6):471-481.\u003c/li\u003e\n\u003cli\u003eDiwan V, Brown L, Gobe GC. Adenine-induced chronic kidney disease in rats. Nephrology (Carlton). 2018;23(1):5-11. \u003c/li\u003e\n\u003cli\u003eFurlan AD.Malmivaara A, Chou R, et al. 2015 Updated method guideline for systematic reviews in the Cochrane back and neck group.Spine.2015;40:1660-1673.\u003c/li\u003e\n\u003cli\u003eStang A. Critical evaluation of the Newcastle-Ottawa scale for the assessment of the quality of nonrandomized studies in meta-analyses. Eur J Epidemiol .2010;25:603-605.\u003c/li\u003e\n\u003cli\u003eHooijmans CR, Rovers MM, de Vries RB, et al. SYRCLE\u0026apos;s risk of bias tool for animal studies. BMC Med Res Methodol. 2014;14:43. \u003c/li\u003e\n\u003cli\u003ePrice PA, Faus SA, Williamson MK. Bisphosphonates alendronate and ibandronate inhibit artery calcification at doses comparable to those that inhibit bone resorption. Arterioscler Thromb Vasc Biol. 2001;21(5):817-824.\u003c/li\u003e\n\u003cli\u003eSynetos A, Toutouzas K, Drakopoulou M, et al. Inhibition of Aortic Valve Calcification by Local Delivery of Zoledronic Acid-an Experimental Study. J Cardiovasc Transl Res. 2018;11(3):192-200. \u003c/li\u003e\n\u003cli\u003eLi H, JIA GL, Wang HC, et al. The effect of alendronate on arterial calcification in rat model.Chin J Intern Med.2006;(06):489-492.\u003c/li\u003e\n\u003cli\u003eJia P,Guan SM,Wang B, et al.Study on the intervention of alendronate on vascular calcification.Chinese Journal of Microcirculation.2012;22(02):9-11.\u003c/li\u003e\n\u003cli\u003eYang LY, Guan SM,Fang X, et al.The effect of alendronate on the expression of osteoprotegerin in rats calcified aorta tissue[J].Chin J Arterioscler,2008,(08):623-627.\u003c/li\u003e\n\u003cli\u003eSynetos A, Toutouzas K, Benetos G, et al. Catheter based inhibition of arterial calcification by bisphosphonates in an experimental atherosclerotic rabbit animal model. Int J Cardiol. 2014;176(1):177-181.\u003c/li\u003e\n\u003cli\u003eGuo LM, Liu Y, Wang YH, et al. Effect of oral and subcutaneous injection of alendronic acid on calcitriol induced vascular calcification in rats with partial nephrectomy. Chin J Nephrol,2008,24(03):202-203.\u003c/li\u003e\n\u003cli\u003ePrice PA, Roublick AM, Williamson MK. Artery calcification in uremic rats is increased by a low protein diet and prevented by treatment with ibandronate. Kidney Int. 2006;70(9):1577-1583.\u003c/li\u003e\n\u003cli\u003eTorregrosa JV, Fuster D, Gentil MA, et al. Open-label trial: effect of weekly risedronate immediately after transplantation in kidney recipients. Transplantation. 2010;89(12):1476-1481.\u003c/li\u003e\n\u003cli\u003eOkamoto M, Yamanaka S, Yoshimoto W, et al.Alendronate as an effective treatment for bone loss and vascular calcification in kidney transplant recipients. J Transplant. 2014;2014:269613.\u003c/li\u003e\n\u003cli\u003eHill JA, Goldin JG, Gjertson D, et al. Progression of coronary artery calcification in patients taking alendronate for osteoporosis. Acad Radiol. 2002;9(10):1148-1152. \u003c/li\u003e\n\u003cli\u003eCutini PH, Rauschemberger MB, Sandoval MJ, et al. Vascular action of bisphosphonates: In vitro effect of alendronate on the regulation of cellular events involved in vessel pathogenesis. J Mol Cell Cardiol. 2016;100:83-92. \u003c/li\u003e\n\u003cli\u003eZhou S, Fang X, Xin H, et al.Effects of alendronate on the Notch1‑RBP‑J\u0026kappa; signaling pathway in the osteogenic differentiation and mineralization of vascular smooth muscle cells. Mol Med Rep. 2013;8(1):89-94.\u003c/li\u003e\n\u003cli\u003eHildebrand S, Cunningham J. Is there a role for bisphosphonates in vascular calcification in chronic kidney disease?. Bone. 2021;142:115751.\u003c/li\u003e\n\u003cli\u003eCaffarelli C, Montagnani A, Nuti R, et al. Bisphosphonates, atherosclerosis and vascular calcification: update and systematic review of clinical studies. Clin Interv Aging. 2017;12:1819-1828. \u003c/li\u003e\n\u003cli\u003eWu L, Zhu L, Shi WH, Zhang J, et al.Zoledronate inhibits the proliferation, adhesion and migration of vascular smooth muscle cells. Eur J Pharmacol. 2009;602(1):124-131. \u003c/li\u003e\n\u003cli\u003ePawade TA, Doris MK, Bing R, et al. Effect of Denosumab or Alendronic Acid on the Progression of Aortic Stenosis: A Double-Blind Randomized Controlled Trial. Circulation. 2021;143(25):2418-2427.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable1.\u0026nbsp;\u003c/strong\u003eCharacteristics of the included human studies.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"1084\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.479224376731302%\" rowspan=\"2\"\u003e\n \u003cp\u003eStudy \u0026nbsp;(year)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.386888273314866%\" rowspan=\"2\"\u003e\n \u003cp\u003eCountry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.109879963065558%\" rowspan=\"2\"\u003e\n \u003cp\u003eDesign\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.648199445983379%\" rowspan=\"2\"\u003e\n \u003cp\u003eFollow-up period \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.97229916897507%\" rowspan=\"2\"\u003e\n \u003cp\u003ePopulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.850415512465373%\" rowspan=\"2\"\u003e\n \u003cp\u003eTreatment group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.61865189289012%\" rowspan=\"2\"\u003e\n \u003cp\u003eComparison group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.172668513388736%\" rowspan=\"2\"\u003e\n \u003cp\u003eSample size\u003cbr\u003e\u0026nbsp;(Treatment group/ Comparion group)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.21791320406279%\" rowspan=\"2\"\u003e\n \u003cp\u003eMean age (years)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.463527239150507%\" rowspan=\"2\"\u003e\n \u003cp\u003eMale\u0026nbsp;\u003cbr\u003e\u0026nbsp;(n,%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.0803324099723%\" rowspan=\"2\"\u003e\n \u003cp\u003eAssessment of \u0026nbsp;VC \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"31\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"NaN%\" height=\"62\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.479224376731302%\"\u003e\n \u003cp\u003eHill\u003cbr\u003e\u0026nbsp;(2002)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.386888273314866%\"\u003e\n \u003cp\u003eAmerica\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.109879963065558%\"\u003e\n \u003cp\u003eMatched- control study\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.648199445983379%\"\u003e\n \u003cp\u003e24 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.97229916897507%\"\u003e\n \u003cp\u003ePatients with osteoporosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.850415512465373%\"\u003e\n \u003cp\u003eAlendronate sodium \u0026nbsp;10 mg po daily for a mean of 24 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.61865189289012%\"\u003e\n \u003cp\u003eNo treatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.172668513388736%\"\u003e\n \u003cp\u003e56\u0026nbsp;\u003cbr\u003e\u0026nbsp;56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.21791320406279%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.463527239150507%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.0803324099723%\"\u003e\n \u003cp\u003eCoronary\u0026nbsp;arterial calcification\u0026nbsp;scores by CT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"77\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.479224376731302%\"\u003e\n \u003cp\u003eTorregrosa （2010）\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.386888273314866%\"\u003e\n \u003cp\u003eSpain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.109879963065558%\"\u003e\n \u003cp\u003eRCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.648199445983379%\"\u003e\n \u003cp\u003e12 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.97229916897507%\"\u003e\n \u003cp\u003eKidney transplant patients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.850415512465373%\"\u003e\n \u003cp\u003eRisedronate 35 mg po weekly,\u0026nbsp;\u003cbr\u003e\u0026nbsp;Calcium \u0026nbsp;and vitamin D\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.61865189289012%\"\u003e\n \u003cp\u003eCalcium and vitamin D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.172668513388736%\"\u003e\n \u003cp\u003e52\u0026nbsp;\u003cbr\u003e\u0026nbsp;49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.21791320406279%\"\u003e\n \u003cp\u003e47.4 \u0026plusmn; 14.1\u0026nbsp;\u003cbr\u003e\u0026nbsp;50.7 \u0026plusmn; 15.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.463527239150507%\"\u003e\n \u003cp\u003e28（53.8） \u0026nbsp; \u0026nbsp;27（55.1）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.0803324099723%\"\u003e\n \u003cp\u003eVC score by abdomen and hand X-ray\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"77\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.479224376731302%\"\u003e\n \u003cp\u003eOkamoto\u003cbr\u003e\u0026nbsp;（2014）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.386888273314866%\"\u003e\n \u003cp\u003eJapan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.109879963065558%\"\u003e\n \u003cp\u003eProspective study\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.648199445983379%\"\u003e\n \u003cp\u003e24 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.97229916897507%\"\u003e\n \u003cp\u003eKidney Transplant patients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.850415512465373%\"\u003e\n \u003cp\u003eAlendronate 35 mg/week for 24 months\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.61865189289012%\"\u003e\n \u003cp\u003eNo treatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.172668513388736%\"\u003e\n \u003cp\u003e5\u003cbr\u003e\u0026nbsp;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.21791320406279%\"\u003e\n \u003cp\u003e52.8 \u0026plusmn; 12.6 52.9 \u0026plusmn; 7.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.463527239150507%\"\u003e\n \u003cp\u003e4（80）\u003cbr\u003e\u0026nbsp;4（57）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.0803324099723%\"\u003e\n \u003cp\u003eAbdominal\u0026nbsp;arterial calcification\u0026nbsp;by CT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"77\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eCharacteristics of the included animal studies.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"1060\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"6.126295947219604%\" rowspan=\"2\"\u003e\n \u003cp\u003eStudy\u003c/p\u003e\n \u003cp\u003e(year)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.880301602262017%\" rowspan=\"2\"\u003e\n \u003cp\u003eCountry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.19981149858624%\" rowspan=\"2\"\u003e\n \u003cp\u003eSpecies（Age，Sex，Weight）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.572101790763432%\" rowspan=\"2\"\u003e\n \u003cp\u003eThe disease of animal model\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.022620169651272%\" rowspan=\"2\"\u003e\n \u003cp\u003eTreatment group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.687087653157398%\" rowspan=\"2\"\u003e\n \u003cp\u003eComparison group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.215834118755891%\" rowspan=\"2\"\u003e\n \u003cp\u003eSample size (Treatment group/ Comparion group)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.142318567389255%\" rowspan=\"2\"\u003e\n \u003cp\u003eFollow-up period\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.15362865221489%\" rowspan=\"2\"\u003e\n \u003cp\u003eOutcome\u003cbr\u003e\u0026nbsp;evaluated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"31\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"NaN%\" height=\"122\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"6.126295947219604%\"\u003e\n \u003cp\u003ePrice（2006）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.880301602262017%\"\u003e\n \u003cp\u003eUSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.19981149858624%\"\u003e\n \u003cp\u003eRat（13 weeks，371.4\u0026plusmn;8.4 g）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.572101790763432%\"\u003e\n \u003cp\u003eUremia model using 0.75% adenine diet for 4 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.022620169651272%\"\u003e\n \u003cp\u003e2.5% protein diet，\u0026nbsp;Ibandronate ic at a dose of 0.25 mg/kg/day beginning 12 days after the start of the adenine diet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.687087653157398%\"\u003e\n \u003cp\u003e2.5% Protein diet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.215834118755891%\"\u003e\n \u003cp\u003e5\u003cbr\u003e\u0026nbsp;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.142318567389255%\"\u003e\n \u003cp\u003e4 weeks after the start of the adenine diet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.15362865221489%\"\u003e\n \u003cp\u003eThoracic aorta calcium and \u0026nbsp;PO\u003csub\u003e4\u003c/sub\u003e determined colorimetrically\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"142\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"6.126295947219604%\"\u003e\n \u003cp\u003eSynetos（2018）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.880301602262017%\"\u003e\n \u003cp\u003eGreece\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.19981149858624%\"\u003e\n \u003cp\u003eNew Zealand rabbits\u003c/p\u003e\n \u003cp\u003e（3 months，male，3.7\u0026plusmn;0.2 kg）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.572101790763432%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eAortic valve stenosis model using vitamin D -enriched atherogenic diet for 3 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.022620169651272%\"\u003e\n \u003cp\u003eLocal delivery of a mixture containing 500 \u0026mu;g/l \u0026nbsp;zoledronate on the cusps of the aortic valve using a dedicated balloon \u0026nbsp; \u0026nbsp; catheter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.687087653157398%\"\u003e\n \u003cp\u003eA placebo mixture administered with the same \u0026nbsp;process\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.215834118755891%\"\u003e\n \u003cp\u003e8\u0026nbsp;\u003cbr\u003e\u0026nbsp;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.142318567389255%\"\u003e\n \u003cp\u003e28 days after local drug delivery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.15362865221489%\"\u003e\n \u003cp\u003eProgression of aortic valve calcification by PET/CT Imaging，aortic valve calcification area by von Kossa staining\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"216\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"6.126295947219604%\"\u003e\n \u003cp\u003eSynetos（2014）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.880301602262017%\"\u003e\n \u003cp\u003eGreece\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.19981149858624%\"\u003e\n \u003cp\u003eNew Zealand rabbits（3.8 \u0026plusmn; 0.5 kg）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.572101790763432%\" valign=\"top\"\u003e\n \u003cp\u003eArterial calcification and atherosclerosis model using vitamin D -enriched atherogenic diet for 3 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.022620169651272%\"\u003e\n \u003cp\u003eLocal delivery of a mixture \u0026nbsp; \u0026nbsp; containing 500 \u0026mu;g/l zoledronate on the vascular wall of the \u0026nbsp;iliac artery, using a dedicated balloon catheter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.687087653157398%\"\u003e\n \u003cp\u003eA placebo mixture administered on the contralateral iliac artery with the same \u0026nbsp;process\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.215834118755891%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.142318567389255%\"\u003e\n \u003cp\u003e28 days after local drug delivery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.15362865221489%\"\u003e\n \u003cp\u003eThe calcium content \u0026nbsp;of \u0026nbsp;iliac artery by computer-assisted histomorphometry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"162\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"6.126295947219604%\"\u003e\n \u003cp\u003eLi Huan（2006）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.880301602262017%\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.19981149858624%\"\u003e\n \u003cp\u003eSD Rat\u003c/p\u003e\n \u003cp\u003e（4 weeks，male）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.572101790763432%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eArterial calcification \u0026nbsp;model using warfarin and vitamin D for 4 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.022620169651272%\"\u003e\n \u003cp\u003e\u003cbr\u003e\u0026nbsp;Alendronate ic at a dose of 1 mg/kg/day starting 4 days before the first warfarin and vitamin D injection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.687087653157398%\"\u003e\n \u003cp\u003eEqual amounts of saline ic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.215834118755891%\"\u003e\n \u003cp\u003e6\u003cbr\u003e\u0026nbsp;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.142318567389255%\"\u003e\n \u003cp\u003e4 days after the first warfarin and vitamin D injection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.15362865221489%\"\u003e\n \u003cp\u003eAortic calcification area by von Kossa staining\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"162\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"6.126295947219604%\"\u003e\n \u003cp\u003ePrice（2001）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.880301602262017%\"\u003e\n \u003cp\u003eUSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.19981149858624%\"\u003e\n \u003cp\u003eRats(42 days，male)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.572101790763432%\"\u003e\n \u003cp\u003eArterial calcifica\u003cbr\u003e\u0026nbsp;tion \u0026nbsp;model using warfarin for 4 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.022620169651272%\"\u003e\n \u003cp\u003e\u003cbr\u003e\u0026nbsp;Alendronate ic at a dose of 0.025 or 0.25 mg/kg/day starting 4 days before the first warfarin injection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.687087653157398%\"\u003e\n \u003cp\u003eNo treatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.215834118755891%\"\u003e\n \u003cp\u003e8\u0026nbsp;\u003cbr\u003e\u0026nbsp;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.142318567389255%\"\u003e\n \u003cp\u003e4 weeks after the first warfarin injection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.15362865221489%\"\u003e\n \u003cp\u003eCarotid PO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003edetermined colorimetrically\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"162\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"6.126295947219604%\"\u003e\n \u003cp\u003eJia peng（2012）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.880301602262017%\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.19981149858624%\"\u003e\n \u003cp\u003eSD Rat\u003c/p\u003e\n \u003cp\u003e（5 weeks，male，150-200g）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.572101790763432%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eArterial calcification \u0026nbsp;model using warfarin and vitamin D for 4 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.022620169651272%\"\u003e\n \u003cp\u003e\u003cbr\u003e\u0026nbsp;Alendronate ic at a dose of 1 mg/kg/day starting 4 days before the first warfarin and vitamin D injection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.687087653157398%\"\u003e\n \u003cp\u003eEqual amounts of saline ic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.215834118755891%\"\u003e\n \u003cp\u003e20\u003cbr\u003e\u0026nbsp;20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.142318567389255%\"\u003e\n \u003cp\u003e8 days after the first alendronate injection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.15362865221489%\"\u003e\n \u003cp\u003eThoracic aorta\u0026nbsp;calcium\u0026nbsp;determined colorimetrically\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"162\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"6.126295947219604%\"\u003e\n \u003cp\u003eYang liyuan\u003c/p\u003e\n \u003cp\u003e(2008)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.880301602262017%\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.19981149858624%\"\u003e\n \u003cp\u003eSD Rat（4 weeks，male，200-250g）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.572101790763432%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eArterial calcification \u0026nbsp;model using warfarin and vitamin D for 4 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.022620169651272%\"\u003e\n \u003cp\u003e\u003cbr\u003e\u0026nbsp;Alendronate ic at a dose of 1 mg/kg/day starting 4 days before the first warfarin and vitamin D injection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.687087653157398%\"\u003e\n \u003cp\u003eEqual amounts of saline ic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.215834118755891%\"\u003e\n \u003cp\u003e6\u003cbr\u003e\u0026nbsp;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.142318567389255%\"\u003e\n \u003cp\u003e8 days after the first alendronate injection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.15362865221489%\"\u003e\n \u003cp\u003eThoracic aorta\u0026nbsp;calcium\u0026nbsp;determined colorimetrically\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"162\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"6.126295947219604%\"\u003e\n \u003cp\u003eGuo liming（2008）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.880301602262017%\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.19981149858624%\"\u003e\n \u003cp\u003eSD Rat（female）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.572101790763432%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eArterial calcification \u0026nbsp;model using subtotal nephrectomy with vitamin D ， \u0026nbsp;high phosphorus and calcium diet for 4 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.022620169651272%\"\u003e\n \u003cp\u003eAlendronate ic at a dose of \u0026nbsp;0.25 mg/kg/day after \u0026nbsp;nephrectomy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.687087653157398%\"\u003e\n \u003cp\u003eNo treatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.215834118755891%\"\u003e\n \u003cp\u003e10\u0026nbsp;\u003cbr\u003e\u0026nbsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.142318567389255%\"\u003e\n \u003cp\u003e4 weeks after \u0026nbsp;nephrectomy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.15362865221489%\"\u003e\n \u003cp\u003eThoracic aorta\u0026nbsp;calcium\u0026nbsp;by automatic biochemical measurement\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"216\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eQuality assessment\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of randomized control trial.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"756\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.798941798941797%\"\u003e\n \u003cp\u003eStudy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.492063492063492%\"\u003e\n \u003cp\u003eRandom sequence generation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.402116402116402%\"\u003e\n \u003cp\u003eAllocation\u003cbr\u003e\u0026nbsp;concealment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.227513227513228%\"\u003e\n \u003cp\u003eBlinding of participants and personnel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.227513227513228%\"\u003e\n \u003cp\u003eIncomplete outcome data\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83068783068783%\"\u003e\n \u003cp\u003eSelective reporting\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.02116402116402%\"\u003e\n \u003cp\u003eOther bias\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.798941798941797%\"\u003e\n \u003cp\u003eTorregrosa （2010）\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.492063492063492%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.402116402116402%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.227513227513228%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.227513227513228%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.83068783068783%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.02116402116402%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe randomized control trial was evaluated using the Cochrane assessment tool.+,low risk of bias;?,unclear risk of bias; -,high risk of bias.\u003c/p\u003e\n\u003cp\u003eTable 4. \u003cstrong\u003eQuality assessment of non-randomized control trial.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"803\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.306351183063512%\"\u003e\n \u003cp\u003eStudies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.046077210460773%\"\u003e\n \u003cp\u003eSelection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.940224159402241%\"\u003e\n \u003cp\u003eComparability\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.672478206724783%\"\u003e\n \u003cp\u003eOutcome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.03486924034869%\"\u003e\n \u003cp\u003eScore\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.306351183063512%\"\u003e\n \u003cp\u003eHill（2002）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.046077210460773%\"\u003e\n \u003cp\u003e\u003cstrong\u003e★★★★\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.940224159402241%\"\u003e\n \u003cp\u003e\u003cstrong\u003e★\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.672478206724783%\"\u003e\n \u003cp\u003e\u003cstrong\u003e★★\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.03486924034869%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.306351183063512%\"\u003e\n \u003cp\u003eOkamoto（2014）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.046077210460773%\"\u003e\n \u003cp\u003e\u003cstrong\u003e★★★★\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.940224159402241%\"\u003e\n \u003cp\u003e\u003cstrong\u003e★\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.672478206724783%\"\u003e\n \u003cp\u003e\u003cstrong\u003e★★\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.03486924034869%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe Cohort studies were evaluated using the Newcastle-Ottawa scale,which are comprised of the study of selection (Representativeness of the exposed group,Representativeness of the non exposed group,Ascertainment of exposure,Demonstration that outcome of interest was not present at start of study), group comparability(Controls for the most important factor,Controls for any additional factor),outcome measures (Assessment of outcome,Was follow-up long enough for outcomes to occur,Adequacy of follow up of cohorts), a total of nine points. \u003cstrong\u003e★\u003c/strong\u003e, 1 point.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable 5. \u003cstrong\u003eQuality assessment of animal experiment\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"764\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e\n \u003cp\u003eStudies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e①\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e②\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e③\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e④\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e⑤\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e⑥\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e⑦\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e⑧\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e⑨\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e⑩\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e\n \u003cp\u003ePrice（2006）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e\n \u003cp\u003eSynetos（2018）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e\n \u003cp\u003eSynetos（2014）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e\n \u003cp\u003eLi Huan（2006）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e\n \u003cp\u003ePrice（2001）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e\n \u003cp\u003eJia peng（2012）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e\n \u003cp\u003eYang liyuan(2008)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e\n \u003cp\u003eGuo liming（2008）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.115183246073299%\"\u003e\n \u003cp\u003e?\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe animal experiment was evaluated using the SYRCLE tool.① allocation sequence generated；② baseline \u0026nbsp;similar；③ allocation concealment ④ random housing animal；⑤ \u0026nbsp;investigator/caregivers blinded；⑥ random outcome assessment；⑦ outcome assessor blinded； ⑧ \u0026nbsp;incomplete outcome adequatedly adressed；⑨ Selective outcome reporting；⑩ other risks of bias; +,low risk of bias;?,unclear risk of bias; -,high risk of bias.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Nitrogen-containing bisphosphonate, Vascular calcification, Animal experiments, Meta-Analysis ","lastPublishedDoi":"10.21203/rs.3.rs-3972838/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3972838/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The purpose of our study was to explore the effect of nitrogen-containing bisphosphonate (N-BP) on vascular calcification (VC) by animal experiments and a meta-analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e In our animal experiments, the SD rats were randomly divided into control group, VC group, low-dose Zoledronic acid (ZOL) group (20ug/kg) and high-dose ZOL group (100ug/kg). The calcification of aortic arch was detected by alizarin red staining. The calcium content of aortic arch was detected. In our meta-analysis, databases including PubMed, EMbase, the Cochrane Library, CNKI , Wanfang database were searched from the inception to December 20th, 2023. Eligible studies comparing N-BP versus no N-BP in the treatment of VC were included.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eIn our animal experiment, compared with the VC group, the red-stained calcification structure in the low-dose ZOL group were slightly reduced, and the red-stained calcification structure in the high-dose ZOL group were significantly reduced. The calcium content in the low-dose ZOL group was slightly lower than that in the VC group, but there was no statistical difference(P \u0026gt; 0.05).The calcium content in the high-dose ZOL group was significantly lower than that in the VC group(P \u0026lt; 0.05). Our meta-analysis from human studies showed that N-BP did not reduce arterial calcification score(P \u0026gt; 0.05). Our meta-analysis from animal studies showed that N-BP did not reduce arterial calcification score significantly(P \u0026gt; 0.05), but N-BP reduce arterial calcification area, arterial calcium and PO4 content(P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eOur animal experiment revealed that high-dose ZOL inhibited VC, but low-dose ZOL did not inhibit VC significantly. Our meta-analysis from humans studies revealed that N-BP was not effective for the treatment of VC, but Our meta-analysis result from animal studies was inclined to supporting the role of N-BP in inhibiting VC.\u003c/p\u003e","manuscriptTitle":"Nitrogen-containing bisphosphonate for vascular calcification: animal experiments and a meta-analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-04 17:46:40","doi":"10.21203/rs.3.rs-3972838/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-26T19:01:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-26T10:04:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282363197943138150277008023116882346976","date":"2024-11-22T20:01:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5144023866835167756196118324947762520","date":"2024-11-19T18:56:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"56232465549017776979347546782899088780","date":"2024-11-18T09:34:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218855929382067048518933924201008890024","date":"2024-11-17T22:54:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-14T22:46:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282906357887489736458695760265735899146","date":"2024-06-14T22:30:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-16T12:48:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-16T06:22:31+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-01T17:16:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-29T16:42:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cardiovascular Disorders","date":"2024-02-20T13:04:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fad62fdf-3ebc-41b1-9063-c91e778cbb99","owner":[],"postedDate":"March 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-03T16:04:48+00:00","versionOfRecord":{"articleIdentity":"rs-3972838","link":"https://doi.org/10.1186/s12872-025-04526-w","journal":{"identity":"bmc-cardiovascular-disorders","isVorOnly":false,"title":"BMC Cardiovascular Disorders"},"publishedOn":"2025-01-31 15:58:16","publishedOnDateReadable":"January 31st, 2025"},"versionCreatedAt":"2024-03-04 17:46:40","video":"","vorDoi":"10.1186/s12872-025-04526-w","vorDoiUrl":"https://doi.org/10.1186/s12872-025-04526-w","workflowStages":[]},"version":"v1","identity":"rs-3972838","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3972838","identity":"rs-3972838","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}