Animal experimentsExperimental protocol

Twenty-four 8-week-old male Sprague-Dawley (SD) rats weighing 250–300 g were purchased from Huachuang Xinuo Pharmaceutical Technology (China). All the rats were randomly divided into four groups, with six rats in each group; the groups included the control group, VC group, low-dose ZOL group and high-dose ZOL group. The VC model was established in the VC group, low-dose ZOL group and high-dose ZOL group. The method for establishing the VC model was as follows [9, 10]. Adenine (450 mg/kg/day) was administered by gavage in the first week, 300 mg/kg/day adenine was administered by gavage in the second to fourth weeks, and high phosphorus feed (1.8% phosphorus, 1% calcium) was provided at the same time. The low-dose and high-dose ZOL groups were given an intraperitoneal injection of 20–100 µg/kg ZOL once a week for 4 weeks. ZOL was administered on the first day of VC modelling. The VC group was given an intraperitoneal injection of the same volume of normal saline. The control group was provided with ordinary feed (0.6% phosphorus, 1% calcium) and administered an equal volume of normal saline by intraperitoneal injection. After 4 weeks, the rats were euthanized by intraperitoneal injection of 150 mg/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 the rats in each group were embedded in paraffin, sectioned, roasted, dewaxed and dehydrated. The tissue sections were incubated with 1% Alizarin red S solution (Solarbio, China) at room temperature for 1 h and then washed with double steaming water. Then, the tissue sections were sealed, and images were acquired by microscopy.

Determination of the calcium content

The calcium contents were determined with a calcium assay kit (Beyotime, China). The aortic tissues that were collected from each group were placed in a centrifuge tube, to which the sample lysate was added. The samples were then homogenized with a homogenizer and centrifuged at 12,000 rpm at 4 °C for 5 min, and then, the supernatants were collected. The samples were subsequently added to the detection buffer and colour developing solution and incubated at room temperature for 10 min. Finally, the absorbance of the samples at 575 nm was measured with a microplate reader, and the calcium content of the samples was calculated by a standard curve.

Statistical analysis

All the data are presented as the means ± SDs. Differences among the groups were compared using one-way ANOVA. All the statistical analyses were performed with SPSS 20.0 software. Graphs were plotted with GraphPad Prism 8.0 software. P < 0.05 was considered statistically significant.

Meta-analysisSearch strategy

Our systematic review and meta-analysis was reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. We searched the PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure (CNKI), and Wanfang databases from their inception to December 20, 2023. The combined text and MeSH terms included (“nitrogen-containing bisphosphonate” or “minodronicacid” or “alendronate” or “risedronate” or “ibandronate” or “zoledronate” or “pamidronate”) and (“vascular calcification”). In addition, the relevant references and cited papers were manually searched to identify additional studies that met the inclusion criteria. There were no language restrictions.

Inclusion and exclusion criteria

The inclusion criteria were the Population, Intervention, Control, and Outcomes (PICO) strategy. The population included patients with VC. The intervention studied involved the use of N-BP. The comparison was no N-BP treatment. The outcomes were the assessment of VC, which included at least one of several indicators, such as the arterial calcification score, arterial calcification area, and arterial calcium or PO4 contents.

The exclusion criteria were (1) case series, comments, and reviews; (2) no control group; and (3) lack of relevant outcome 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 data such as the first author, year of publication, location, study design, population, specific methods used in the experimental and control groups, follow-up period, sample size, mean age, sex, weight, and 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 nonrandomized studies [12]. The Systematic Review Centre for Laboratory Animal Experiments (SYRCLE) tool was used to assess the quality of the 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 < 0.05 was considered statistically significant. We used the I2 statistic to assess heterogeneity among studies. We considered I2 > 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.