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. 2024 Jul 20;25(14):7940.
doi: 10.3390/ijms25147940.

Effect of Acetylsalicylic Acid on Biological Properties of Novel Cement Based on Calcium Phosphate Doped with Ions of Strontium, Copper, and Zinc

Tamara Vlajić Tovilović  1 , Sanja Petrović  1 , Miloš Lazarević  1 , Aleksandar Pavić  2 , Nikola Plačkić  2 , Aleksa Milovanović  3 , Miloš Milošević  3 , Vesna Miletic  4 , Djordje Veljović  5 , Milena Radunović  1
Affiliations

Effect of Acetylsalicylic Acid on Biological Properties of Novel Cement Based on Calcium Phosphate Doped with Ions of Strontium, Copper, and Zinc

Tamara Vlajić Tovilović et al. Int J Mol Sci. .

Abstract

This study aimed to compare the biological properties of newly synthesized cements based on calcium phosphate with a commercially used cement, mineral trioxide aggregate (MTA). Strontium (Sr)-, Copper (Cu)-, and Zinc (Zn)-doped hydroxyapatite (miHAp) powder was obtained through hydrothermal synthesis and characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy dispersive X-ray spectrometry (EDX). Calcium phosphate cement (CPC) was produced by mixing miHAp powder with a 20 wt.% citric acid solution, followed by the assessment of its compressive strength, setting time, and in vitro bioactivity. Acetylsalicylic acid (ASA) was added to the CPC, resulting in CPCA. Biological tests were conducted on CPC, CPCA, and MTA. The biocompatibility of the cement extracts was evaluated in vitro using human dental pulp stem cells (hDPSCs) and in vivo using a zebrafish model. Antibiofilm and antimicrobial effect (quantified by CFUs/mL) were assessed against Streptococcus mutans and Lactobacillus rhamnosus. None of the tested materials showed toxicity, while CPCA even increased hDPSCs proliferation. CPCA showed a better safety profile than MTA and CPC, and no toxic or immunomodulatory effects on the zebrafish model. CPCA exhibited similar antibiofilm effects against S. mutans and L. rhamnosus to MTA.

Keywords: antibiofilm; calcium phosphate; dental pulp stem cells; hydroxyapatite; zebrafish.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Scanning electron micrographs (SEM) of miHAp powder, (A) prior and (C) after calcination, and corresponding XRD patterns (B,D).
Figure 2
Figure 2
Surface of CPC after 15 days of incubation in SBF, showing the presence of HAp.
Figure 3
Figure 3
The viability of hDPSC cells at various cement extract concentrations (12.5%, 25%, 50%, 75%, and 100%) assessed by MTT assay; * <0.05 (ANOVA followed by Bonferroni post hoc assay). The dotted line represents 80% of cell viability. Values above 80% indicate that the material is not cytotoxic.
Figure 4
Figure 4
Evaluation of the toxicity of CPCA, CPC, and MTA in the zebrafish model. The effects of the different concentrations of the applied material on (A) AB embryos’ survival and teratogenicity and (B) morphology are shown. CPC prevented the swim bladder from inflating (arrow).
Figure 5
Figure 5
The biocompatibility of dental materials CPC, CPCA, and MTA assessed in the transgenic Tg (mpx:GFP) i114 zebrafish line with fluorescently labeled neutrophils. (A) Neutrophil occurrence and (B) fluorescence intensity is shown. No statistically significant difference between the control (untreated) and treated embryos was detected (p > 0.5, ANOVA and Bonferroni post hoc assay).
Figure 6
Figure 6
The effect of dental materials CPC, CPCA, and MTA on the intersegmental vessel (ISV) development assessed in the transgenic zebrafish line Tg(fli1:EGFP) with fluorescently labeled vasculature. The effect on the ISV development was investigated by analyzing (A) the frequency of embryos affected in the ISVs (not developed, reduced in size, wrong patterning) (n = 10 embryos), (B) the number of ISVs affected (n = 5 embryos), and (C) the effects on the ISV growth. (D) The morphology of the transgenic embryos indicating the affected ISVs (arrow). Statistically significant differences between the treated and untreated groups were determined using ANOVA and Bonferroni test (* p < 0.5, *** p < 0.001).
Figure 7
Figure 7
Antibiofilm effect on (A) L. rhamnosus and (C) S. mutans on discs, and antimicrobial effect on (B) L. rhamnosus and (D) S. mutans in surrounding medium. (One-Way ANOVA followed by Bonferroni test * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001).
Figure 8
Figure 8
Isolation and cultivation of hDPSC: (A) third molar extraction and transportation to the laboratory; (B) tooth was cleaned with PBS with antibiotic and antimycotic; (C) tissue pulverizer was used for exposing the pulp tissue, which was removed with endodontic instruments; (D) dental pulp was cut in small fragments and transported to the T25 flask; (E) cultivation of hDPSCs.
Figure 9
Figure 9
A graphic representation of the in vivo experiments performed in the various zebrafish lines in the different time frames.
See this image and copyright information in PMC

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