949 D3000 new imprint Word template Vol 13, No 1 (2025) ISSN 2167-8677 (online) DOI 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu Evaluating the Citotoxicity and Antibacterial Activity of Nano Zinc- Glyde Mixture for Intracanal Irrigation Shammaa Anees Sahib AlAnsari1, Maitha Sameer Kadhim2, Hayder Hamed Abed3 1College of Den*stry, Ibn Sina University, Baghdad, Iraq 2Al-Rafidain University College, Baghdad, Iraq 3College of Den*stry, Mustansiriyah University, Baghdad, Iraq Abstract Objec8ve: In this research, we evaluated the anHbacterial efficiency and cytotoxicity of Glyde including its Zinc NPs preparaHons in three different raHos comparing them with the conven- Honal Glyde. Material and Methods: AnHbacterial efficiency was evaluated using inhibiHon zone method against three common gram-posiHve pathogens Enterococcus faecali, Lactoba- cillus and Streptococcus. Three different irrigant preparaHons were evaluated, GI: Glyde gel alone as a control group, GII: experimental gel (3% Zinc NPs-Glyde), GIII: (4% Zinc NPCs-Glyde gel), GIV: (5% Zinc NPCs-Glyde gel). We evaluated the cytotoxicity by the MTT test. assessing the cells’ viability as soon as possible a]er 24h, 48h, and 72h. A]er a color change assess- ment, spectrophotometric analysis with wavelength ranged from 190-780 nm was per- formed. The spectrum analysis was performed for diluted mixtures in solvent. Data were plo^ed and recorded for each wavelength. Kruskal–Wallis (p < 0.05) and post-hoc Bonferroni pairwise (p < 0.05) were used for staHsHcal analysis. Results: All the groups a reducHon of the three types of bacteria (p > 0.05) was seen, with inhibiHon zone increasing with increasing addiHon of zinc NPs up to 5%. For cytotoxicity, it seems that cell bioavailability remained for 24h, 48h, and declined at 72h. Data were not correlated with addiHon of zinc nano parHcles, especially within the visible light range. Conclusion: The addiHon of zinc nano- parHcles has acceptable anHbacterial properHes and cytotoxic features, and Glyde gel may be used for root canal dis- infecHon without remarkable color change. Open Access Cita%on: AlAnsari SAS, et al. (2025) Evalua%ng the Ci- totoxicity and An%bacterial Ac%vity of Nano Zinc-Glyde Mixture for Instracanal Irriga%on. Den%stry 3000. 1:a001 doi:10.5195/d3000.2025.949 Received: May 24, 2025 Accepted: June 26, 2025 Published: August 13, 2025 Copyright: ©2025 AlAnsari SAS, et al. This is an open ac- cess ar%cle licensed under a Crea%ve Commons AVribu- %on Work 4.0 United States License. Email: shammaa.alansary@ibnsina.edu.iq Introduc)on In most cases of irreversible pulpitis and pulp necrosis, pathogens get into the pulp through mainly caries, periodontal disease or trauma [1,2]. Dahlén et al. [3] suggested that this kind of inAlammation is often caused by speciAic bacterial strains such as lactoba- cilli, streptococci, and gram-negative obligate anaerobes. The root canal system is shaped and cleaned chemically and mechanically for the removal of inAlamed and/or necrotic pulp tissue, and thus, of established bacteria. Chemical cleaning has been recommended for the root canal system [4]. Sodium hypo- chlorite works well at amounts between 0.5 and 5.25% [5-7]. Zinc oxide nanoparticle (ZnO NP) is an odor- less white powder found in bone, muscle, skin, and teeth. Its molecular weight is 81.38 g/mol and the U.S. Food and Drug Admin- istration (FDA) recognized it as a safe sub- stance (GRAS). The use of zinc oxide nano- particles (ZnO NPs) in biomedicine is safe, show no systemic toxicity, and are cost-effec- tive based on their increased speciAic surface area and enhanced particle surface activity [7, 8]. Jowkar et al. found that adding ZnO to an EDTA solution for irrigation enhanced the fracture resistance of the roots [9,10]. A study by Aguiar et al. discovered that these NPs helped with alkalization and acted against E. faecalis when mixed with calcium hydroxide NPs and chlorhexidine [11]. Material and Methods Chemicals and reagents Glyde Gel (Dentsply-Sirona, Switzerland) was used as a base material in this experi- ment. The nano zinc with high purity of 99.9 %, 35-45 nm, metal base applied in this work was supplied from US Research Nano- material’s, Inc. 3302 Twig Leaf Lane, Hou- ston, TX77084, USA. Glyde-nano zinc mixture was prepared in three different loading percentages 3%, 4 % and 5 %. All preparations were performed under sterile conditions to avoid any sam- ples contaminations. The three experimental mixtures (group II: III, and group IV) were prepared by weighting zinc nanoparticles following by vigorous mixing with glyde to prepare exactly the required percentage of EvaluaHng the Citotoxicity and AnHbacterial AcHvity of Nano Zinc-Glyde Mixture for Intracanal IrrigaHon Vol 13, No 1 (2025) DOI 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 2 mixture of Zn nano-Glyde. Group I represent the glyde material without any additive nano particles to serve as comparison. Antibacterial activity test Three different human pathogenic bacteria, Enterococcus faecali, (a gram-positive, facul- tative anaerobic coccus), Lactobacillus (Gram-positive, Obligate anaerobes rods) and Streptococcus (Gram-positive Faculta- tive anaerobes cocci) were investigated as tested organisms. A clean cotton swab was used to slide some of the bacterial solution around on the Muller-Hinton agar medium and make sure it was spread out evenly. Af- ter that, the mix was left for 10 minutes. In the layer of agar that came before, 3 wells with a thickness of 5 mm were made. Each well got 50 microliters of puriAied and crude EPS, and D.W. was put in the middle well as a control. After that, the agar plates were taken out. For 18 hours, the plates were kept at 37 °C. Then, the inhibition zones' widths were recorded. The well diffusion experiment was used to see if glyde-zinc nanoparticles could kill pathogens. In each well of the Petri plates, three different amounts of Nano zinc mix were added: 3%, 4%, and 5%. MTT assay The neutral red (NR) and tetrazolium MTT in vitro cytotoxicity assays were compared for prepared mixtures of glyde – nano zinc mix- tures at maximum loading (5%) and for the glyde only on Aibroblast cell line as the bio in- dicator, at three-time intervals (24hr, 48h, and 72h). There was good agreement (r = 0.9052) in how the test agents were ranked based on their median cytotoxicity values (NR50 and MTT50), even though the tests were based on different physiological end- points. Spectroscopic analysis Glyde-Nano zinc mixtures were scanned us- ing spectrophotometer. The wavelength ranged from 190-780 nm. The expert data were plotted and record in each wavelength. Statistical evaluation The three groups were compared using the Kruskal–Wallis test (p < 0.05): GI (glyde alone), GII (glyde plus 3% zinc), GIII (glyde plus 4% zinc), and GIV (glyde plus 5% zinc). We used Post-hoc Bonferroni pairs (p < 0.05) to Aind the means that are very different from each other. Results Table 1 shows the inhibition zones as anti- bacterial test against Lactobacillus (Gram- positive, obligate anaerobes rods). There was a signiAicant difference depending on the added zinc nanoparticles with different ra- tios of glyde as intracanal material (p > 0.05). When using post hoc Bonferroni, we found a signiAicant difference between GII and GIV. In Table 2, inhibition zone diameters (mm) of Enterococcus faecali, (a gram-positive, fac- ultative anaerobic coccus) by antibacterial action of Glyde groups showed a signiAicant difference among tests groups by adding zinc nano particles (p>0.05). Post-hoc Bonferroni pairwise comparisons showed a signiAicant difference between Glyde group alone and group IV (5% zinc). In Table 3, there was a signiAicant difference among tests groups (p>0.05) according to the inhibition zones diameters (mm) of Streptococcus (Gram-positive Facultative anaerobes cocci) by the effect of Glyde alone and Glyde mixed zinc nano particles. Using Post-hoc Bonferroni pairwise comparisons showed a signiAicant difference between Glyde group alone and group IV (5% zinc). To evaluate cytotoxic, a human normal Aibro- blast cell line (BJI) was used. The MTT essay was performed at 24h, 48h and 72h. At 24h, the result revealed that Glyde alone IC50 was at a dose of 85.6mg/ml. After adding zinc na- noparticles within the same interval, the IC50 was 83.12mg/ml. Cell viability was 92.66% at a dose of 50mg/ml, while at the same dose cell viability was 77.16% with Glyde. Increasing the dose of MTT up to 1000mg/ml decreased cell viability for both Glyde and Glyde zinc mix (10.94% and 11.15%, respectively) (Figures 1 and 2). At 48h the result revealed that Glyde alone IC50 was at a dose of 85.6mg/ml. After add- ing zinc nanoparticles within the same time, the IC50 was 83.12mg/ml. Cell viability was 92.66% at a dose of 50mg/ml for Glyde only, while at the same dose, cell viability was 77.16% for Glyde zinc mix. Increasing the dose of MTT up to 1000mg/ml decreased cell viability for both Glyde and Glyde zinc mix (10.94% and 11.15%, respectively) (Figures 3 and 4). At 72h, the result revealed that IC50 was 44.71 mg/ml for Glyde alone and 7.71mg/ml for Glyde zinc mix. On the other hand, cell vi- ability at a dose of 50mg/ml was 40.12% for Glyde alone and 27.17% for Glyde zinc mix. When the dose was increased up to 500mg/ml, only 7.25% viability was seen for Glyde only and 5.85% viability was seen at a dose of 1000mg/ml (Figures 5 and 6). Spectroscopic analysis The UV-VIS scanning recorded very similar values, without any intense abnormal peaks, especially in visible range. Discussion Nanoparticles are often used to combat mi- crobial infections because they are less cyto- toxic [12,13]. To test the antibiotic effects of added zinc nanoparticles to Glyde, Entero- coccus faecali was picked because it is one of the types most often found in individuals who have had persistent root canal infec- tions. Another reason, E. faecalis can live in harsh conditions with few nutrients and for a long time inside canals, even after treat- ment [14,15]. It can also survive at very hot temperatures or acidic conditions [16]. In our study, selection of Streptococcus and lactobacillus species was due to their viru- lence and involvement in rapid progression of periodontal diseases, endoperio lesions, both acidogenic and aciduric environments, enamel demineralization leading to dental caries, and initiation of bioAilm containing Streptococcus [17-19]. The antibacterial effect of Glyde gel depends on the oxygen bubbling which are liberated from carbamide peroxide through the re- moving of pulp tissue [20]. As shown in Ta- bles 1, 2, and 3 there was a signiAicant differ- ence among tests groups (p>0.05) according to the inhibition zones diameters (mm) for the three bacterial types. Results indicated that as we increase the percentage of zinc na- noparticles in the mixtures, the more inhibi- tion zones size appeared in diameter for the three types of bacteria in comparison with the Glyde alone or even low zinc percent. These results may relate to electrostatic forces between the positive bacterial cell wall and the positive NPs that enhance the antibacterial activity [21]. Positively charged nano particles showed higher antibacterial activity against both S. mutans and E. faecalis. While for the Airst time zinc nano mixtures was investigated for its antibacterial efAi- ciency, many other metal nano particles re- vealed same higher activity against different types of bacteria when added to Glyde [22]. Or they Ailled the canals with nanoparticles as a kind of irrigant [16]. Our study looked at the possibility of harmful cytotoxicity of a Glyde material (after 24 h, 48 h, and 72 h) with and without 5% zinc nanoparticles as the highest loading percent. To get to a level that is useful in medicine, we used the MTT method to look for deadly activity in the hu- man normal Aibroblast cell line (BJI). Color was used in the MTT test to show how meta- bolically active cells are and how dangerous they are. This is by turning a yellow color called 3-(4, 5-dimethylthiazol-2-yl)-2, 5-di- phenyl tetrazolium bromide that dissolves in water into purple formazan crystals that do not dissolve in water [23]. Material consid- ers as cytotoxic if it kills more than half of the cell’s lysis, which is a score of ≤ 21. As per ISO 10993-5, the test extract was harmful if less than 70% of the cultures survived compared to control cultures that were not handled with it [24,25]. EvaluaHng the Citotoxicity and AnHbacterial AcHvity of Nano Zinc-Glyde Mixture for Intracanal IrrigaHon Vol 13, No 1 (2025) DOI 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 3 Immediately after 24h, the Aindings revealed that there was a remarkable difference for adding zinc Nano particles to Glyde material when IC50 diminished from 220.1 to 56.03 mg/ml but cell viability was still acceptable for both Glyde and Glyde zinc mix, even when decreased, mostly due to the immedi- ate role of zinc particles. Up to 48 h, we saw that both Glyde and its zinc additive mixture reached close to IC50 results with cell viabil- ity at 50 mg/ml. MTT dose of 92.66473274%, 77.16150079%, which is suitable biocompatibility. While increasing time up to 72h, we noticed cell viability in- creasing by adding zinc nano particles com- pared with Glyde alone. This could be due to suppression of cytotoxicity by interaction between Glyde EDTA and zinc nano particles [26]. Considering the increasing demands for aesthetics, biomaterials should be chro- matically stable, present optical properties like dental structures and not exert staining effects to hard dental tissues [27]. Biomateri- als should be color stable, have visual fea- tures like tooth structures, and not damage hard mouth tissues. The zinc mixture used in this study could not change color and zinc, a small metal, seen to cause staining. Zinc may even reverse discoloration and greatly re- duces the darkening of teeth caused by SDF [28]. Conclusions The study results revealed that zinc nano particles, when added to Glyde, signiAicantly increased the antibacterial activity against three types of human pathogens: Enterococ- cus faecali, Lactobacillus and Streptococcus. It also showed it is antibacterial efAiciency is dose dependent. However, this offers a promising material to be used during rotary instrumentation as a lubricant and antibac- terial material at the same time. These exper- imental mixtures revealed favorable cell via- bility results when tested by MTT assay and they did not induce remarkable color change. References 1. Seltzer S, Bender IB, Ziontz M. The interrelation- ship of pulp and periodontal disease. Oral Surg Oral Med Oral Pathol. 1963; 16:1474–90. 2. Andreasen FM, Kahler B. Pulpal response after acute dental injury in the permanent dentition: clinical implications—a review. J Endod. 2015; 41:299–308. 3. Dahlén G. Microbiology and treatment of dental abscesses and periodontal—endodontic lesions. 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Valdivia-Tapia, Guilherme Roncari Rocha, Yan Wu, Xinyue Mao, Nora Alomeir, Danielle Benoit, Anderson T. Hara, Tong Tong Wu, Jin Xiao, Yihong Li. Effect of Zinc on improving silver diamine gluoride-derived tooth discoloration in vitro. BMC Oral Health (2024) 24:1410. EvaluaHng the Citotoxicity and AnHbacterial AcHvity of Nano Zinc-Glyde Mixture for Intracanal IrrigaHon Vol 13, No 1 (2025) DOI 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 4 Kruskal-Wallis Test Post-hoc Bonferroni pairwise comparisons Lacto + Glyde mixed groups Group N Median (Range) P-value Sample 1 + Sample 2 Test Statis- tics Standard Er- ror P- value Lacto+ GII 3 21 (1) 0.02 Lacto +G II Lacto +GI - 0.3 2.9 1.0 Lacto+ GIII 3 24 (2) Lacto+ GII Lacto +GIII - 4.6 2.9 0.6 Lacto+ GIV 3 28 (1) Lacto +GII Lacto +GIV - 7.6 2.9 0.05 Lacto+GI 3 22 (2) Lacto +GI Lacto +GIII 4.3 2.9 0.8 Lacto +GI Lacto +GIV 7.3 2.9 0.07 Lacto + GIII Lacto +GIV - 3.0 2.9 1.0 Table 1. Inhibition zone diameters in mm of Lactobacillus bacteria by Glyde alone and mixed groups. EvaluaHng the Citotoxicity and AnHbacterial AcHvity of Nano Zinc-Glyde Mixture for Intracanal IrrigaHon Vol 13, No 1 (2025) DOI 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 5 Table 2. Inhibition zone diameters in mm of Enterococcus faecali bacteria by Glyde alone and mixed groups. Enterococcus faecali +Glyde mixed groups Group N Median (Range) P- value Sample 1 – Sample 2 Test Statis- tics Stand- ard Er- ror P-value Entero+ GII 3 20 (2) 0.03 Entero + GI Entero + GII 2.1 2.8 1.0 Entero + GIII 3 22 (3) Entero + GII Entero + GIII - 1.6 2.9 1.0 Entero + GIV 3 25 (1) Entero + GII Entero + GIV - 5.8 2.9 0.2 Entero + GI 3 20 (1) Entero + GI Entero + GIII 3.8 2.9 1.0 Entero +GI Entero +GIV 8.0 2.9 0.02 Entero +GIII Entero +GIV - 4.1 2.9 0.8 EvaluaHng the Citotoxicity and AnHbacterial AcHvity of Nano Zinc-Glyde Mixture for Intracanal IrrigaHon Vol 13, No 1 (2025) DOI 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 6 Strepto+Glyde mixed groups Group N Median (Range) P-value Sample 1 – Sample 2 Test Statis- tics Standard Error P-value Strepto+ GII 3 15 (1) 0.01 Entero +GI Entero +GII 2.1 2.8 1.0 Strepto + GIII 3 22 (1) Entero +GII Entero+ GIII - 1.6 2.9 1.0 Strepto + GIV 3 24 (1) Entero +GII Entero + GIV - 5.8 2.9 0.2 Strepto + GI 3 13 (1) Entero + GI Entero + GIII 3.8 2.9 1.0 Entero +GI Entero +GIV 8.0 2.9 0.02 Entero +GIII Entero + GIV - 4.1 2.9 0.8 Table 3. Inhibition zone diameters in mm of Streptococcus bacteria by Glyde alone and mixed groups. EvaluaHng the Citotoxicity and AnHbacterial AcHvity of Nano Zinc-Glyde Mixture for Intracanal IrrigaHon Vol 13, No 1 (2025) DOI 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 7 Figure 1. Cell availability of Glyde alone at 24h. Figure 2. Cell availability of Glyde – zinc mix at 24h. Figure 3. Cell availability of Glyde alone at 48h. Figure 4. Cell availability of Glyde-Zinc mix at 48h. Figure 5. Cell availability of Glyde alone at 72h. Figure 6. Cell availability of Glyde-Zinc mix at 72h. EvaluaHng the Citotoxicity and AnHbacterial AcHvity of Nano Zinc-Glyde Mixture for Intracanal IrrigaHon Vol 13, No 1 (2025) DOI 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 8 Glyde only Plus 3% zinc Plus 4% zinc Plus 5% zinc Min in UV 0.106630904 At 364 nm. 0.295807 At 358 nm. 0.177871 At 380 nm. 0.052597 at 372 nm Max in UV 4.3346711 At 202 nm. 2.200893 At 192 nm. 4.835236 At 224 nm. 4.492694 At 208 nm. Min in visible 0.106996147 At 390 nm. 0.296713 At 424 nm. 0.165798 At 602 nm. 0.0453 at 426 nm, Max in visible 0.131435706 At 780 nm. 0.309136 At 772 nm. 0.178722 At 404 nm. 0.0528 at 382 nm. Table 4. Minimum and maximum values of wavelength absorption according to Glyde and its additive mixtures.