Glass ionomer cement particles pre-reacted with chlorhexidine: Physical/chemical properties and antimicrobial activity

Glass ionomer cement particles pre-reacted with chlorhexidine: Physical/chemical properties and antimicrobial activity

1. GOOD MORNING 1

2. Glass ionomer cement particles pre-reacted with chlorhexidine: Physical/chemical properties and antimicrobial activity Gomes FS, Campos-Ferreira PV, Macedo RFC, Costa-Oliveira BE, Bauer J. Glass ionomer cement particles pre-reacted with chlorhexidine: Physical/chemical properties and antimicrobial activity. J Mech Behav Biomed Mater. 2024 Oct;158:106678. doi : 10.1016/j.jmbbm.2024.106678. Epub  2024 Jul 31. 2

3. CONTENTS Introduction​ Aim Null hypothesis Materials and methods ​Statistical analysis Results ​Discussion ​ Conclusion References 3

4. INTRODUCTION 4

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6. COMPOSITION : 6

7. AIM The objective of the current study is to evaluate the Cohesive strength , Modulus of elasticity , Microhardness , Surface roughness , Ion release (Ca+2,PO4−,and F−), Setting time , and Antibacterial activity 7

8. Null hypothesis (1) the incorporation of pre-reacted particles with chlorhexidine (CHX) in glass ionomer cement (GIC ) would not impair the physical/chemical properties. (2) the incorporation of pre-reacted particles with chlorhexidine (CHX) in glass ionomer cement (GIC) would not enhance the antibacterial properties 8

9. MATERIALS AND METHODS 1 . Loading of CHX into GIC: The commercial GIC used in this study was Bioglass R ( Biodinamica , Parana, Brasil ). An aqueous solution of 20% chlorhexidine digluconate ( Naturalle - compounding pharmacy) was used. For every 1 ml of CHX, 10 mg of GIC powder was employed. The solution was mixed using a magnetic stirrer ( Heidolph instruments, Schwabach , Germany) for 1 h. 9

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11. Afterwards, the obtained solutions were centrifuged (4000 rpm) for 10 min and the resulting material was stored in an oven at 37 ◦C for 7 days. Experimental GIC powders containing concentrations of 1%, 2.5% and 5% of particles functionalized with chlorhexidine. Functionalised GIC + Non functionalized GIC Experimental GIC 11

12. The portion corresponding to the liquid of the material was not changed and the powder/liquid proportion for preparing the material followed the manufacturer’s recommendations. O GIC containing non-functionalized particles was used as a control. 12

13. 2 . Fourier-transform infrared spectroscopy (FTIR) powders analyze: To characterize the functionalization of glass ionomer particles with chlorhexidine, FTIR analyses were conducted before and after the loading process. 13

14. 3. Cohesive strength and modulus of elasticity: Hourglass-shaped specimens (10 mm length, 2 mm width, and 1 mm thickness) with a cross-sectional area of 1 were fabricated for each group. The materials were inserted into a polymeric matrix, covered with a polyester strip until complete setting.   14

15. 4. Microhardness: The specimens resulting from the cohesive strength tests were fixed onto an acrylic base and polished for the measurement of the Initial Vicker Microhardness. The HMV-G20 microhardness was used. 15

16. 5. pH test: Disk specimens were prepared and are manipulated with a plastic spatula for GIC. The specimens were placed in vials containing 5 mL of distilled water for 28 days. pH readings were then taken at the following intervals: 15 min, 30 min, 1h, 2h, 24h, 48h, 7days, 14days, and 28days. The solutions were frozen for subsequent analysis of , , and ion release.   16

17. 6. Ions release Ca+2 , PO4 − and F− : 17 Chlorhexidine-Functionalized GICs :

18. 7. Setting time: For the setting time evaluation, a Gilmore needle was used. 18

19. 8. Surface roughness: To verify the roughness of the material a Surface Roughness Tester was used. Twelve disc-shaped specimens (10 mm × 1 mm) were prepared for each group (n = 12), and subjected to a polishing sequence. The force applied by the needle was 5N. 19

20. 9. Antibacterial activity The experimental GIC discs were placed in a sterile 24-well culture plate, and sterilized using ultraviolet light for 30 minutes. 20

21. 10. Statistical analysis: Statistical analysis was performed using SigmaPlot software. All data was subjected to the Shapiro-Wilk test to determine normality. Cohesive strength, modulus of elasticity, microhardness, ions release and surface roughness data were submitted to ANOVA (One-way) and Holm- Sidak . 21

22. RESULTS 1. Fourier-transform infrared spectroscopy (FTIR) powders analyze : Demonstrated the presence of a band at 1580 cm⁻ 1 , indicating protonation of the N–H⁺ group of chlorhexidine. This band suggests direct interaction with the particle surfaces. 22

23. 2. COHESIVE STRENGTH Statistical analysis indicates that functionalization of particles with chlorhexidine digluconate increases cohesive strength compared to the control group (Table 2). 23

24. 3. MICROHARDNESS The results indicate an increase in microhardness as the concentration of chlorhexidine increases (Table 2). 4. pH TEST The pH of all groups showed an acidic behavior in all periods evaluated. 24

25. 5. Ions release Ca+2 , PO4 − and F− The means and standard deviations of fluoride, calcium, and phosphate ion release are presented in Fig. 4. The incorporation of prereacted CHX particles did not alter the release of fluoride ions, calcium ions, and phosphate ions compared to the control group. 25

26. 6. SETTING TIME The setting time readings indicate that chlorhexidine functionalization did not alter the property of the material (Table 2). 7. SURFACE ROUGHNESS The surface roughness results showed no statistically difference between the tested groups (Table 2). 26

27. 8. ANTIBACTERIAL ACTIVITY: Statistical analysis indicated a significant difference in CFU counts between the tested groups. GIC-CHX 0.12%(positive control), 2.5%,5% > GIC-CHX 1% and control group. 27

28. DISCUSSION Null Hypothesis : The study initially posited that incorporating chlorhexidine (CHX) into glass ionomer cement (GIC) would not impair its properties; this was accepted . Rejection of Other Null Hypotheses : The hypotheses suggesting that CHX would not affect antimicrobial activity and mechanical properties were rejected . 28

29. Cohesive strength Ion release Microhardness Surface roughness Antibacterial activity Setting time Durability of restoration pH Rheology 29 POSITIVE OUTCOMES NO CHANGES Therefore, while the incorporation of CHX did result in certain enhancements, it did not affect other crucial properties of GIC.

30. The incorporation of GIC cement particles functionalized with chlorhexidine has emerged as a promising strategy to improve the clinical performance of restorations made with glass ionomer, especially in atraumatic restorative treatments (ART’s). ART is recommended for clinical cases involving poor oral hygiene due to conditions like dementia and physical limitations , requiring effective cavity-sealing materials. ( Frencken et al., 2014) 30

31. Importance of Antimicrobial Properties : It is crucial for these materials to not only seal cavities effectively but also prevent microorganism proliferation to avoid recurrent carious processes and potential pulp involvement. (Duque et al., 2017) Hospitalized patients require oral health care while in bed, with chlorhexidine being the most commonly used material for oral hygiene. The most used material in special needs patients is glass ionomer through ART. Therefore, an association of the two materials is strongly desirable. 31

32. The successful loading of chlorhexidine onto glass ionomer cement can be attributed to the presence of silicates in the GIC particles, which facilitate strong binding interactions between the alkaline portions of chlorhexidine (NH) molecules and the silanol (Si–OH) groups present on the surface of the particles.(Moritz and Geszke -Moritz, 2015) Reduction in Bacterial Counts : Studies report a reduction in bacterial counts during certain evaluation periods. 32

33. Bactericidal properties : The experimental groups GIC-CHX 2.5% and GIC-CHX 5% exhibited significant bactericidal properties. Mechanism of Action : Chlorhexidine releases through the natural degradation of the ionomer surface and ion exchange, leading to bacterial cell membrane disruption and ultimately bacterial death. Findings by Jedrychowski et al. (1983) warn that GIC may deteriorate if CHX is added at concentrations greater than 5% . 33

34. Recently, a systematic review and meta-analysis revealed that there was no significant difference in the survival of ART restorations when CHX was used as a pretreatment. (Takahashi et al., 2006). The divergent results regarding the mechanical properties resulting from the incorporation of CHX into the GIC can be attributed to the different methods of adding CHX, which can be in powder (chlorhexidine diacetate) or liquid (chlorhexidine digluconate ) potentially altering the powder/liquid ratio or the setting reaction of the material. 34

35. Greater cohesive strength : improves the material's ability to withstand shear and traction forces, reducing risk of fracture . Durability : microhardness suggest enhanced durability and longevity of GIC dental restorations. Surface roughness and setting time remained unchanged, which are important for preventing bacterial biofilm accumulation and minimizing patient chair time. 35

36. The well-established property of ion release exhibited no significant alterations between the control and the other experimental groups, suggesting that the pre-reacted particles with CHX, bound to silanol, are permeable, facilitating the continuous release of ions. Similarly, pH variation showed no discernible difference from the control group. 36

37. These findings highlight the promise of using functionalized GIC particles at high concentrations as a viable strategy for improving the mechanical properties and durability of dental restorations without compromising other critical aspects of the material’s performance. Brito et al has evaluated the effect of chlorhexidine-functionalized  bioactive glass  (45S5) on mechanical and  bioactive properties  of experimental self-etching adhesives. 37 Brito, A.C.R., Ferreira, P.V.C., Cardoso, S.M.N.R., Guimar ˜ aes , S.J.A., Gomes, F.S., Cavaleiro-de-Macedo, R.F., Oliveira, B.E.C., De Oliveira, T.J.L., Dos Santos, A.P.S.A., Bauer, J., 2023. Chlorhexidine-Loaded bioactive glass for incorporation into adhesive systems: mechanical properties, antibacterial activity, cell viability, and hydroxyapatite precipitation. Int. J. Adhesion Adhes . 124, 103384. https://doi.org/ 10.1016/j.ijadhadh.2023.103384

38. 38 MATERIALS:

39. Results:- The 45S5 CHX group (20%) presented the lowest values of mechanical properties. On the other hand, only groups containing 45S5 previously functionalized with  chlorhexidine were able to prevent biofilm formation. SEM and  EDS analyses  revealed the functionalization of 45S5  bioactive glass  with  chlorhexidine  did not impair the  bioactivity  of the newly developed adhesive systems. 39

40. Cristiane Duque et al   evaluated the effects of incorporating chlorhexidine (CHX) in the in vitro biological and chemico -mechanical properties of GIC and in vivo clinical/microbiological follow-up of the ART with GIC containing or not CHX. 40 Duque C, Aida KL, Pereira JA, Teixeira GS, Caldo -Teixeira AS, Perrone LR, Caiaffa KS, Negrini TC, Castilho ARF, Costa CAS. In vitro and in vivo evaluations of glass-ionomer cement containing chlorhexidine for Atraumatic Restorative Treatment. J Appl Oral Sci. 2017 Sep-Oct;25(5):541-550. doi : 10.1590/1678-7757-2016-0195

41. Antimicrobial activity of GIC was analyzed using agar diffusion and anti-biofilm assays . A randomized controlled trial was conducted on 36 children that received ART either with GIC or GIC with CHX. Saliva and biofilm were collected for mutans streptococci (MS) counts and the survival rate of restorations was checked after 7 days, 3 months and one year after ART. 41 Duque C, Aida KL, Pereira JA, Teixeira GS, Caldo -Teixeira AS, Perrone LR, Caiaffa KS, Negrini TC, Castilho ARF, Costa CAS. In vitro and in vivo evaluations of glass-ionomer cement containing chlorhexidine for Atraumatic Restorative Treatment. J Appl Oral Sci. 2017 Sep-Oct;25(5):541-550. doi : 10.1590/1678-7757-2016-0195

42. Results:  Incorporation of 1.25% and 2.5% CHX improved the antimicrobial activity of GIC, without affecting F release and mechanical characteristics, but 2.5% CHX was cytotoxic . Survival rate of restorations using GIC with 1.25% CHX was similar to GIC. A significant reduction of MS levels was observed in saliva and biofilm samples 7 days after treatment. 42 Duque C, Aida KL, Pereira JA, Teixeira GS, Caldo -Teixeira AS, Perrone LR, Caiaffa KS, Negrini TC, Castilho ARF, Costa CAS. In vitro and in vivo evaluations of glass-ionomer cement containing chlorhexidine for Atraumatic Restorative Treatment. J Appl Oral Sci. 2017 Sep-Oct;25(5):541-550. doi : 10.1590/1678-7757-2016-0195

43. Luana Mafra Marti has evaluated the porosity , surface roughness and anti-biofilm activity of a glass-ionomer cement (GIC) after incorporation of different concentrations of chlorhexidine (CHX) gluconate or diacetate. Marti LM, Becci AC, Spolidorio DM, Brighenti FL, Giro EM, Zuanon AC. Incorporation of chlorhexidine gluconate or diacetate into a glass-ionomer cement: porosity, surface roughness, and anti-biofilm activity. Am J Dent. 2014 Dec;27(6):318-22 . 43

44. MATERIALS: 44

45. Results:  Regarding GIC porosity, the ANOVA showed that the presence of CHX increased the porosity proportionally to the increase in concentrations, without however, presenting interaction between material and concentration. The surface roughness test demonstrated no statistically significant effect. Anti-biofilm activity analysis pointed out a significant effect in reducing microorganisms with CHX diacetate. 45 Marti LM, Becci AC, Spolidorio DM, Brighenti FL, Giro EM, Zuanon AC. Incorporation of chlorhexidine gluconate or diacetate into a glass-ionomer cement: porosity, surface roughness, and anti-biofilm activity. Am J Dent. 2014 Dec;27(6):318-22.

46. LIMITATIONS In Vitro Nature : The study was conducted in vitro, which may not fully reflect clinical conditions Chlorhexidine Release Profile : The release profile of chlorhexidine was not analyzed, limiting insights into the duration of its antimicrobial activity. Single Type of GIC : Only one type of glass ionomer cement (GIC) was used for loading chlorhexidine, which may affect the generalizability of the findings. 46

47. CONCLUSION The pre-reacted CHX in GICs was able to confer antimicrobial activity , improve cohesive strength , microhardness , and did not impair ion release, setting time, and roughness. 47

48. REFERENCES Brito, A.C.R., Ferreira, P.V.C., Cardoso, S.M.N.R., Guimar ˜ aes , S.J.A., Gomes, F.S., Cavaleiro-de-Macedo, R.F., Oliveira, B.E.C., De Oliveira, T.J.L., Dos Santos, A.P.S.A., Bauer, J., 2023. Chlorhexidine-Loaded bioactive glass for incorporation into adhesive systems: mechanical properties, antibacterial activity, cell viability, and hydroxyapatite precipitation. Int. J. Adhesion Adhes . 124, 103384. https://doi.org/ 10.1016/j.ijadhadh.2023.103384. Carvalho, N.K., Barbosa, A.F.A., Coelho, B.P., Gonçalves, L.S., Sassone , L.M., Silva, E.J.N. L., 2021. Antibacterial, biological, and physicochemical properties of root canal sealers containing chlorhexidine-hexametaphosphate nanoparticles. Dent. Mater. 37 (5), 863–874. https://doi.org/10.1016/j.dental.2021.02.007. 48

49. de Castilho, A.R.F., Duque, C., Negrini , T.C., Sacono , N.T., de Paula, A.B., De Souza Costa, C.A., Spolidorio , ´ D.M., Puppin-Rontani , R.M., 2013. In vitro and in vivo investigation of the biological and mechanical behaviour of resin-modified glassionomer cement containing chlorhexidine. J. Dent. 41, 155–163. https://doi.org/ 10.1016/j.jdent.2012.10.014. Chen, L., Wang, Q., Xiong, L., 2017. Molecular dynamics study on structure stability, lattice variation, and melting behavior of silver nanoparticles. J. Nanoparticle Res. 19 https://doi.org/10.1007/s11051-017-4003-7. Chen, J., Zhao, Q., Peng, J., Yang, X., Yu, D., Zhao, W., 2020. Antibacterial and mechanical properties of reduced graphene-silver nanoparticle nanocomposite modified glass ionomer cements. J. Dent. 96, 103332. https://doi.org/10.1016/j. jdent.2020.103332. 49

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