Fluoride Release and Inhibition of Caries Around Restoration by Ion Releasing Restorative Materials: An In Vitro Study

doi:10.21203/rs.3.rs-4125468/v1

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Fluoride Release and Inhibition of Caries Around Restoration by Ion Releasing Restorative Materials: An In Vitro Study

https://doi.org/10.21203/rs.3.rs-4125468/v1

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Objectives: To evaluate and compare fluoride release of three ion releasing restorative materials and assess their inhibitory effect on secondary caries induced by cariogenic bacteria.

Materials and Methods: Three ion releasing restorative materials, self-adhesive hybrid composite (group A), ion releasing flowable composite liner (group B), and alkasite restorative material (group C), were examined. Twenty-two discs were fabricated from each material for fluoride release test, conducted on days 1, 7, and 14. For assessing secondary caries inhibition, sixty-six sound molar teeth were used and standardized class V cavities were prepared. Teeth were divided into three groups according to each material, followed by 2000 cycles of thermocycling. Subsequently, teeth were immersed in a solution containing cariogenic bacteria for 30 days. After that, teeth were sectioned bucco-lingually and analyzed using a polarized light microscope to measure inhibition area, outer lesion depth, and extension. Data was statistically analyzed using different tests.

Results: Statistically significant differences in fluoride release existed among materials. Self-adhesive hybrid composite exhibited the highest fluoride release, followed by alkasite restorative material and ion releasing flowable composite liner.

Lesion extension and depth were statistically significantly greater next to ion releasing flowable composite liner, while next to alkasite restorative material, they exceeded those next to Self-adhesive hybrid composite. The inhibition areas next to self-adhesive hybrid composite were statistically significantly larger than alkasite restorative material and ion releasing flowable composite liner.

Conclusion: All three ion releasing restorative materials displayed fluoride release and potential to inhibit secondary caries formation. Self-adhesive hybrid composite demonstrated the highest fluoride-releasing potential and the greatest ability to inhibit secondary caries. Conversely, ion releasing flowable composite liner exhibited the least fluoride release with minimal secondary caries inhibition. Increasing fluoride release correlated with larger inhibition areas and reduced outer lesion depth and extension.

New dental restorative materials prioritize bioactivity, facilitating ion release for bonding between the material and dental tissue. This promotes Apatite-like material formation, reducing microleakage and postoperative sensitivity.⁽¹⁾ Fluoride's anticariogenic effects involve mechanisms like demineralization reduction, remineralization enhancement, microbial growth suppression, and glucose metabolism inhibition. Using topical fluoride in toothpaste and varnish forms is the most effective way to prevent caries formation, creating acid-resistant fluorapatite crystals and causing bacterial death via glucose metabolism disruption. These mechanisms are termed physical-chemical and biological anticariogenic approaches.⁽²⁾

Adding fluoride to various restorative materials, such as Glass Ionomer Cements (GIC), resin-modified GICs (RMGIC), and resin composite, enhances their ability to prevent caries and promote natural tooth remineralization. These materials increase fluoride levels in saliva, plaque, and tooth tissues while releasing fluoride into the oral environment, contributing to their anticariogenic properties.⁽³⁾ The development of fluoride-releasing products for restorative materials, lining cements, sealants, and orthodontic cements has gained significant attention. These products have proven effective in reducing the occurrence of secondary caries.⁽⁴⁾

Oral cavity has more than 1000 species of microorganisms.⁽⁵⁾ The oral environment, with various surfaces like teeth and mucosa, fosters a diverse microorganism population, including bacteria and fungi, due to its suitable growth conditions.⁽⁶⁾ Dental plaque is the main cause for dental caries that is the most prevalent disease in humans.⁽⁷⁾ Dental caries is influenced by tooth susceptibility, bacteria, and carbohydrates. Cariogenic bacteria produce lactic acid, leading to enamel demineralization and potential dentin involvement if untreated.⁽⁸⁾ Secondary caries is the main reason for the failure of dental restorations.⁽⁹⁾ New dental materials have been developed to minimize the destruction of tooth structure when replacing restorations. These materials are designed to interact with cariogenic bacteria by inhibiting their adhesion, growth, or metabolism.⁽¹⁰⁾

Recent research has focused on incorporating antibacterial agents like bioactive glass into dental materials. This aims to not only inhibit secondary caries formation but also leverage additional benefits offered by these materials.⁽¹¹⁾ According to the literature review, the three ion releasing dental materials included in this research (self-adhesive hybrid composite, ion releasing flowable composite liner and alkasite restorative material) have few studies regarding their fluoride release potential and their ability to inhibit secondary caries formation. Therefore, there is an increasing need for further studies.

This study employed three ion releasing materials, self-adhesive hybrid composite, ion releasing flowable composite liner and alkastie restorative material. (Table 1)

Table 1

Materials used in the study.
Material	Manufacturer	Type	Composition	Batch Number
Surefil One	Dentsply, Konstanz, Germany	Dual-curing, Self-adhesive composite hybrid	MOPOS, BADEP, acrylic acid, stabilizer, .water, reactive glass filler, non-reactive glass filler and initiator.	2104000853
Re-Gen Flowable Composite	Vista Apex, USA	Light-curing, Flowable composite liner	Barium glass, 2-Propenoic acid, 2-methyl-, (1-methylethylidene) bis[4,1-phenyleneoxy(2-hydroxy-3,1-propanediyl)]ester, TEGDMA, Submicron Silica, Benzoic acid, 4-(dimethylamino)-, ethyl ester.	90648
Cention Forte	Ivoclar Vivadent, Liechtenstein	Self-curing Alkasite restorative material	UDMA, initiator, pigments, glass filler.	740832WW
	Vista Apex, USA	Universal Adhesive	2-Propenoic-acid,2-methyl-,(1-methylethylidene)bis[4,1-phenyleneoxy(2-hydroxy-3,1-propanediyl)]ester,1,2,4,5-BTC,1,4-bis[2-[(2-methyl-1-oxo-2-propen1-yl)oxy]-1-[[(2-methyl-1-oxo-2-propen-1-yl)oxy]methyl]ethyl] ester, 10-MDP, Benzoic acid, 4-(dimethylamine)-, ethyl ester, 2-HEMA, Ethyl alcohol.	90637
Clean & boost	Vista Apex, USA	Dentin and enamel cleanser	2-hydroxyethyl methacrylate, Propan-2-ol, Nitric acid.	90417
Cention Primer	Ivoclar Vivadent, Liechtenstein	self-etching and self-curing primer	2-HEMA, Bis-GMA, ethanol, MA-PA-Ester, 1,10-decandiol dimethacrylate, MA-MO-PAA, camphor quinone, 2-DMAM	740832WW
Tryptic Soy Broth	Difco-Becton, USA	Soybean-casein Digest Medium	Bacto™ Trypton, Bacto Soytone, Glucose, Sodium Chloride, Dipotassium Hydrogen Phosphate.	8141955
Abbreviations: MOPOS: Modified polyacids, BADEP: Bifunctional acrylate, TEGDMA: Triethylene glycol dimethacrylate, UDMA: Urethane Di methacrylate, BTC: Benzenetricarboxylic Acid, MDP: Methacryloyloxydecyl dihydrogen phosphate, HEMA: Hydroxyethylmethacrylate, Bis-GMA: bisphenol A-glycidyl methacrylate, MA-PA: methacrylated phosphoric acid, MA-MO-PAA: methacrylate modified polyacrylic acid, DMAM: dimethylaminoethyl methacrylate.

Sample size

Sample size was calculated using G*Power software (v3.1.9.7),^(12–15) Universität, Kiel, Germany with an anticipated large effect size (f = 0.4) for fluoride release and anti-cariogenic effects among three groups. Each group comprised 22 samples in a one-way ANOVA, totaling 66 units. This achieved 82% power to detect mean differences at a 0.0500 significance level, with an effect size (f) of 0.4000.⁽¹⁶⁾ Fluoride Release Test

Sixty-six discs (2mm thickness, 10 mm diameter) were prepared from Surefil One, Re-Gen Flowable Composite, and Cention Forte. Discs were fabricated by molds placed on a mylar strip and a glass slab and then another mylar strip and glass slap were used to exert adequate pressure and remove excess material.⁽¹⁷⁾ A total of 22 discs were made for each material. Surefil One composite capsule was activated and placed in a capsule mixer for 10 seconds, dispensed into the mold, and allowed to set for 6 minutes. Re-Gen Flowable Composite was injected into the mold and light-cured for 20 seconds. Cention Forte capsules were activated, mixed for 15 seconds, injected into the mold, and allowed to self-cure for 6 minutes. After complete setting of the materials, the discs were removed from molds and polished using impregnated aluminum oxide discs (Sof-Lex, 3M ESPE). Sequence of coarse, medium, fine and superfine grain disks attached to low-speed handpiece (NSK, Tokyo, Japan), was done.⁽¹⁸⁾

Each disc was placed in a separate glass container containing 5mL of deionized water, stored at 37°C in an incubator (Imperial II Incubator, Lab Line Instruments, Inc.) for 24 hours, and the water was changed every 24 hours.⁽¹⁹⁾ After 24h, each container was agitated, and water was removed for examination. Discs were replaced in 5mL of new deionized water. Fluoride release was measured at day 1, 7 and 14.(Fig. 1) The water removed from each container was combined with 0.5 mL of Total Ionic Strength Adjustment Buffer III solution (TISAB III). This was done to regulate the pH, prevent the creation of fluoride complexes, and enable accurate determination of the overall fluoride concentration.⁽²⁰⁾ Fluoride was measured in water using the SPADNS colorimetric method (SM4500 F standard method for examination of water and wastewater 24th edition 2023).

The analysis was based on the reaction between fluoride and a zirconium-dye lake. To create the SPADNS reagent, 958 mg of SPADNS (Sulfanilic acid azochromotrop, Sigma Aldrich, USA) was dissolved in deionized water and diluting it to 500 ml deionized water. Zirconyl-acid reagent was prepared by dissolving 133 mg zirconyl chloride octahydrate, ZrOCl2·8H2O (Sigma Aldrich, USA), in about 25 mL deionized water, adding 350 mL conc HCl, and diluting to 500 ml with deionized water. Acid zirconyl-SPADNS reagent was prepared by mixing equal volumes of SPADNS solution and zirconyl-acid reagent. Standard solutions with fluoride concentrations (0.0, 0.2, 0.4, 0.6, 0.8, 1.0 ppm) were prepared from a certified reference material of fluoride (Merck KGaA, Darmstadt, Germany) and used for calibration. The absorbance of the standards was determined at a wavelength of 570 nm using a UV-visible double beam spectrophotometer (LAMDA 365, PerkinElmer, USA). Samples were diluted by a ratio of 1:50 and measured against the prepared standards to obtain results.⁽²¹⁾

Evaluation of Inhibition of Secondary Caries Caused by Cariogenic Bacteria

Teeth Selection

Sixty-six sound human molar teeth were collected from periodontal disease patients at the surgery department, Faculty of Dentistry, Mansoura University. These teeth were devoid of calculus, cavities, or defects and underwent thorough cleaning using an ultrasonic scaler. Afterward, they were stored in 0.5% chloramine-T for 48 hours and preserved in regularly changed distilled water. All procedures followed infection control standards that had been approved by the Faculty of Dentistry Ethical Committee. The molars were divided into three main groups (n = 22) based on the chosen restorative material.⁽²²⁾

Teeth Preparation

Class V cavities were prepared on the middle third of buccal and lingual surfaces of each tooth,⁽²²⁾ measuring 4mm-long mesiodistally, 2mm-wide occluso-cervically, and 2mm deep.⁽²³⁾ The occlusal and gingival margins were situated on enamel. Preparation was done using a diamond bur (SF-31SC, Mani, INC, Japan) in a high-speed handpiece (Sirona, Germany) with copious air-water cooling spray, and burs were replaced every fifth preparation.⁽²⁴⁾ Cavity dimensions were verified with a periodontal probe for accuracy,⁽²⁵⁾ and all preparations were conducted by one operator. After the cavities were prepared, they were cleaned using aqueous slurry of pumice, rinsed, and then subjected to macroscopic examination with magnification to detect any enamel defects.⁽²²⁾

Restorative Techniques

Regarding Group A, Surefil One capsule was activated and placed in a capsule mixer for 10 seconds. The material was dispensed into the cavity and it was gradually withdrawn. A translucent matrix (Hawe, soft transparent cervical matrices, Kerr, 67771940) was applied to aid in contouring, and the material was left for 6 minutes to set.

In Group B, the Re-gen Flowable Composite application involved several steps. First, Clean & Boost was applied to the tooth surface, including both dentin and enamel, agitated for 10 seconds, rinsed thoroughly, and left moist. Following this, the Re-Gen Universal Adhesive System was applied. This involved applying one drop of the adhesive to the mixing well, followed by using a brush to coat one layer of the adhesive over the cavity preparation and surrounding tooth structure. It was left for 20 seconds without drying, and two more coats were applied using the same brush, with the remaining material in the well. The surface was then dried until a thin immobile layer was achieved. Subsequently, the adhesive was light-activated for 10 seconds using a curing light (C02-C, Premium Plus, USA). Finally, Re-Gen Flowable Composite Liner was applied into the cavity preparation, aided using a translucent matrix for contouring. The restoration was completed by light-activating it for 20 seconds.

For Group C, the procedure involved conditioning of the cavity using Cention Primer. One drop of self-etching self-adhering and self-curing Cention Primer was dispensed into a dipping dish, and a Cention Primer single-use applicator coated with the catalyst was used to stir the liquid for 5 seconds. The primer was then applied to the cavity, starting from the enamel and moving towards the rest of the cavity. The primer was agitated for 10 seconds, dispersed with water and oil-free compressed air until a glossy thin immobile layer remained. Next, the Cention Forte capsule was mixed by a capsule mixer for 15 seconds, and the material was injected into the cavity using a capsule applicator. Contouring was facilitated using a translucent matrix, and the restoration was allowed to self-cure for 6 minutes.

Following the restoration process in all groups, additional steps included finishing and polishing the restorations using impregnated aluminum oxide discs (Sof-Lex, 3M ESPE), were done.⁽²⁶⁾ Sequence of coarse, medium, fine and superfine grain disks attached to low-speed handpiece (NSK, Tokyo, Japan). Each disc was applied to the restoration for 10 seconds under water cooling. The same sequence was done for all restorations.⁽²⁷⁾ Teeth underwent 2000 thermocycles at 5°C and 55°C distilled water baths.⁽²⁸⁾ And an acid-resistant nail varnish was applied to the restored teeth, leaving a 2mm free zone around the cavity margins. Steel wires were affixed to the root portions of every tooth.⁽²⁹⁾ The tooth/wire units were sterilized using Ultraviolet Radiation in MaXtreamTM V (DAIHAN Scientific, Seongbuk-gu, Seoul, Korea) at faculty of Medicine Mansoura University.

Bacterial system

A standard strain of Streptococcus Mutans (ATCC 25175),⁽³⁰⁾ obtained from Microbiological Resources Centre (Cairo MIRCEN) at faculty of Agriculture, Ain Shams University, was used for this in vitro study to induce recurrent caries. The purified isolates were underwent short-term and long-term preservation procedures.⁽³¹⁾ After that, bacteria was incubated (37°C for 24 h) in Tryptic Soy Broth (TSB) (Difco-Becton, Dickinson, and Company - Sparks, MD, USA) with 5% sucrose to stimulate bacterial growth. The bacteria were then plated in a solid culture medium (TSA, Difco-Becton, Dickinson, and Company - Sparks, MD, USA) to obtain isolated colonies and then incubated (37°C) for 24 h. Afterwards, the formed colonies were transferred to tubes containing TSB and 5% sucrose, starting a preconditioning of the bacteria with sucrose for 6 days. The tube containing the inoculum broth was obtained with approximately 1–2 ×10⁶ colony-forming units (CFU)/ml using portable spectrophotometer (Biochrom, Colorwave WPA, CO7500) at a wavelength of 600 nm.⁽²⁴⁾

Measuring Culture Density

Cuvettes were cleaned with deionized water. A control solution (blank) was prepared with 1 mL of TSB (with 5% sucrose) in one cuvette. In another cuvette, 1 mL of inoculum broth was loaded (Sample). The spectrophotometer's wavelength was set to 600nm,⁽³²⁾ and readings were recorded for the blank and sample cuvettes. The inoculum broth was diluted with TSB (with 5% sucrose) until the culture density reached 0.1.⁽³³⁾ Sixty-six falcon tubes were prepared, each containing a tooth immersed in 5 mL TSB + 5% sucrose and 200 µL of inoculum broth. These tubes were then placed in an anaerobic jar,⁽³⁴⁾ and incubated for 48 hours. Fresh falcon tubes with the same solution were prepared every 48 hours, and teeth were transferred to them. Additionally, random old falcon tubes underwent Gram-Stain contamination tests during the one-month procedure.

Contamination Test (Gram Stain)

For Gram staining, the necessary equipment comprises a Bunsen burner, alcohol-cleaned microscope slide, microscope, and inoculation loop. Essential reagents include Crystal violet (primary stain), Gram's iodine solution (mordant), alcohol (decolorizer), saffranine (counterstain), and water. The slide smear preparation involved placing a drop of the solution from the falcon tube onto a microscope slide using an inoculation loop, ensuring an even film thickness over a 15 mm diameter circle. Heat was applied to the slide over a low flame, rotating it to prevent overheating or ring pattern formation. Crystal violet stain was applied to the fixed culture for 30 seconds, followed by washing with water. The dye was fixed with iodine solution for 60 seconds, and after washing, excess water was removed. Alcohol was added to the slide for 5 seconds, followed by thorough washing. Saffranine was applied for 5 minutes, washed away, and the slide was left to dry. Microscopic examination involved adding oil to the slide, using a X40 objective lens for smear evaluation, and a X100 oil immersion lens (Labomed Lx300, Labomed America) to examine all areas. The Streptococcus Mutans strain stained violet, indicating it is Gram-positive.⁽³⁵⁾

The measurement of culture density and contamination tests using Gram stain were repeated every 48 hours for 30 days. Following this duration, the teeth were removed from the bacterial system, and the steel wires were taken off. Then, each tooth was embedded in epoxy resin. After that, the teeth were sectioned bucco-lingually passing through both restorations, using an Isomet 4000 linear precision cutting machine (Buehler, Lake Bluff, IL, USA), resulting in sections of 500 µm in thickness.

Two random sections were further processed by hand grinding to approximately 100 µm thickness using sandpaper numbered 150, 220, 400, and 600 (Qingdao Shangli Abrasives Co., Ltd.) (Fig. 1). After that, sections were embedded in water and examined under a polarized light microscope (Olympus BX50; Olympus, Tokyo, Japan) coupled with a digital camera (Olympus Imaging Crop, Model No. E420 DC 7.4 V) to capture and process images. Measurements were recorded in both the occlusal and cervical regions, encompassing Extension (E) from one outer lesion margin to the opposite margin, Depth (D) at the lesion's deepest point, and Inhibition Area (IA), which represents the distance between the lesion and the restoration margin.⁽²⁴⁾

The collected data were analyzed using the statistical package for social science (SPSS) version 25 (Armonk, NY: IBM Corp) on a compatible IBM computer. Qualitative data were presented as numbers and percentages, while quantitative data were represented as mean ± SD (standard deviation). The analysis of variance (ANOVA) test (F test) was employed for comparing three or more groups with quantitative variables. (LSD). Bivariate correlations were established using Spearman's coefficient. A p-value of < 0.05 was considered statistically significant, and < 0.001 was considered highly significant for two-tailed tests.

Fluoride Release Test

Two-way ANOVA test followed by post hoc test (LSD) was used for comparison between the fluoride test results in the three groups on 1st ,7th, 14th day and showed that fluoride test results in 1st, and 7th days in group A were statistically significantly higher than groups B and C, while in group B, they were statistically significantly lower than group C. Regarding the fluoride test results at the 14th day, in group A were statistically significantly higher than groups B and C, while there was no statistically significantly difference between group B, and group C. The test also showed that there was a significant decrease in fluoride test results across days where the 1st day test results were the highest and the 14th day test results were the lowest in each group of the three groups (Table 2), (Fig. 2).

Table 2

Comparison between three groups as regards fluoride test results at 1st ,7th, 14th day.
Group	A (Surefil one self-adhesive composite)			B (Re-Gen Flowable composite)			C (Cention Forte)			P-value
	Min	-	Max	Min	-	Max	Min	-	Max
	Mean	±	S. D	Mean	±	S. D	Mean	±	S. D
Day 1	24.990		60.830	1.090		14.110	5.500		17.460	P1 = < 0.001* P2 = < 0.001* P3 = 0.027*
Day 1	41.961	±	10.568	5.838	±	4.474	10.579	±	3.629	P1 = < 0.001* P2 = < 0.001* P3 = 0.027*
Day7	6.270		25.000	0.670		2.820	2.260		9.110	P1 = < 0.001* P2 = < 0.001* P3 = 0.023*
Day7	16.994	±	5.821	1.723	±	0.699	4.194	±	1.685	P1 = < 0.001* P2 = < 0.001* P3 = 0.023*
Day 14	2.380		7.120	0.220		1.630	1.090		3.230	P1 = < 0.001* P2 = < 0.001* P3 = < 0.001*
Day 14	5.135	±	1.298	0.738	±	0.533	1.856	±	0.547	P1 = < 0.001* P2 = < 0.001* P3 = < 0.001*
	P4 = < 0.001* P5 = < 0.001* P6 = < 0.001*			P4 = < 0.001* P5 = < 0.001* P6 = < 0.001*			P4 = < 0.001* P5 = < 0.001* P6 = < 0.001*
P1: Group A vs Group B, P2: Group A vs Group C, P3: Group B vs Group C
P4: Day 1 vs Day 7, P5: Day 1 vs Day 14, P6: Day 7 vs Day 14

Evaluation of Inhibition of Secondary Caries Caused by Cariogenic Bacteria

The contamination tests that were run during the 1-month period were negative. No other species were seen in the randomly chosen samples. Only Gram-positive (violet) Streptococcus Mutans were observed. The detected oval-shaped cells were mutant streptococci (Fig. 3). Polarized light microscopy (2.5X) was used to analyze photomicrographs of lesions next to restorations (Fig. 4).

Outer lesions and inhibition areas were seen next to restorations. No wall lesions were observed. One-way ANOVA followed by a post hoc test was done and showed that the extension and depth of the lesion in group B were statistically significantly higher than groups A and C. For group C, they were statistically significantly higher than group A. The inhibition area in group A was statistically significantly higher than groups B and C. However, in group C, it was statistically significantly higher than group B (Table 3).

Table 3

Comparison between three groups as regards lesion extension, depth, and inhibition area (mm).
Group	A (Self-adhesive hybrid composite)			B (Re-Gen flowable composite )			C (Alkasite restorative material)			P-value
	Min	-	Max	Min	-	Max	Min	-	Max
	Mean	±	S. D	Mean	±	S. D	Mean	±	S. D
Extension	1.367		1.655	1.636		1.840	1.417		1.699	P1 = < 0.001* P2 = < 0.001* P3 = < 0.001*
Extension	1.458	±	0.065	1.737	±	0.054	1.541	±	0.080	P1 = < 0.001* P2 = < 0.001* P3 = < 0.001*
Depth	0.157		0.311	0.489		0.616	0.376		0.464	P1 = < 0.001* P2 = < 0.001* P3 = < 0.001*
Depth	0.257	±	0.041	0.555	±	0.031	0.407	±	0.023	P1 = < 0.001* P2 = < 0.001* P3 = < 0.001*
Inhibition area	0.029		0.043	0.009		0.096	0.018		0.033	P1 = < 0.001* P2 = 0.002* P3 = 0.032*
Inhibition area	0.036	±	0.004	0.018	±	0.018	0.025	±	0.004	P1 = < 0.001* P2 = 0.002* P3 = 0.032*
P1: Group A vs Group B, P2: Group A vs Group C, P3: Group B vs Group C

On the first day, there was no significant correlation between the fluoride released and lesions extension, depth, and inhibition area. However, on the seventh and fourteenth days, there was a significant negative correlation between fluoride test and lesions extension (r=-0.417, p = 0.001; r=-0.381, p = 0.002), and depth (r=-0.308, p = 0.012; r=-0.286, p = 0.020). A positive correlation was obtained between fluoride test and lesions inhibition area (r = 0.352, p = 0.004; r = 0.252, p = 0.041) as shown in Table 4.

Table 4

Correlation between lesion Extension, Depth, and Inhibition area of the three groups with fluoride test results at 1st ,7th, 14th day.
Fluoride test	Extension		Depth		Inhibition area
Fluoride test	r	P	r	P	r	P
Day 1	-0.369	0.232	-0.240	0.052	0.184	0.138
Day7	-0.417	0.001*	-0.308	0.012*	0.352	0.004*
Day 14	-0.381	0.002*	-0.286	0.020*	0.252	0.041*

Ion-releasing dental materials combat bacteria and aid tooth remineralization, unlike bioinert resins composites.⁽¹⁹⁾ Composite restoration failures occur due to biofilm buildup on resin surfaces and weak tooth-restorative interfaces, increasing secondary caries risk.^{(36, 37)} Therefore, dental materials' bioactivity inhibits cariogenic bacteria and supports tooth bonding for biomineralization.⁽³⁷⁾

The study utilized a novel self-adhesive hybrid composite, an advancement from RMGIC, with high molecular weight polyacrylic acid, polymerizable groups, polyalkenoate acid copolymer, silanized non-reactive fillers, and FAS fillers. This material supports water and ion exchange in the oral cavity, releasing fluoride, aluminum, calcium ions, and potentially others.⁽³⁸⁾

Alkasite restorative materials are analogous to GIC and RMGIC, where it has high fluoride liberation, as the company claims. Also, it acts as an esthetic filling material. Alkasite restorative material devours an alkaline filler, which may deliver acid neutralizing particles.⁽³⁹⁾

The ion-releasing flowable composite contains Bio glass 45S5, releasing calcium, phosphate, and fluoride for tooth remineralization.⁽⁴⁰⁾ Low-viscosity flowable composites are ideal for small cavities, with easy syringe application that may reduce polymerization shrinkage through better stress relaxation.^{(41, 42)}

To assess fluoride release, deionized water was chosen over saliva or pH-cycling due to its simplicity and ability to provide accurate fluoride release measurements without the potential interference of organic molecules or nutrients that might be present in saliva or pH-cycling solutions.^{(42, 43)} SPADNS colorimetric method was used in this investigation to measure fluoride release, as it is a simple and rapid technique with high accuracy.⁽⁴⁴⁾

The significant initial release of fluoride from the Self-adhesive hybrid composite group on the first day might be attributed to an early discharge of fluoride from the glass components. This abrupt release of fluoride from the external surface into the surrounding liquid, coupled with the interaction between polyalkenoate acid and the glass components containing fluoride during the setting process, may elucidate the rapid burst in fluoride release.⁽⁴⁵⁾ The decline in fluoride release over the following days could result from the initial burst of fluoride being released from the glass particles as they dissolve within the polyalkenoate acid during the setting reaction.⁽⁴⁶⁾ Furthermore, the reduction in fluoride release can be attributed to the diminished dissolution of glass particles within the material's pores.⁽⁴⁷⁾

The alkasite restorative material contains alkaline fillers that generate particles capable of neutralizing acids. When in a mixed state, the material consists of 23.5% alkaline glass by weight, potentially leading to an initial rise in fluoride release.⁽⁴⁸⁾ Based on the results of the present study, it was observed that the alkasite restorative material exhibited lower fluoride release compared to the self-adhesive composite at all tested time intervals. This consistent fluoride release pattern in alkasite, without a burst effect, can be attributed to a higher powder-to-liquid ratio and a significant presence of alkaline glass in its final form.

This is consistent with a study that found that GICs emitted more fluoride than alkasite restorative material throughout all measurement intervals.⁽⁴⁹⁾ Another study evaluated the cumulative fluoride release in RMGI with varying concentrations and reported that the largest cumulative fluoride release was in Self-adhesive hybrid composite, followed by alkasite restorative material.⁽⁵⁰⁾

H Abouelleil etal,⁽⁴⁹⁾ found that the fluoride released amount was significantly higher for Self-adhesive hybrid composite on 14 days than alkasite restorative material which is in agreement with the current study. In contrast, another study has concluded that alkasite restorative material (self-cure) has the highest fluoride ion release and alkalizing potential in acidic pH as compared to GIC.⁽⁵¹⁾

Ion releasing flowable composite liner might have had lowest fluoride release because it lacks initial fluoride burst. Furthermore, unlike alkasite restorative material, it lacks alkaline fillers which might contribute to increased fluoride release. No previous studies were found for the Ion releasing flowable composite liner until the end of this study.

The study used closed batch culture for antibacterial assessment due to its ease, affordability, and minimal chemical requirements. Streptococcus Mutans, which is commonly used in caries models, was employed.⁽⁵²⁾ Previous studies have demonstrated that the use of a Streptococcus Mutans-based caries model for evaluating the demineralization-inhibiting effects of biomaterials was beneficial.^{(53, 54)} Selecting the best species and their relative abundances in multi-species models is a difficult task. Additionally, the use of a single-species model reduces the possibility of misleading aspects.^{(38, 55)} The bacterial approach to in vitro artificial caries production has been employed in the current investigation because it allows the demineralization of dental tissue and enables the examination of various restorative material anti-cariogenic qualities or microleakage surrounding restorations. The bacterial approach is thought to replicate in vivo circumstances closely.⁽⁵⁵⁾ Furthermore, biological tests appear to be the most relevant tests of the in vitro experiments because clinical caries are most likely dependent on biofilm formation.⁽⁵⁶⁾ Teeth underwent two thousand thermocycles to replicate thermal changes that might occur intraorally.⁽⁵⁷⁾

The statistical analysis model employed in this study accounted for four distinct crown sites: occlusal, cervical, buccal, and lingual regions of the restorations. This approach was adopted due to the influence of various anatomical landmarks on the crown, which can impact the quality of the margin.⁽⁵⁸⁾ Polarized light microscope is employed to evaluate the inhibition area, lesion depth, and extension of enamel because it allows for better visualization of the histological features of enamel due to its inherent birefringence property. This characteristic, which is not effectively observed using a transmitted light microscope.⁽⁵⁹⁾

Gram stain confirmed Streptococcus Mutans as the sole microorganism affecting the tested teeth. This microbiological technique distinguishes Gram-negative from Gram-positive bacteria based on dye retention due to cell wall properties. Streptococcus Mutans, a Gram-positive species, retained violet stain.⁽²⁴⁾

The Self-adhesive hybrid composite showed the smallest outer lesion depth, and extension could be because the fluoride released affected outer (primary) surface lesions that occurred on enamel surfaces next to the restorations. When the fluoride is released from the adjacent restoration, the amount of minerals lost from the outer lesion is decreased. Lesion depth and mineral loss have a linear relationship with the total amount of fluoride produced over time.⁽⁶⁰⁾ Fluoride is known to decrease the production of bacterial acids and glucans by Streptococcus mutans, which is widely acknowledged as the primary causative factor behind the development of carious lesions.⁽⁶¹⁾

Alkasite restorative material presented higher resistance to secondary caries than Ion releasing flowable composite liner, as it released more fluoride and might have released more fluoride in the presence of bacterial acids and decreased pH. Alkasite restorative material might have released more fluoride in the existence of low pH due to dissolution of its surface-unaffected layer, which exposed more matrix for fluoride release.⁽⁵¹⁾

Ion releasing flowable composite liner had the least or no inhibition areas with largest lesion depth and extension, maybe due to the low amount of fluoride released at all time intervals. These results are in acceptance with a study has shown that inhibitory effects of fluoride on the growth of Streptococcus Mutans biofilms either in the early or mature stage were related to the concentration of the fluoride.⁽⁶²⁾ Another study have shown different results.⁽⁶³⁾ It demonstrated that demineralization inhibitory effect of alkasite restorative material is more than that of Self-adhesive hybrid composite, both in depth and in the degree of demineralization. These different results may be due to different techniques used in that investigation and different periods of time in which the study was conducted.

Chau etal.⁽⁶⁴⁾ clearly demonstrate a negative correlation between the rate of fluoride release and various factors associated with the acid-producing capabilities, dry weight, bacterial bio-volume, and the amount of extracellular polysaccharides within Streptococcus mutans biofilms. These results strongly suggest that the release of fluoride from ion-releasing materials could be a pivotal factor in inhibiting the metabolic activity of cariogenic bacteria within biofilms. This suggests that the development of restorative materials capable of releasing higher concentrations of fluoride ions may lead to even more potent anti-cariogenic biofilm activity. Furthermore, such materials could potentially provide more efficient inhibition of subsequent secondary caries formation.

In a study Comparing fluoride-releasing sealants to non-fluoride-containing sealants, a notable decrease in the occurrence of wall lesions was observed. The mean outer lesion depths in enamel adjacent to fluoride-releasing sealants were considerably lower compared to non-fluoride-containing sealants.⁽⁶⁵⁾

The evaluation of restorative materials in the laboratory setting (in vitro study) has its limitations, as it does not fully replicate the dynamic conditions present in the oral cavity. Factors such as salivary flow characteristics, the presence of plaque, oral hygiene practices, and dietary habits specific to individual patients can all influence outcomes and may yield results different from those observed in the current study. Therefore, it is crucial to emphasize the need for further research involving clinical trials to better understand how these materials perform intraorally.

Within the limitations of this in vitro study, it could be concluded that:

The three ion releasing restorative materials used in the current study exhibited fluoride release and prevention of secondary caries formation.
The Self-adhesive hybrid composite was the material with the highest fluoride-releasing potential at each time interval. Also, it was the material with the highest ability to inhibit secondary caries induced by cariogenic bacteria.
The Ion releasing flowable composite liner showed the least fluoride release with no or minimal secondary caries inhibition.
All materials have shown decrease in fluoride release by time.
Increasing amount of fluoride release has led to increase the size of inhibition areas, while decrease the outer lesion depth and extension.

Ethical approval and consent to participate

All procedures performed in this study were carried out in accordance with relevant ethical guidelines and regulations of Helsinki Declaration. All experimental protocols were approved by the Ethics Committee, Faculty of dentistry, Mansoura University with reference number “M16020822”. Written consent was obtained from all participants.

Consent for Publication

Not applicable

Competing Interest

The authors declare that they have no competing interest.

Funding

None

Acknowledgement

The authors would like to express their thanks to the Microbiology department, Faculty of Pharmacy, Mansoura University for their valuable assistance in solutions preparation and conducting the cariogenic challenge test. Also, the authors would like to thank the water company in Mansoura, Egypt for their valuable help in fluoride measurements.

Author Contribution

N.Z planned and supervised the study. E.H supervised the microbiological part of the study. M.E helped with the statistical planning and analysis. E.E took the lead in doing all the study procedures and writing the manuscript. All authors discussed the results, provided critical feedback, and helped shape the research, analysis and manuscript and have all contributed to the final version of the manuscript. All authors have read and approved the final manuscript.

Availability of Data and Materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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Fluoride Release and Inhibition of Caries Around Restoration by Ion Releasing Restorative Materials: An In Vitro Study

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Abstract

Figures

Introduction

Materials and Methods

Sample size

Evaluation of Inhibition of Secondary Caries Caused by Cariogenic Bacteria

Teeth Selection

Teeth Preparation

Restorative Techniques

Bacterial system

Measuring Culture Density

Contamination Test (Gram Stain)

Results

Fluoride Release Test

Evaluation of Inhibition of Secondary Caries Caused by Cariogenic Bacteria

Discussion

Conclusion

Declarations

References

Additional Declarations

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