The effect of Chlorohexidine, Er:YAG Laser and Diode Laser 980 nm as Dental Cavity Disinfectants on Dentine Morphology and Microleakage of Composite Restoration: An In Vitro Study

doi:10.21203/rs.3.rs-7345490/v1

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The effect of Chlorohexidine, Er:YAG Laser and Diode Laser 980 nm as Dental Cavity Disinfectants on Dentine Morphology and Microleakage of Composite Restoration: An In Vitro Study

https://doi.org/10.21203/rs.3.rs-7345490/v1

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Introduction

Residual microorganisms may remain even after thorough mechanical cavity preparation, leading to secondary caries. Additionally, the smear layer generated during this process can impair the adhesion between composite resin and dentine and limit the penetration of disinfectants into dentinal tubules. This study aimed to compare the effects of 2% chlorhexidine (CHX), 980 nm diode laser, and Er:YAG laser as cavity disinfectants on dentine morphology, mineral content, and microleakage of composite restorations.

Materials and Methods

Forty extracted sound human primary molars were randomly assigned to four groups (n = 10): Group I (negative control, no disinfection), Group II (2% CHX application), Group III (980 nm diode laser, 1 W, continuous wave), and Group IV (Er:YAG laser, 1.2 W). Dentine morphology and restoration microleakage were examined via Scanning Electron Microscopy: (SEM), while mineral content was evaluated using Energy-Dispersive X-ray Spectroscopy (EDX).

Results

SEM analysis showed that CHX (Group II) left smear layer residues with narrowed tubules, while the diode laser (Group III) partially removed the smear layer. Er:YAG laser (Group IV) resulted in complete smear layer removal, wider tubules. EDX revealed significantly higher mineral content in Group IV compared to Groups II and group III (p < 0.001), with no significant difference between Groups II and III. Microleakage was highest in the control group and lowest in the Er:YAG group (p < 0.001).

Conclusion

The Er:YAG laser showed superior outcomes in improving dentine morphology, increasing mineral content, and minimizing microleakage, making it the most effective disinfectant tested.

Health sciences/Health care/Dentistry

Health sciences/Health care/Dentistry/Paediatric dentistry

Erbium:YAG laser

diode laser

chlorhexidine

dentin morphology

mineral content

microleakage

cavity disinfection

composite restoration

Dental caries is a highly prevalent condition globally that represents a multifactorial disease arising from the complex interplay between cariogenic bacteria, dietary sugars, and host-related factors. These microorganisms metabolize dietary sugars, generating acids that gradually demineralize the tooth's hard tissues, ultimately resulting in tooth decay [1]

Cervical caries is a common form of tooth decay, often resulting from poor oral hygiene, and a highly cariogenic diet. Residual microorganisms can persist even after thorough mechanical preparation, particularly in cases of incomplete caries removal. [2]

These bacteria may penetrate the restoration-tooth interface, promoting microleakage and increasing the risk of secondary or recurrent caries⁽¹¹⁾. To minimize this risk, disinfecting the dentin surface is recommended following cavity preparation and before placing any restorative material [3].

Additionally, a smear layer formed during cavity preparation can be a barrier preventing adequate bonding between composite resin restoration and dentine and disinfectants from effectively penetrating the dentinal tubules [2].

Dentin, a mineralized tissue, is composed of both organic and inorganic components. Its inorganic portion mainly consists of hydroxyapatite crystals containing calcium (Ca) and phosphorus (P). The typical calcium-to-phosphorus (Ca/P) ratio in dentin hydroxyapatite is approximately 1.67, which reflects a stable and consistent mineral composition. However, this ratio can vary depending on crystal type, calcium availability, anatomical location, and measurement method [4]

Chemical agents used during dental procedures may alter the Ca/P ratio, leading to changes in the structural and chemical integrity of dentin, including its permeability and solubility which can consequently disrupt the balance between dentin’s organic and inorganic components and negatively impact its physical properties such as micro-hardness and roughness. [5]

Furthermore, these chemical agents can lead to a reduction in mineral content that may compromise the mechanical strength and have a direct impact on bonding between dentine and restorative material which can affect the long-term durability of the restoration [6]

So, Microleakage, defined as the undetectable seepage of fluids, bacteria, or ions between the tooth structure and restoration, is a leading cause of clinical failure [7]. It may lead to tooth discoloration, postoperative sensitivity, recurrent decay, and pulpal complications. Therefore, optimizing disinfection protocols and improving adhesive bonding are crucial for reducing bacterial contamination, enhancing marginal integrity, and ensuring the longevity of composite restorations [8]

Chlorhexidine (CHX) is widely regarded as the gold standard for cavity disinfection due to its strong antimicrobial properties and its ability to bind to bacterial amino acids. Studies have shown that concentrations of up to 10% are considered safe for use on living tissues [9]. However, Chlorhexidine (CHX) lacks the ability to dissolve tissue and effectively remove the smear layer. As a result, the residual smear layer can act as a barrier, reducing the contact time between the irrigant and the dentin [10]

In the recent years, lasers have gained popularity in dentistry due to their broad range of applications; one of these applications is dental cavity disinfection. In addition to effectively penetrating and eliminating bacteria, they can also seal dentinal tubules, thereby preventing potential pathways for bacterial reinfection. Lasers have the ability of removal of smear layer, consequently increasing the bonding strength between restorative material and dentine. [11]

Among the lasers that are widely used as cavity disinfectants; Diode 980nm and Erbium YAG 2840nm. "Diode lasers offer the advantage of effectively removing the smear layer and inducing melting of the dentin surface with partial to complete obliteration of dentinal tubules [2]

Low-energy Er:YAG laser treatment modifies the dentin surface by efficiently eliminating debris and revealing open dentinal tubules. Research indicates that the heat produced by the Er:YAG laser can neutralize free radicals and alter the dentin, resulting in a surface more favorable for bonding [12]

According to our knowledge, there is no study compared the effect of chlorohexidine, diode laser 980 nm and Er:YAG laser when used for dentine disinfection, on dentine morphology and mineral content. Therefore, this study was conducted to assess the morphological changes and mineral content of dentine using a scanning electron microscope and EDX and to evaluate microleakage of composite resin restoration using SEM.

2.1. Study Design

This in vitro experimental study adhered to the CRIS Guidelines (Checklist for Reporting In-vitro Studies) published in 2014 to ensure methodological transparency and research quality [13]. A total of 40 freshly extracted human primary molars were used. To minimize structural variability, extractions were performed at or near the natural exfoliation period.

2.2. Ethical Considerations

The authors bear full accountability for all aspects of the research, guaranteeing that any issues related to the accuracy or integrity of any component are rigorously investigated and appropriately resolved. All procedures were executed in full compliance with the ethical principles set forth in the Declaration of Helsinki. Informed consent was secured from all participants prior to their involvement in the study. The experimental protocol was reviewed and approved by the Medical Research Ethics Committee of the National Research Centre (approval no1234052022.). 0n 7/4/2022

2.3. Sample Size Calculation

Sample size was determined based on a previous study by Jamel and Taher (2024), which evaluated the antibacterial efficacy of a 940 nm diode laser on Streptococcus mutans and other cariogenic bacteria. Their study reported mean ± SD of CFU as 34 ± 5.8 in Group I and 25 ± 3.6 in Group II, yielding an effect size of 1.86. Using a power of 0.9 and a significance level (α) of 0.05, the minimum required sample size was calculated to be 8 per group. To account for a potential 20% dropout, this was increased to 10 per group. The calculation was conducted using a t-test in G*Power software (version 3.1.4.9) [14]

2.4. Randomization and Allocation

All 40 extracted teeth were labeled numerically (1–40) and stored in a sterile, sealed, opaque container. Random allocation into four equal groups (n = 10) was carried out using a computer-generated random sequence from www.random.org on November 12, 2024, applying a 1:1 allocation ratio.

2.5. Blinding

A double-blind design was adopted. Both the SEM and EDX examiners and the statistician performing the data evaluation were blinded to the group assignments.

2.6. Specimen Selection and Preparation

2.6.1. Inclusion and Exclusion Criteria

Inclusion criteria: Sound freshly extracted primary molars with at least 50% of root structure intact.
Exclusion criteria: Teeth with cracks, restorations, deep caries, pulpal involvement, structural anomalies, or prior dental treatment were excluded.

2.6.2. Collection and Storage

Teeth were collected from the outpatient pediatric dentistry clinic of the NRC. An informed parental consent was obtained before extraction. Parents were briefed about the study involving their child’s extracted teeth. Following extraction, the teeth were rinsed with tap water, residual soft tissues were carefully removed using a scalpel, and the crowns were gently cleaned with a soft-bristled brush (Sulcus, Oral-B, Mexico). Prior to analysis, the specimens were stored in deionized water at 4°C [15]. The structural integrity of all samples was first assessed through visual inspection, followed by examination under a stereoscope (Olympus Optical Co., Ltd., Japan).

2.6.3. Cavity Preparation

Standardized Class V cavities were created on the buccal surfaces of each tooth using a high-speed handpiece equipped with a diamond bur (Horico Diament, Germany) under continuous water cooling. Dimensions were fixed at 3 mm (mesiodistal) × 2 mm (occlusogingival) × 1.5 mm (depth), with the occlusal margin 1 mm above the cementoenamel junction. Measurements were verified using a digital caliper. A new bur was used after every five cavities [16] eeth were sterilized by autoclaving at 121°C for 15 minutes and stored individually in sterile, sealed test tubes [17]

2.7. Experimental Grouping

Teeth were randomly assigned to four groups:

Group 1 (Negative Control): No disinfection applied.
Group 2 (Positive Control): In Group 2, the chlorhexidine group (positive control), disinfection was performed using a 2% chlorhexidine gluconate solution (Grace for dental industries, Egypt) applied at a flow rate of 10 mL/min for 60 seconds. Following application, the cavity was rinsed with sterile saline and air-dried to remove any residual solution. [18]
Group 3 (Diode Laser): the diode laser group, a 980 nm diode laser (LASOTRONIX, Poland) was employed at a power of of 1 Watt in continuous mode. The cavity surface was irradiated for a total of 60 seconds, divided into two 30-second cycles. A 8mm tip was used, positioned 1 mm from the cavity in a perpendicular orientation, and moved in a scanning motion to ensure even coverage., spot size 8mm [9]
Group 4 (Er:YAG Laser): the Er:YAG laser group, was treated using a 2940 nm Er:YAG laser (Fotona: AT Fidelies Ljubljana, Slovenia) with settings including a pulse energy of 120 mJ, power output of 1.2 Watt, frequency of 10 Hz, water and air levels set to 4, and SP (short pulse) mode with a pulse duration of 300 microseconds. The irradiation was delivered using an R02 tipless handpiece spot size 0.9mm [12]. The power of Er: YAG laser was selected according to a pilot study conducted before the beginning of the main study. The range between 50 to 150 mj is considered safe regarding dentine morphology and mineralization .the pilot study was done using 50, 100, 120, 150 mj. Both 120 and 150 mj energy showed the highest antibacterial efficiency against streptococcus mutans bacteria with no statistically significant difference between them; so the energy of Er:YAG laser chosen was 120 mj.

2.8. Assessment of the effects of different treatment modalities:

2.8.1. Assessment of morphological changes of dentine via Scanning Electron Microscopy (SEM) Analysis

Imaging was performed at base line to image the normal dentin then after application of each treatment modality to examine the morphological changes of dentine in each group.

Samples were examined using a Quanta 250 FEG SEM equipped with an EDX unit. Imaging was performed at 30 kV with magnifications of ×2000 and ×6000. The resolution of the electron gun reached 1 nm.

2.8.2 Assessment of mineral content changes of dentine via Energy Dispersive X-ray (EDX):

Samples were examined using EDX device attached to Quanta 250 FEG SEM for analyzing the elemental and quantitative composition of dentin.

3. Restoration Procedures:

Following disinfection and examination using Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDX), the cavities were etched with Prime Dent Etch (USA) for 20 seconds, rinsed with water for 10 seconds, and gently air-dried using an air syringe.

Subsequently, a universal bonding agent (Prime and Bond Universal, Dentsply Sirona, Germany) was applied using a microbrush for 20 seconds, air-thinned for 5 seconds, and then light-cured for 10 seconds.

The restoration procedure was carried out using Spectra ST HV Composite (Dentsply Sirona, Germany), applied in increments of 2 mm thickness. Each increment was individually light-cured for 20 seconds [19].

4. Thermocycling Process

The samples were immersed in distilled water at 37°C for 24 hours, followed by thermocycling for 78 seconds per cycle (30 seconds at 55°C, 10 seconds at ambient pause, 30 seconds at 5°C, and 8 seconds for the transition back to the initial condition). A total of 500 thermal cycles were performed using a TC-300 thermocycler (Vafai Factory) [20].

5. Microleakage Testing

5.1. Sample Preparation for Dye Penetration

Following thermocycling, all specimens were thoroughly dried, and their apical regions were sealed with sticky wax to prevent dye penetration. Two consecutive layers of nail varnish were then applied, leaving an uncoated gingival margin of approximately 1 mm and maintaining a 2 mm clearance from the tooth–restoration interface, thereby ensuring dye infiltration occurred exclusively at the interface region \[9]. The specimens were subsequently immersed in 2% methylene blue solution (Sparks, USA) for 24 hours at room temperature. After immersion, the teeth were rinsed, dried, and sectioned longitudinally along the buccolingual axis using a microtome (MTI Corporation, Richmond, CA)[21].

5.2. Microleakage Assessment via Scanning Electron Microscopy (SEM) Analysis

Assessment of linear dye penetration at tooth- restoration interface both occlusal and gingival was done using the Quanta 250 FEG SEM to study the degree of microleakage in micrometers.

Statistical analysis:

Mean and standard deviation values were calculated for each group under all test conditions. The data distribution was assessed for normality using the Kolmogorov-Smirnov and Shapiro-Wilk tests, confirming a parametric (normal) distribution. For comparisons involving more than two independent groups, one-way analysis of variance (ANOVA) was applied, followed by Tukey’s post hoc test for pairwise comparisons. The paired sample test was employed to analyze differences between two related groups. A significant level of p ≤ 0.05 was adopted for all statistical tests. Data analysis was conducted using IBM® SPSS® Statistics software, version 20 (IBM Corp., Armonk, NY, USA).

1) Morphological changes of dentine via SEM Analysis:

SEM examination of dentine surface after cavity preparation and before application of different disinfection modalities (baseline) of all groups revealed relatively partial occlusion of dentinal tubules with presence of a smear layer figures (1,3and 5)

SEM examination of dentine surface after application of CHO 2% showed an apparent narrowing of dentinal tubules with precipitation of some particle with lack of removal of the smear layer figure (2).

SEM image of dentine after diode laser application showed an apparent widening of dentinal tubules with partial removal of the smear layer figure (4).

SEM image of dentine after Er:YAG laser application showed an obvious widening of dentinal tubules with removal of the smear layer and melting of dentine figure (6).

A) Chlorohexidine group:

B) Diode laser 980 nm group:

C)Er:YAG laser group:

2) Mineral content changes of dentine via EDX:

I) Ca content changes:

i. Relation between Pre and Post treatment values among all groups:

As shown in table (1) and figure (7), A statistically significant difference was observed between the Pre and Post groups, with *p* < 0.001. The highest mean value was recorded in the Pre group, whereas the lowest mean value was observed in the Post group.

Relation between different groups according to Ca content:

As presented in table (1) and figure (7), No statistically significant difference was observed between (CHX), (Diode laser) and (Er-YAG laser) where (p=0.372) in the pretreatment values of Ca content which means that the Ca content was standardized.

There was a statistically significant difference between (CHX), (Diode laser) and (Er-YAG laser) post-treatment values where (p<0.001).

A statistically significant difference was observed between the Er:YAG laser group and both the CHX and Diode laser groups (*p* < 0.001 for each comparison). No statistically significant difference was detected between the CHX and Diode laser groups (*p* = 0.941). The highest mean value was recorded in the Er:YAG laser group, whereas the lowest mean value was observed in the CHX group.

Table (1): The mean, standard deviation (SD) values of Ca of different groups.

Variables	Ca
	Pre		Post		p-value
	Mean	SD	Mean	SD	p-value
CHX	58.89	1.18	42.90	0.82	<0.001*
Diode laser	57.58	1.12	43.17	0.49	<0.001*
Er-YAG laser	59.52	0.42	52.95	0.33	<0.001*
*p-value*	0.372ns		<0.001*

*; significant (p<0.05) ns; non-significant (p>0.05)

Relation between different groups according to Ca percentage of change:

As shown in table (2) and figure (8), A statistically significant difference was observed among the CHX, Diode laser, and Er:YAG laser groups (*p* < 0.001). Pairwise comparisons revealed significant differences between the Er:YAG laser group and both the CHX and Diode laser groups (*p* < 0.001 for each). No statistically significant difference was detected between the CHX and Diode laser groups (*p* = 0.073). The highest mean value was recorded in the CHX group, whereas the lowest mean value was observed in the Er:YAG laser group.

Table (2): The mean, standard deviation (SD) values of percentage of change of Ca of different groups.

Variables	Ca Percentage of change
Variables	Mean	SD
CHX	27.12%	0.69
Diode Laser	24.92%	0.82
Er-YAG laser	11.02%	0.45
*p-value*	<0.001*

*; significant (p<0.05)

II) P content changes:

i. Relation between Pre and Post treatment values among all groups:

As shown in table (3) and figure (9), A statistically significant difference was observed between the Pre and Post groups, with *p* < 0.001. The highest mean value was recorded in the Pre group, whereas the lowest mean value was observed in the Post group

Relation between different groups according to P content

As presented in table (3) and figure (9), no statistically significant difference was observed between (CHX), (Diode laser) and (Er-YAG laser) where (p=0.719) in the pretreatment values of P content which means that the P content was standardized.

There was a statistically significant difference between (CHX), (Diode laser) and (Er-YAG laser) where (p<0.001).

A statistically significant difference was observed between Er-YAG laser group and both CHX and Diode laser groups (*p* <0.001) for each comparison.No statistically significant difference was detected between the CHX and Diode laser groups where (*p* =0.950).The highest mean value was recorded in Er-YAG laser group, whereas the least mean value was observed in CHX group

Table (3): The mean, standard deviation (SD) values of P of different groups.

Variables	P
	Pre		Post		p-value
	Mean	SD	Mean	SD	p-value
CHX	20.80	0.12	17.45	0.13	<0.001*
Diode laser	20.49	0.39	17.58	0.44	<0.001*
Er-YAG laser	20.71	0.23	19.93	0.22	<0.001*
*p-value*	0.719ns		<0.001*

*; significant (p<0.05) ns; non-significant (p>0.05)

Relation between different groups according to P percentage of change:

As shown in table (4) and figure (10), A statistically significant difference was observed among the CHX, Diode laser, and Er:YAG laser groups (*p* < 0.001). Pairwise comparisons revealed significant differences between the Er:YAG laser group and both the CHX and Diode laser groups (*p* < 0.001 for each). No statistically significant difference was detected between the CHX and Diode laser groups (*p* = 0.050). The highest mean value was recorded in the CHX group, whereas the lowest mean value was observed in the Er:YAG laser group.

Table (4): The mean, standard deviation (SD) values of percentage of change of P of different groups

Variables	P Percentage of change
Variables	Mean	SD
CHX	16.09%	0.45
Diode Laser	14.29%	0.64
Er-YAG laser	3.76%	0.32
*p-value*	<0.001*

*; significant (p<0.05)

II) Ca/P ratio changes:

i. Relation between Pre and Post treatment values among all groups:

As shown in table (5) and figure (11), A statistically significant difference was observed between the Pre and Post groups, with *p* < 0.001. The highest mean value was recorded in the Pre group, whereas the lowest mean value was observed in the Post group

Relation between different groups according to Ca/P ratio changes:

As presented in table (5) and figure (11), there was no statistically significant difference observed between CHX group, Diode laser group and Er-YAG laser group where (*p* =0.785) in the pretreatment values of Ca/P ratio which means that the Ca/P ratio was standardized.

Statistically significant difference was observed between the CHX, Diode laser and Er-YAG laser groups (*p* =0.030).

A statistically significant difference was detected between Er-YAG laser group and each of CHX and Diode laser groups (*p* =0.040) and (*p* =0.043).

No statistically significant difference was detected between CHX l group and Diode laser group (*p* =0.996).The highest mean value was recorded in Er-YAG lase group, while the lowest mean value was observed in CHX group.

Table (5): The mean, standard deviation (SD) values of Ca/P ratio of different groups.

Variables	Ca/P ratio
	Pre		Post		p-value
	Mean	SD	Mean	SD	p-value
CHX	2.83	0.06	2.46	0.06	<0.001*
Diode laser	2.82	0.08	2.47	0.07	<0.001*
Er-YAG laser	2.88	0.03	2.66	0.03	<0.001*
*p-value*	0.785ns		0.030*

*; significant (p<0.05) ns; non-significant (p>0.05)

Relation between different groups according to Ca/ P ratio percentage of change:

As shown in table (6) and figure (12), A statistically significant difference was observed among the CHX, Diode laser, and Er:YAG laser groups (*p* < 0.001). Pairwise comparisons revealed significant differences between the Er:YAG laser group and both the CHX and Diode laser groups (*p* < 0.001 for each). No statistically significant difference was detected between the CHX and Diode laser groups (*p* = 0.712). The highest mean value was recorded in the CHX group, whereas the lowest mean value was observed in the Er:YAG laser group.

Table (6): The mean, standard deviation (SD) values of percentage of change of Ca/P of different groups.

Variables	Ca/P Percentage of change
Variables	Mean	SD
CHX	13.14%	0.63
Diode Laser	12.38%	0.94
Er-YAG laser	7.55%	0.34
*p-value*	<0.001*

*; significant (p<0.05)

3) Microleakage Assessment via SEM:

Microleakage results were summarized in table (7) and Figure (13).

Figures (14, 15, 16 and 17) Illustrate SEM images of dye penetration along the tooth-restoration interface.

At occlusal margin of the cavity:

A Statistically significant difference was observed among the Control, CHX, Diode laser and Er-YAG laser groups (*p* <0.001). Comparisons revealed significant differences between the Control group and both of Er-YAG laser and Diode laser groups (*p* <0.001), meanwhile no statistically significant difference was observed between the Control and CHX groups (*p* =0.999).

A statistically significant difference was observed between the CHX group and each of Er-YAG laser and Diode laser groups (*p* <0.001).

Also, a statistically significant difference was observed between the Er-YAG laser and Diode laser groups (*p* =0.006).

The highest mean value was recorded in the Control, group whereas the lowest mean value was observed in the Er-YAG laser group.

2-At gingival margin of the cavity:

A Statistically significant difference was observed among the Control, CHX, Diode laser and Er-YAG laser groups (*p* <0.001). Comparisons revealed significant differences between the Control group and each of CHX, Er-YAG laser and Diode laser groups (*p* <0.001),

A statistically significant difference was observed detected CHX group and each of Er-YAG laser and Diode laser groups (*p*<0.001) and (*p*=0.008).

Also, a statistically significant difference was observed between Er-YAG laser and Diode laser groups (*p*<0.001).

The highest mean value was recorded in the Control group whereas the lowest mean value was observed in the Er-YAG laser group.

3-Average of microleakage both occlusally and gingivally:

A Statistically significant difference was observed among the Control, CHX, Diode laser and Er-YAG laser groups (*p* <0.001).

Comparisons revealed significant differences between the Control group and each of CHX, Er-YAG laser and Diode laser groups (*p*<0.001).

A statistically significant difference was detected between the CHX group and each of Er-YAG laser and Diode laser groups (*p* <0.001).

Also, a statistically significant difference was found between Er-YAG laser and Diode laser groups (*p*<0.001).

The highest mean value was recorded in the Control group whereas the lowest mean value was observed in the Er-YAG laser group.

Table (7): The mean, standard deviation (SD) values of microleakage of different groups.

Variables	Microleakage
	Occlusal		Gingival		Average
	Mean	SD	Mean	SD	Mean	SD
Control	7.45	0.35	15.17	0.25	11.31	0.11
CHX	7.44	1.14	6.60	1.10	7.02	0.02
Diode laser	4.96	1.30	5.69	0.27	5.33	0.79
Er-YAG laser	3.52	0.51	1.34	0.24	2.44	0.22
*p-value*	<0.001*		<0.001*		<0.001*

*; significant (p<0.05)

Contemporary caries removal techniques do not reliably eradicate all microorganisms from the prepared cavity. Several studies have shown that bacteria may persist within the dentin even following the application of caries detector dyes. Furthermore, research indicates that fermentative microorganisms can survive beneath restorations lacking antiseptic properties for up to 139 days. Consequently, the incorporation of cavity disinfection as an adjunct to conventional caries removal procedures is recommended to eliminate residual microbial contamination. This approach may help reduce the risk of secondary caries, postoperative pulp sensitivity, and pulpal inflammation prior to definitive restoration [22].

The infiltration of Bacterial cells, nutrient compounds, or hydrogen ions derived from dental plaque into the interface between the tooth and restorative material can result in complications such as marginal discoloration, fractures, secondary caries, corrosion, sensitivity, and pulpal inflammation [23]

Numerous studies have advocated for various cavity disinfection methods, encompassing chemical agents, lasers, ozone therapy, and other modalities [9].

Chlorhexidine digluconate (CHX) is a potent antimicrobial agent recognized for its effective inhibition of streptococcal growth, rendering it a promising candidate for the prevention of dental caries. Upon application to dentin surfaces, CHX exerts a prolonged antibacterial effect by the sustained release of positively charged molecules, a phenomenon known as substantive antimicrobial activity (SAA) [23].

2% concentration of chlorhexidine digluconate (CHX) was employed in this study due to its widespread use in both clinical dentistry and research. This aqueous formulation is generally considered biocompatible and exhibits an acceptable toxicological profile, rendering it a reliable and effective choice for cavity disinfection [24].

Although CHX has a widely proven effect Haralur et al., found that it has a negative effect on dentine regarding Ca content and hardness [5] also it negatively impacts the bonding and sealing of adhesive restorations to dentin, potentially leading to increased microleakage [3].

Laser has been widely used as an efficient dental cavity disinfectant; it has a potent antibacterial effect with avoidance of the side effects of using chemicals and it has some positive effects on dentine such as removal of smear layer which consequently leads to improvement of bonding between dentine and restoration [4, 12, 25, and 26]

The interaction between the laser and tissue is greatly influenced by the laser's wavelength and the power density applied [4].

Diode lasers are available in four main wavelengths: 810–830 nm, 940 nm, 980 nm, and 1064 nm. Their antibacterial effectiveness is largely attributed to the thermal effects and the corresponding rise in temperature generated during irradiation. Diode laser 980 nm was chosen in our study because of its potent antibacterial efficacy beside its ability to remove the smear layer [25]

Low-energy Er:YAG laser treatment modifies the dentin surface by efficiently removing debris and exposing open dentinal tubules. Researches indicated that the heat generated by the Er:YAG laser can neutralize free radicals and alter the dentin surface, thereby enhancing its suitability for bonding [7].

Operating at a wavelength of 2.94 µm, the Er:YAG laser has demonstrated bactericidal effects against Streptococcus mutans. In this study, a low energy output of 1.2 watts was utilized to minimize thermal damage to dentin and avoid potential reductions in dentin hardness. This parameter selection is supported by Du et al., who found that power settings between 1 and 1.5 watts effectively reduce S. mutans levels without compromising dentin integrity [27].

Scanning electron microscopy (SEM) was employed in this study to evaluate the ultrastructural changes on the dentin surface following the application of the tested treatments, owing to its capability to provide both qualitative and quantitative assessment of morphological features [28].

Scanning electron microscopy analysis of dentin treated with 2% chlorhexidine demonstrated irregular dentinal tubules characterized by partial occlusion and the presence of residual smear layer. The findings corroborate those presented in prior research by Lapinska et al. [29], who observed multiple and singular deposits irregularly distributed across the dentine surface, located both within pits created by mechanical preparation and on the smooth areas of the sample. Similarly, Siwnata et al. [30] reported that 2% chlorhexidine gluconate was insufficient for complete removal of the smear layer during cavity cleansing, resulting in the persistence of smear plugs that impeded the full opening of dentinal tubules.

In the present study, dentin treated with diode laser at a power setting of 1 W exhibited an irregular surface with an apparent opening of dentinal tubules with partial removal of smear layer. Our findings are in line with prior research outcomes by Behniafar et al., who also observed that the application of a diode laser at 1 W power output and a wavelength of 980 nm did not result in a significant increase in pulpal temperature or cause structural damage such as fissures or cracks on the dentin surface with partial removal of smear layer [31].

These outcomes contradict earlier reported data of Jhingan et al. [25] and El Tayeba et al. [4] who stated that total smear layer and debris removal, accompanied by morphological changes such as dentin melting, these findings may be attributed to the higher power used in their study as both of them employed diode laser at 2 W and 1.5 respectively.

Another study conducted by Abdou et al. [28] achieved effective smear layer removal and complete opening of dentinal tubules using a diode laser at 1.5 W. although the previously used parameters produced an efficient smear layer removal but they may produce harmful effects on the pulp according to Jaine et al who found that both 1 W and 2W had similar antibacterial effects when used for dentine disinfection but found that 1 W was safer than 2 W for the pulp which produced higher temperature rise of the pulp [32]

In our study dentine surface treated with erbium yag laser showed an obvious widening of dentinal tubules with total removal of smear layer. These findings were in alignment with Vieira et al., and Wang et al., who reported that dentin surfaces treated with Erbium:YAG laser exhibited open dentinal tubules and were free of smear layer, resulting in a retentive surface pattern that can improve bonding to dentin [16, 26].

A study conducted by found Burlat et al. hat using Er:YAG laser with a power range between 250–300 mj led to surface cracks and disintegration of dentine but these findings may be the result of the high laser power used in his study, this was in agreement with another study conducted by Wanderley et al., [33, 34]

Calcium (Ca) and phosphorus (P), which are found in hydroxyapatite crystals, make up the primary inorganic components of dental hard tissues. Changes in the Ca/P ratio can disrupt the natural balance between the organic and inorganic components, possibly altering the structural properties of dentin, including its permeability and solubility. [35]

In our study, Edx was used to the change in mineral content of dentine after different disinfection modalities, it was chosen because of its accuracy and sensitivity in measuring mineral content [28].

EDX values of Ca and P of all experimental groups exhibited lower Ca/P weight ratios compared to their control (untreated) samples.

The observed decrease in calcium and phosphorus levels following chlorhexidine (CHX) application aligns with the findings reported by Haralur et al. and Kimyai et al. [5]. This decrease Can be ascribed to the cationic nature of CHX, which enables it to bind readily to anionic molecules, such as the phosphate groups present in hydroxyapatite resulting in the displacement and subsequent release of calcium ions (Ca) from dentin ;these findings suggest that CHX may contribute to calcium ion removal through its interaction with phosphate [3, 5, 36].

Regarding diode laser Statistical analysis indicated a significant difference between values recorded before and after treatment regarding calcium and phosphorous content, this was in accordance with Azmy et al who found that diode laser as a surface treatment resulted in observable changes in the mineral content of radicular dentin [37].

In contrast with our findings, Abdou et al, claimed that no chemical changes in dentin components following diode laser application. These results may be attributed to the use of distilled water as an irrigant immediately before the application of the 980 nm diode laser on the dentin surface which may lead to absorbance of heat produced by the photothermal effect of diode laser thereby preserving the inorganic content of the dentin without causing any alteration in the mineral content [28].

Er:YAG laser treatment resulted in the smallest percentage change in calcium and phosphorus content. These findings are consistent with those of Soares et al. [47], who reported that Er:YAG laser irradiation at an energy setting of 100 mJ caused only minimal alterations in the elemental composition of the treated surface, along with a slight reduction in the calcium-to-phosphorus (Ca/P) ratio.

On the contrary, Moosavi [39] has reported that Er:YAG laser irradiation led to an increase in microhardness and calcium ion content in dentine; however, these differences were not statistically significant when compared to their respective control groups.

Microleakage is defined as the infiltration of oral fluids, molecules, bacteria, and ions at the interface between the cavity walls and the restorative material. Preventing microleakage is crucial for ensuring the longevity and clinical success of dental restorations. [11].

As a matter of fact, an ideal cavity disinfectant should offer effective antimicrobial activity while maintaining the sealing integrity of restorative materials. Compromising this seal can lead to marginal leakage, which may reduce the longevity of the restoration by allowing bacteria and fluids to infiltrate the interface between the tooth and the restorative material [14].

Microleakage can be evaluated using various techniques, among these, dye penetration technique which was used in our study, is one of the most commonly used methods in recent research due to its practicality—dye solutions are readily available, the method does not involve reactive chemicals or radiation, and it is both highly feasible and easily reproducible. [31]

Thermal cycling is widely employed in in vitro studies. This technique is particularly valuable in microleakage research, as it effectively simulates the clinical aging process of dental restorations in the oral cavity. In the present study, all specimens in our study were subjected to 5,000 thermal cycles between 5°C and 55°C prior to microleakage assessment [15]

The group treated with chlorhexidine exhibited a lower level of microleakage at the tooth–restoration interface when compared to control group. These findings are consistent with the studies conducted by Ramezanian et al., [38] which reported that chlorhexidine significantly reduces microleakage both immediately following restoration and over time. Similarly, a review by Satpute highlighted chlorhexidine’s role in enhancing the longevity of restorative materials [39]

However, The study outcomes disagree with a study by Mutluay et al. who reported that chlorhexidine had no significant effect on microleakage compared to the control group. This discrepancy may be attributed to differences in experimental protocols, particularly the duration of chlorhexidine application and the type of restorative material used; their study employed a giomer-based restoration, whereas composite resin was used in the present study [2].

In our study, diode laser 980 nm group exhibited a lower microleakage value when compared with control and chlorohexidine groups. These findings were consistent with of El Mansy et al., who reported that the use of a 980 nm diode same power and time laser resulted in the lowest statistically significant microleakage levels when compared to CHX and control groups. In contrast, Ipek et al. found no significant improvement in microleakage following diode laser treatment, this discrepancy may be attributed to variations in laser parameters used across studies which are employing a different application time and utilizing a different restorative material [9].

In relation to the Er:YAG laser, our results indicated that it produced the lowest levels of microleakage among all groups tested

A study by Emilie Luong and Amir Shayegan demonstrated that Er:YAG laser conditioning of enamel and dentin surfaces beneath resin composite has a great potential to reduce microleakage [40]

A study by Sharafeddin and Tabrizi found that the Er:YAG laser had a favorable effect in reducing microleakage at both occlusal and gingival margins when compared to CO2 laser [7]. Similarly, a study conducted by Ipek Aslan et al. found that the Er,Cr:YSGG laser group demonstrated superior performance in reducing nanoleakage compared to the diode laser and chlorhexidine groups. [17].

. According to our knowledge, there is no previous study that compared Er:YAG laser with diode laser or chlorohexidine for cavity disinfection or compared between their effect on the morphology of dentine.

Within the limitations of this study, it can be concluded that the Erbium:YAG laser demonstrated the most favorable outcomes in terms of preserving and enhancing both the morphological and chemical composition of dentin. Moreover, it resulted in the lowest microleakage values among all tested groups, indicating superior sealing ability and potential for long-term restoration success.

Both the diode laser and 2% chlorhexidine (CHX) showed beneficial effects compared to the control, with no significant difference between them

CHX exhibited the highest microleakage values, suggesting a less favorable impact on the marginal integrity of restorations.

Competing interests:

The authors declare no conflicts of interest related to this study.

Funding:

This research was conducted without external funding and was entirely self-financed by the authors

Availability of data and materials:

The datasets generated and/or analyzed during the present study are available from the corresponding author upon reasonable request. All measures were taken to ensure the protection of participants’ privacy.

Matar LAR, Dowidar KML, Talaat DM, Kholeif DA, Abdelrahman HH. Effectiveness of chlorhexidine as a cavity disinfectant in atraumatic restorative treatment in primary teeth: A randomized controlled clinical trial. Alexandria Dent J. 2020;46(2C):178.
Mutluay AT, Mutluay M. Effects of different disinfection methods on microleakage of giomer restorations. Eur J Dent. 2019;13(4):569–573. doi:10.1055/s-0039-1698370
Kimyai S, Mohammadi N, Bahari M, Pesyanian E, Pesyanian F. Effect of cavity disinfection with chlorhexidine on marginal gap of Class V composite restorations bonded with a universal adhesive using self-etch and etch-and-rinse bonding strategy. Front Dent. 2020; 17:3. doi: 10.18502/fid.v17i1.3963. PMID: 33615301; PMCID: PMC7882204.\
El Tayeba EAA, Moharrum HS. Evaluation of various laser irradiations on the dentin tissue permeability. Int J Health Sci. 2022; 6(S5):992–1012. doi:10.53730/ijhs.v6nS5.9052
Haralur SB, Alqahtani MM, Alqahtani RA, Shabab RM, Hummadi KA. Effect of dentin-disinfection chemicals on shear bond strength and microhardness of resin-infiltrated human dentin in different adhesive protocols. Medicina (Kaunas). 2022;58(9):1244. doi: 10.3390/medicina58091244. PMID: 36143921; PMCID: PMC9501625.
Sacramento PA, de Castilho AR, Banzi EC, Puppi-Rontani RM. Influence of cavity disinfectant and adhesive systems on the bonding procedure in demineralized dentin: a one-year in vitro evaluation. J Adhes Dent. 2012;14(6):575–83. doi:10.3290/j.jad.a24533
Sharafeddin F, Fadaei Tabrizi A. Evaluation of the microleakage of class V composite restoration after cavity treatment with Erbium, CO2 lasers, Papain, and Bromelain enzymes. Clin Exp Dent Res. 2023 Nov 22. doi:10.1002/cre2.822
Bin-Shuwaish M, AlHussaini A, AlHudaithy L, AlDukhiel S, AlJamhan A, Alrahlah A. Effects of different antibacterial disinfectants on microleakage of bulk-fill composite bonded to different tooth structures. BMC Oral Health. 2021;21:348. doi: 10.1186/s12903-021-01717-7
Mansy MM, Sabry S, Tadros T, Saleh RS. Comparative evaluation on the effect of different cavity disinfectant nano gels; Chlorohexidine, Propolis, Liquorice versus Diode Laser in terms of composite microleakage (comparative in vitro study). BDJ Open. 2023;9(1). doi: 10.1038/s41405-023-00176-2.
Mahapatra KK, Varshney S, Nadish, Verma S, Gupta D, Mynam RS. Antibacterial efficacy of different irrigants used during endodontic surgery: a comparative study. J Neonatal Surg. 2025;14(26s):903. Available from: https://www.jneonatalsurg.com
. Sadony DM, Abozaid HE. Antibacterial effect of metallic nanoparticles on Streptococcus mutans bacterial strain with or without diode laser (970 nm). Bull J Natl Res. Cent. 2020;44:2
Wang JH, Yang K, Zhang BZ, Zhou ZF, Wang ZR, Ge X, Wang LL, Chen YJ, Wang XJ. Effects of Er:YAG laser pre-treatment on dentin structure and bonding strength of primary teeth: an in vitro study. BMC Oral Health. 2020;20:316. doi: 10.1186/s12903-020-01315-z.
Krithikadatta J, Gopikrishna V, Datta M. CRIS Guidelines (Checklist for Reporting In-vitro Studies): A concept note on the need for standardized guidelines for improving quality and transparency in reporting in-vitro studies in experimental dental research. J Conserv Dent. 2014;17(4):301–4.
Jameel NM, Taher HJ. Antibacterial efficacy of 940 nm diode laser against cariogenic bacteria. Baghdad Sci J. 2024;21(8):2722. doi:10.21123/bsj.2024.9041
Bettero FCBS, Lopes CCA, Guerra GJ, Novais VR. Impact of solutions and storage time on the chemical and mechanical properties of human dentin. J Clin Exp Dent. 2025;17(4):e374–e381. doi:10.4317/jced.62433
Ipek A, Ozgul B, Tamer T, Fatih E, Aykut C, Mehmet KF. The effects of cavity disinfection on the nanoleakage of compomer restorations: an in vitro study. J Eur Oral Res. 2020;54:16–24.
Sancakli HS, Siso SH, Yildiz SO, Gökçe YB. Antibacterial effect of surface pretreatment techniques against Streptococcus mutans. Niger J Clin Pract. 2018;21(2):183–8. doi:10.4103/njcp.njcp_98_16
Hubbezoğlu İ, Alici O. The efficacy of four cavity disinfectant solutions and two different types of laser on the micro-shear bond strength of dentin adhesives. Cumhuriyet Dent J. 2018;21(1):9–17. doi:10.7126/cumudj.389990
Neves, P., Pires, S., Marto, C. M., Amaro, I., Coelho, A., Sousa, J., Ferreira, M. M., Botelho, M. F., Carrilho, E., Abrantes, A. M., & Paula, A. B. (2022). Evaluation of microleakage of a new bioactive material for restoration of posterior teeth: An in vitro radioactive model. Applied Sciences, 12(22), Article 11827. https://doi.org/10.3390/app122211827
Nassaja AE, Ghadimi S, Seraj B, Chiniforush N. Effect of photodynamic therapy on microleakage of class V composite restorations in primary teeth. J Photodiagnosis Photodyn Ther. 2020;32:101964.
AlHadad M, AlGharrawi H, AlHashemi J. Microleakage evaluation of SonicFill, silorane-based and nanofilled methacrylate-based composites: a comparative study. J Oral Dent Res. 2017;4:120–9.
Arslan I, Baygin O, Tuzuner T, Erdemir F, Canakci A, Korkmaz FM. The effects of cavity disinfection on the nanoleakage of compomer restorations: an in vitro study. Eur Oral Res. 2020;54(1):16–24. doi:10.26650/eor.20200053.
Bin-Shuwaish M, AlHussaini A, AlHudaithy L, AlDukhiel S, AlJamhan A, Alrahlah A. Effects of different antibacterial disinfectants on microleakage of bulk-fill composite bonded to different tooth structures. BMC Oral Health. 2021;21:348. doi:10.1186/s12903-021-01717-7.
atpute TS, Mulay SA. Chlorhexidine in operative dentistry – A review. J Int Clin Dent Res Organ. 2021;13(2):80–5. doi:10.4103/jicdro.jicdro_2_21.
Jhingan P, Sandhu M, Jindal G, Goel D, Sachdev V. An in–vitro evaluation of the effect of 980 nm diode laser irradiation on intra–canal dentin surface and dentinal tubule openings after biomechanical preparation: scanning electron microscopic study. Indian J Dent. 2015 Apr–Jun;6(2):85–90. doi: 10.4103/0975-962X.155889
Vieira AA, Silva ACN. Effects of erbium laser radiation on the dentin organic matrix. Lasers in Dental Science. 2021;5(2):69–78. doi:10.1007/s41547-021-00122-1
Du Q, Ge L, Zhang S, Zhang Q. Effects of erbium: yttrium–aluminum–garnet laser irradiation on bovine dentin contaminated by cariogenic bacteria. Photobiomodul Photomed Laser Surg. 2019;37(5):305–311. doi:10.1089/photob.2018.4586
Abdou SA, Moharrum HS, Eltayeb EA. Comparative assessment of antibacterial effect of two types of laser and their effect on morphology and mineral content of dentin. J Arab Soc Med Res. 2023;18(2):117–127. doi:10.4103/jasmr.jasmr_17_23
Lapinska B, Klimek L, Sokolowski J, Lukomska-Szymanska M. Dentine surface morphology after chlorhexidine application—SEM study. Polymers (Basel). 2018;10(8):905. doi:10.3390/polym10080905. PMID: 30960830; PMCID: PMC6403839.
Siwinata M, Zubaidah N, Soetojo A. The effectivity of cavity cleanser chlorhexidine gluconate 2% and saponin 0.78% of mangosteen peel. Conserv Dent J (Surabaya). 2020;10(1):19–22. doi:10.20473/cdj.v10i1.2020.19–22.
Behniafar B, Noori F, Chiniforoush N, Raee A. The effect of lasers in occlusion of dentinal tubules and reducing dentinal hypersensitivity: a scoping review. BMC Oral Health. 2024;24(1):1407. doi:10.1186/s12903-024-05182-w. PMID: 39563326; PMCID: PMC11575069.
Jain S, Mathur S, Jhingan P, Sachdev V. Evaluation of temperature rise and efficacy of cavity disinfection with diode laser: an in vivo study. J Conserv Dent. 2020;22(6):583–587. doi:10.4103/JCD.JCD_78_19. PMID: 33088070; PMCID: PMC7542083.
Burlat N, Leforestier E, Rocca JP, et al. Shear bond strength of self-etching adhesive systems to Er:YAG laser-prepared dentine with and without pulpal pressure simulation. Photomed Laser Surg. 2008;26(6):579–83. doi:10.1089/pho.2007.2150.
Wanderley RL, Monghini EM, Pecora JD, Palma-Dibb RG, Borsatto MC. Shear bond strength to enamel of primary teeth irradiated with varying Er:YAG laser energies and SEM examination of the surface morphology: an in vitro study. Photomed Laser Surg. 2005;23(3):260–7. doi:10.1089/pho.2005.23.260.
Soares LES, Martin OCL, Moriyama LT, Kurachi C, Martin AA. Relationship between the chemical and morphological characteristics of human dentin after Er:YAG laser irradiation. J Biomed Opt. 2013;18(6):068001. doi:10.1117/1.JBO.18.6.068001.
Elgawish A, Tawfik H, El Gendy A, George R, Bakr MM. The impact of different irrigation regimens on the chemical structure and cleanliness of root canal dentin. Iran Endod J. 2023;18(4):224–232. doi:10.22037/iej.v18i4.38004. PMID: 37829828; PMCID: PMC10565995.
Azmy NH, Shalaby YA, Al-abbassy FH, Alhassan RG. Evaluation of chemical components changes in radicular dentin after different final surface treatments. Alexandria Dent J. 2023;47:19. doi:10.21608/ADJALEXU.2022.273604.
Ramezanian I, Baradaran E, Majidinia S, Ramezanian S, Jafari M. Effect of chlorhexidine and ethanol on microleakage of composite resin restoration to dentine. Chin J Dent Res. 2017;20(3):161–8. doi:10.3290/j.cjdr.a38771.
Satpute TS, Mulay SA. Chlorhexidine in operative dentistry—A review. J Int Clin Dent Res Organ. 2021;13:80–5. doi:10.4103/jicdro.jicdro_2_21.
Luong E, Shayegan A. Assessment of microleakage of Class V restored by resin composite and resin-modified glass ionomer and pit and fissure resin-based sealants following Er:YAG laser conditioning and acid etching: in vitro study. Clin Cosmet Investig Dent. 2018;10:83–92. doi:10.2147/CCIDE.S153989.

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The effect of Chlorohexidine, Er:YAG Laser and Diode Laser 980 nm as Dental Cavity Disinfectants on Dentine Morphology and Microleakage of Composite Restoration: An In Vitro Study

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Version 1

Abstract

Figures

Introduction

Materials and Methods

2.1. Study Design

2.2. Ethical Considerations

2.3. Sample Size Calculation

2.4. Randomization and Allocation

2.5. Blinding

2.6. Specimen Selection and Preparation

2.6.1. Inclusion and Exclusion Criteria

2.6.2. Collection and Storage

2.6.3. Cavity Preparation

2.7. Experimental Grouping

2.8. Assessment of the effects of different treatment modalities:

2.8.1. Assessment of morphological changes of dentine via Scanning Electron Microscopy (SEM) Analysis

2.8.2 Assessment of mineral content changes of dentine via Energy Dispersive X-ray (EDX):

3. Restoration Procedures:

4. Thermocycling Process

5. Microleakage Testing

5.1. Sample Preparation for Dye Penetration

5.2. Microleakage Assessment via Scanning Electron Microscopy (SEM) Analysis

Statistical analysis:

Results

Discussion

Conclusion

Declarations

Competing interests:

Funding:

Availability of data and materials:

References

Additional Declarations

Status:

Version 1