An in vitro assessment of the residual dentin after using three minimally invasive caries removal techniques

doi:10.21203/rs.3.rs-3556818/v1

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An in vitro assessment of the residual dentin after using three minimally invasive caries removal techniques

https://doi.org/10.21203/rs.3.rs-3556818/v1

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published 26 Mar, 2024

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To evaluate the efficiency and effectiveness of three minimally invasive MI caries removal techniques for removing deep dentin carious lesions. Forty extracted carious molars were treated by conventional rotary excavation (control), chemomechanical agent (Brix 3000), ultrasonic abrasion (Woodpecker, Guilin, China); and Er, Cr: YSGG laser (Biolase™ San Clemente, CA). The assessments include; the excavation time, Diagnodent pen, Raman spectroscopy, Vickers microhardness, and scanning electron microscope combined with energy dispersive X-ray spectroscopy (SEM-EDX). The rotary method recorded the shortest excavation time (p < 0.000), Brix 3000 gel was the slowest. All techniques removed the infected tissues completely as the Diagnodent pen readings were < 20 and laser-ablated surfaces recorded the lowest reading. The bur-excavated dentin showed higher phosphate and lower matrix contents with higher tissue hardness that were comparable to sound dentin indicating the non-selectiveness of this technique in removing the potentially repairable dentin tissue. In contrast, the MI techniques exhibited lower phosphate and higher organic contents associated with lower microhardness in the deeper dentin layers. This was associated with smooth residual dentin without smearing and patent dentinal tubules. This study supports the efficiency of these MI methods to be a conservative alternative to rotary excavation, providing a promising strategy for the clinical dental practice.

Biological sciences/Biotechnology

Health sciences/Medical research

The management of deep dentin carious lesions have been directed towards more conservative and biological directions¹, as the traditional rotary excavation causes adverse biological reactions to the dentin-pulp complex. This is attributed to the heat and vibration that associated with the procedure, added to the non-selective behavior in removing both healthy and diseased tissues².

Finding effective minimally invasive MI techniques to overcome the drawbacks of bur excavation is an objective for dental researchers, in order to remove the superficial, bacterially-contaminated and denatured dentin while preserving the partially demineralized tissue that can be sealed with suitable therapeutic restorations³. This will maintain the integrity of healthy and mineralizable tooth tissue, while maximizing the reparative potential of dentin-pulp complex⁴. These methods include laser ablation⁵, air abrasion⁶, sono-abrasion⁷, and the chemomechanical agents CMCR⁸.

BRIX 3000® (Brix medical science, S.R.L. of Argentina) is the latest CMCR agent that launched in Argentina in 2016, it is a papain-based gel with high enzyme activity (3.000 U/mg) due to utilizing the encapsulation buffer emulsion technology, that confers the stability of enzyme and thus reactivity⁹. The chemical method is considered to be effective in dissolving the infected tissues facilitating their removal while preserving the sound dentin structure¹⁰.

The ultrasonic abrasion can remove carious lesions and unsupported hard structure when used at high frequency (20–40 kHz). It relies on the kinetic energy of water molecules that transferred into tooth surface by high-speed oscillated diamond-coated cutting tip accompanied with sonic air-scaler device¹¹. However, Neves et al., (2011)¹⁰ found that the sonic tungsten-carbide tips (Cariex system) is efficient to remove the caries at the cavity floor in a micro-CT study while still residual caries was left at the walls, which agreed with a previous study that revealed a tendency towards under preparation with sono-abrasion¹². However, the utilization of advanced systems with different parameters might showed different caries removal efficiency.

The Erbium, Chromium-doped Yttrium, Scandium, Gallium and Garnet (Er, Cr: YSGG) exhibited promising results in caries removal when proper parameters are applied. The lasers transfer high energy into the tooth through water causing photo-ablation of hard tissues, as it produces rapid subsurface expansion of trapped water causing a massive volume expansion leading to an explosion of the tissue from the surface¹³. However, Neves et al., (2011)¹⁰ suggested that the Er: YAG-laser cannot be considered as a MI technique when equipped with a LIF-feedback system. It showed low MI potential due the excessive dentin removal in inactive and stained lesions that affects LIF readings leading to an over excavation.

Raman spectroscopy, is a non-invasive quantitative technique that can chemically characterize healthy and carious dentin through their specific molecular vibrational energy signatures^14,15. It can provide a molecular information regarding the mineral and matrix components of biologically compromised tissues¹⁶. The integrity of collagen triple helix in type I collagen membrane was previously measured^15,17 in which the peak ratio of amide III to C-H bond of the pyrrolidine ring (1235 cm^− 1 :1450 cm^− 1) indicates the integration of collagen if it is close to 1. The microhardness remains the gold standard measure for the mineral contents of dental hard tissues and the enhanced hardness reflects better crystalline and denser apatite structures¹⁸. Accordingly, this study evaluated and compared the efficiency of three MI caries removal techniques (Brix 3000, ultrasonic abrasion, and Er, Cr: YSGG laser) in comparison to the conventional rotary method (control). The assessments include; the excavation time, the chemical, mechanical and morphological properties of residual dentin by Raman spectroscopy, Vickers microhardness and Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX). The proposed null hypothesis was; these MI methods are not selective in removing the dentin caries in comparison the non-selective conventional rotary method.

Sample preparation.

Forty human carious molars extracted for periodontal reason were selected in the present study using an ethics protocol approved by the health research committee (Ref No. 754, 28/12/2022). In which the natural caries extends through dentin without pulp exposure (score 4 following the International Caries Detection and Assessment System (ICDAS-II)¹⁹. An access cavity preparation was prepared through enamel in each tooth (2 mm depth) using tungsten carbide bur (Komet, Lemgo, Germany) at a high-speed air turbine handpiece (300k rpm) to expose the carious lesion in dentin. In the 1st group (Rotary excavation, n = 10), the carious lesion was removed by a tungsten- carbide round bur (size #12, Komet, Lemgo, Germany) in a slow-speed handpiece with circular light brush strokes (Fig. <link rid="fig1">1</link>, A-1, A-2). In the 2nd group, the caries was removed chemomechanically by the application of Brix 3000® (Brix S.R.L., Area Industrial, Los Arces, Santa Fe, Argentina) following the manufacture’s instruction. The gel was dispensed into the cavity and agitated immediately using round-head excavator hand instrument (1.3 mm diameter). After 2 min the cavity scraped away using the blunt end of excavator in a pendulum movement without pressure until a clinically hard cavity surface was obtained. The gel was washed away periodically prior to check the hardness of the remaining dentin with a sharp probe. This procedure was repeated two to three times until the soft caries was completely removed (Fig. <link rid="fig1">1</link>, B-1, B-2).

In 3rd group (Ultrasonic abrasion), the soft caries was removed using the diamond-coated tips SB1 (Woodpecker, EMS Type, Guilin, China) with 1.5 mm diameter attached to woodpecker air-scaler handpiece (UDS-A LED), and ultrasonic dental scaler (WOODPECKER UDS A LED, Guilin, China). The vibration excursion was ≤ 100µm with 28 ± 3 kHz vibration frequency following manufacturer’s instructions. The procedure was done using copious water until hard dentin was reached (Fig. <link rid="fig1">1</link>, C-1, C-2). The carious tissues, in the 4th group were removed by a Waterlase iPlus Er,Cr:YSGG Laser (Waterlase iPlus, Biolase Technology, Inc., California, USA) by using Quartz tips (MZ- 8, 800 µm diameter, and 6 mm length) attached to a contra-angle gold handpiece (Biolase, Inc., California, foothill Ranch, USA). The wavelength of the laser is 2780 nm used in H mode, 15 Hz frequency, 3 W power output, < 150 mJ energy value. This was accompanied with copious amount of water as recommended by the manufacturer. Caries was excavated by moving the fiber tip gently in a circular or brushing motion in and-out motion, in a1.5 mm distance away from the cavity (a non-contact method)²⁰ (Fig. <link rid="fig1">1</link>, D-1, D-2).

The excavation endpoint was determined when the remaining dentin appeared yellow to light brown in color. On probing, the dentin exhibited either firm sensation or leathery/scratchy in sound or caries-affected dentin areas CAD^21,22. The surfaces were then examined by DIAGNOdent™ detection pen (655-nm diode laser, KaVo, Germany). This device was calibrated following the manufacturer’s instructions. The fluorescence of sound area at each cavity was recorded as a baseline reading. Three points were recorded within each cavity and averaged. The accepted mean readings ranged between 0–20 that indicate the remaining tissue is either sound or CAD²³. The excavation time in all techniques was recorded in minutes using a digital stopwatch including the times for washing, drying, and checking the remaining dentin surfaces.

After caries excavation, each tooth was hemi-sectioned longitudinally in a mesiodistal direction (Isomet 1000, Buehler, Lake Bluff, IL, USA) through the prepared surface with water-cooled diamond blade (330-CA/RS-70300, Struers, Detroit Rd. OH, USA). The buccal halves were mounted in epoxy resin molds exposing the prepared surfaces that kept hydrated throughout the experiments. To get flat and smooth surfaces, the assessed substrates received a sequential polishing (P1200 for 10 s, P2500 for 10 s and P4000) followed by 10 minutes ultrasonic cleaning¹⁵.

Raman Spectroscopy.

A high-resolution confocal Raman microscope (Senterra, Bruker Optics) operating in line scan mode was used to examine the residual dentin after caries excavation (n = 8 per group). To standardize the areas of measurements, a straight line was drawn from the mesial to the distal outline of the excavated cavity AB as shown in (Fig. 2). Another line (X) descends from the center toward the cavity floor representing the 1st reference point (X1). Two further lines were drawn from X bisecting the 90° angles regarded as the 2nd and 3rd reference points (X2 and X3). Six points were scanned by Raman spectroscopy in each sample, 50 µm and 100 µm away from X1-X3, (Fig. 2). The peak value was the average of three reading at the superficial and deeper layers. A control measurement was taken in an area of clinically sound dentin 800 µm away from the dentin-enamel junction (sound reference). Spectra acquisition was performed using a 785 nm near-infrared diode laser and a 400 line/mm diffraction grating. An Olympus 20X/0.40 NA objective lens with a spot size of about 5 µm. The spectra were measured over the range of 200–3600 cm^− 1 with 100 mW of laser power on each point with 30 sec integration time. Baseline correction was performed by Raman processing software (OPUS, Bruker Optics). Four Raman spectroscopic peaks were assessed. The phosphate peak intensity v1 vibration at 960 cm^− 1, amide I peak intensity at 1650 cm^− 1, amide III at 1235 cm^− 1, and C-H bond of the pyrrolidine ring at 1450 cm^− 1. The peak intensities were calculated and averaged at each area (50 µm and 100 µm, n = 6 per sample). The collagen integrity was assessed by calculating the absorbance ratio of 1235 cm^− 1:1450 cm^{− 1 15,16}.

Vickers microhardness.

The microhardness of the remaining and sound dentin was measured by Vickers microhardness tester (TH714, Obsnap Instruments Sdn Bhd, Selangor, Malaysia) using a square-based pyramid diamond-shaped indenter and 300 gf load for 15 sec²⁴. Six indentations were made in each excavated cavity using the same points that were previously assessed by Raman spectroscopy (Fig. 2) The Vickers hardness number (VHN) was the average of three indentation per cavity at each area 50µm and 100µm from the excavated margins. The values were recorded automatically using the manufacturer’s software.

Scanning electron microscopy- energy dispersive X-ray spectroscopy (SEM-EDX).

Five representative teeth from each group were sectioned horizontally at the level of the excavated cavity using a low-speed water-cooled diamond saw microtome (Isomet 1000, Buehler, IL, USA) and cleaned ultrasonically to remove surface debris. Surfaces were carbon-coated and viewed under scanning electron microscope (Inspect F50, FEI Company, Eindhoven, The Netherlands) with an accelerating voltage of 30kV at three magnification powers (x1500, x3000, and x6000). The calcium and phosphorus atomic percentages and the calcium to phosphate ratios were measured using the EDX. The recorded values are the average of three points of the remaining dentin with a sound reference.

Statistical analyses

The statistical analyses were performed using SPSS software version 25 (IBM, Chicago, IL, USA). Shapiro-Wilk test was used to evaluate the normality of data distribution. The excavation time (min), Diagnodent readings and the Ca/P ratios were analyzed by One-way ANOVA test, while the Raman peak intensities (A.U.) and VHN were assessed by Multivariant ANOVA (MANOVA). Both tests were followed by Tukey post-hoc multiple comparisons (p > 0.05).

Ethical approval. The Research Ethical Committee of College of Dentistry, University of Baghdad granted the ethical approval for the current study (Ref No. 754, 28/12/2022). All procedures performed in the study were in accordance with relevant guidelines and regulations of the local institutional committee in accordance with 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The authors confirm that informed consent forms for all extracted teeth were obtained from all patients and/or their legal guardian. The ethical standards were followed precisely during this study. At any stage of the research, authors confirm: (1) No person was exposed to any component of the materials or methods used in the research without any potential harm to be occurred for them (2) The authors didn't use any live plants or cytotoxic materials in this investigation (3) Components or materials were not used in the research in a manner or concentration that would cause direct or indirect harm to the individuals carrying out the research or those in charge of the various measurement processes. (4) All the tools used in the research were dealt with in a scientific, healthy and accurate manner, which entails the safety of individuals and places in accordance with the governing local rules and laws.

Clinical relevance. The minimally invasive methods in caries removal are a conservative alternative to rotary excavation in preserving the remineralizable dentin tissues, providing a promising strategy for the clinical dental practice.

The excavation times

The rotary excavation method recorded the lowest caries removal time (p<0.000) among all methods, while the longer time was observed in Brix 3000 group (p<0.05), with statistically no significant difference between the ultrasonic abrasion and laser ablation (p=0.80), Table 1.

Diagnodent pen readings

The mean readings were <20 in the remaining dentin after all excavation methods which indicate a complete removal of the caries-infected dentin. The laser ablation recorded the lowest reading among all methods (p>0.000), while there were statistically no significant differences between other methods (p>0.05), Table 1.

The calcium to phosphate (Ca:P) ratio

The Ca:P ratio of the remaining dentin was statistically significantly higher in the ultrasonic excavation group (2.27± 0.05) in comparison to all methods and the related sound reference (p<0.05). The rotary excavation showed the lowest ratio (1.91 ± 0.05) among groups; however, the difference was only statistically significant from the corresponded sound reference (p=0.012), Table 1.

Biochemical Analysis of the remaining dentin after caries removal

The relative Raman band intensities (Mean ± SD) with statistical correlations of the remaining superficial and deep dentin layers (50 µm and 100 µm) after caries removal are presented in Table 2. Multivariant ANOVA test showed the presence of statistically significant differences between groups at each area and between areas within the same group in all Raman peaks (p<0.05). Further analysis using Tukey post-hoc test revealed that the phosphate band intensities were statistically significantly lower at the superficial remaining dentin layer (50 µm) after all excavation methods in comparison to their relative values at the deeper layer (100 µm) and their sound reference (p<0.000). The highest phosphate peak intensity was noticed in the rotary excavation in comparison to other methods that showed statistically no significant differences between them at both layers (p>0.05), Table 2.

For the organic components (Amide I, Amide III, and C-H bond of pyrrolidine ring), the Raman peak intensities at 1650 cm⁻¹, 1235 cm⁻¹ and 1450 cm⁻¹, respectively, were higher in the superficial layers of the remaining dentin after all minimally invasive caries removal methods in comparison to their alternative values at the deeper dentin layers and sound reference (p<0.000). Whilst, the rotary excavation showed the lowest organic content (p<0.000) which were statistically not significant between areas and from sound reference (p>0.05). The highest organic peak intensities were observed in the ultrasonic and laser groups at both layers (p=0.000). However, the differences in values of Amide III and C-H bond of pyrrolidine ring were statistically not significant from Brix 3000 (p>0.05) at the superficial and deep dentin layers.

Regarding the collagen integrity, the absorbance ratio of amide III bands at 1235 cm^-1 to the pyrrolidine ring at 1450 cm^-1, ranged from 0.9 -1.4 in all groups which are close to sound dentin reference (1.0). This might indicate the lack of collagen denaturation in the remaining dentin after all excavation techniques. The higher ratios were seen in the ultrasonic and laser groups due to the high absorbance band of the amide III bands at 1235 cm^-1 (amide III), (Fig. 3).

Vickers Microhardness

At the superficial remaining dentin, the mean VHN value was the highest in the rotary excavation which was statistically significant from Brix 3000 (p<0.000) which in turn was close to ultrasonic application (p=0.061). The laser-ablated surfaces showed higher mean VHN value than Brix 3000 (p<0.000) but similar to the ultrasonic group (p=0.526). Similarly at the deeper dentin layer, the surface hardness was the highest in the rotary method (p=0.000) with statistically no significant differences between the MI methods (p=0.999). Generally, the VHN values of the remaining dentin after all caries removal methods were statistically significantly lower than their corresponded sound dentin references (Fig. 4).

The ideal caries-excavation method must have the ability to selectively remove the irreversibly damaged tissue, while preserving the potentially remineralizable dentin that can be healed and repaired by the dentin-pulp complex or the use of bioreactive materials^3,21. However, this cannot easily achieved clinically, even when the currently available caries excavation techniques are claimed to be specific in removing the infected tissues without sacrificing sound or potentially remineralizable tissues. This study examined the effectiveness and efficiency of three MI caries removal techniques (Brix 3000, ultrasonic, and laser) regarding the chemomechanical and morphological properties in comparison to rotary excavation. The proposed hypotheses were rejected as there were statistically significant differences in the properties of minimally-excavated dentin from the bur-excavated surfaces and sound dentin (p<0.05).

The excavation end was determined by visually and by a sharp explorer. However, the experience of the operator and nature of carious lesions may affect the evaluation of the remaining tissues. Accordingly, double-blind examiners helped in deciding the residual dentin. Nevertheless, these clinical methods are efficient to evaluate the residual dentin in comparison to use of caries detector dye or laser fluorescence²⁵. Further examination was accomplished by applying DIAGNOdent pen that can quantify the fluorescence induced by the bacterial metabolites in infected tissues²⁶. The mean readings were comparable between methods 16-17.5 which indicate complete removal of the infected dentin from the excavated lesions¹⁰. However, the laser ablation recorded the lowest reading among methods (p<0.05, Table 1), which inconsistent with Eberhard et al., (2005)²⁷ who observed a two to three-fold increase in the fluorescence after laser excavation due to the dehydration of dentin that induces higher fluorescence. Even though, the visual and tactile methods with DIAGNOdent pen are considered satisfactory to judge the remaining dentin clinically after caries excavation ^25,28.

In agreement with the literature^{13, 29,30} the bur excavation was the quickest among all techniques (7 ± 1min). This is attributed to lack of sensitivity of tactile feedback during tooth cutting which leads to gross and rapid removal of carious and non-carious tissues with less control over the whole process. In contrast, the chemomechanical agent was the slowest among techniques (13 ± 1.6 min, p<0.05). This attributed to the multiple applications of the gel until complete caries removal, added to the variations in the size and activity of the carious lesions which are difficult to be controlled or expected. This finding was supported by previous studies^31,32,33. Even though, the CMCR is widely accepted since it reduces pain in fearful and young patients³⁴ with a reported selectivity⁵in removing the damaged carious tissues while preserving sound dentin. The time taken by Er,Cr:YSGG Laser was longer than the rotary excavation, which was comparable to the ultrasonic caries removal (≃11 min). This finding in agreement with Celiberti et al., (2006)³⁵ but inconsistent with Shigetani et al., (2002) ³⁵who found no significant difference in the caries excavation time between rotary excavation and Er: YAG laser. The dental laser can also reduce pain and stress during cavity preparation and reduce the loss of dentin when the cavity dimensions were measured before and after caries excavation in comparison bur method²⁷.

Biochemically, the mineral component in the residual dentin represented by the characteristic phosphate (v1 PO₄ ^3-) Raman peak at 960 cm^-1, which the most intense peak in dental hard tissue. It was significantly reduced (p<0.05) after all excavation methods in comparison to sound dentin which also varied in different depths based on the degree of tissue demineralization indicating the presence of CAD as reported in previous studies^15,37. The highest phosphate peak intensity was noticed in the bur-excavated surfaces which is located within more highly mineralized dentin. This supports the non-selectiveness of this technique that was proved in previous studies^38,39. Whist, in the minimally-treated dentin (Brix 3000, ultrasonic, and laser), the overall phosphate content was initially lower than sound dentin, but still, they contain more residual mineral in comparison to the reported values for the CAD in a previous study¹⁵. This relatively high mineral level after all excavation methods might affect the subsequent mineral deposition rate and distribution that will help in situ generation of prenucleation clusters, succeeding further dentin remineralization⁴⁰.

The intensity of the organic functional groups can quantitively indicate the biochemical alteration in dentin during carious process which directly affect tissue repair and adhesion. The dentin collagen matrix features were found at 1650 cm⁻¹(Amide I), 1235 cm⁻¹ (Amide III), and at 1450 cm⁻¹ (C-H bond of pyrrolidine ring). These bands intensities were higher in the superficial layer of the minimally-excavated dentin in comparison to bur-excavated and sound dentin. This might indicate that the presence of demineralized tissues with high organic contents, as they are higher in CAD in comparison to sound dentin^40-42. It results from the degradation of extracellular organic matrix after the dissolution of hydroxyapatite. The intensity of Amide I was the highest in the ultrasonically and laser excavated-surfaces (p<0.000), indicating the presence of structural alterations in the remaining collagen⁴³. This might support the selective ability of both technique as this altered organic matrix can regulate the growth and maturation of apatite crystals and thus the mineralization process³⁷. The ultrasonic does not physically cut the dentin but abrades by a diamond-coated tip oscillating at 28 kHz vibration frequency. This oscillation was transformed into vibration at the dentin surface that may have a compacting effect on the carious dentin giving false indication for the clinical excavation endpoint. The chemical dissolution of carious tissues by Brix 3000 also maintained high organic content in the residual dentin especially the Amide III and the C-H bond of pyrrolidine ring in comparison to rotary excavation and sound dentin (p<0.000). This referred to the action of papain enzyme that breakdown the partially degraded collagen and help in the disintegration of the fibrin mantle generated by the carious process while preserving the unimpaired collagen fibrils. However, it is difficult to clearly distinguish between the dissolution effect of the gel from the assisted mechanical removal. The preservation of the organic components in the minimally treated surfaces can provide a suitable scaffold for the biomimetic dentin remineralization and induce tissue healing⁴⁴. This contradicts with Pai et al., (2009)⁴⁵ who found no significant difference in the organic contents of the remaining tissues between rotary and Carisolv, which might be due to the chlorination effect of the chloramine T that may dissolve sound and demineralized tissues⁴⁶. The ratio of the intensity of Amide III to the C-H bond of pyrrolidine ring elucidates the integrity of the collagen fibril with a triple helical structure that may undergo micro-structural changes during the caries process if the ratio below 0.8^15,16. The ratios in the remaining dentin after all excavation methods ranged between 0.9-1.3 which means that even with the presence of changes in the organic structure of dentin, the ratio was close to that reported for CAD and sound dentin (1.0), as shown in Figure 3.

The bur-excavated tissues exhibited higher hardness values, however, at the superficial layer, the value was comparable to that of ultrasonic and laser ablation (p>0.05), which was significantly increased (p>0.000) in the deeper dentin layer. This might be attributed to the higher mineral contents in the residual dentin represented by the higher Raman phosphate intensity and the higher Ca and P atomic % (12.2, 6.6 % respectively) even if the Ca:P ratio was the lowest among groups, table (1). This supports the non-selectiveness of this technique in comparison to the other used method, especially the chemomechanical caries removal technique (Brix 3000 group) that showed the lowest hardness values. Hamama et al., (2013)³⁹ reported lower hardness values of the residual dentin after Carisolv and Papacarie in comparison to the rotary excavation, but Lima Santos et al., (2020)²⁹ did not find any significant difference in microhardness between the CMCR agents (Brix 3000 and Papacarie Duo) and rotary excavation. However, the selective ability of chemomechanical methods was proved in a previous study¹⁰. The hardness of the remaining tissues in Brix 3000 group was similar to the ultrasonic and laser methods (p=0.999) that also showed reduced values as compared to bur excavation and sound dentin at the deeper layer (p<0.000). This indicates the presence of partially demineralized tissues left after caries removal, as the hardness and elastic modulus of CAD is lower than sound dentin, because of the reduced number and size of the apatite crystals in the intertubular dentin after tissue demineralization ^14,15,40. However, the Ca/P ratios in all groups are comparable to sound dentin (2.0-2.2) which falls within the reported ratios ^39,47. Sakoolnamarka et al., (2005)⁴⁸ found the presence of a correlation between the Ca:P ratio and microhardness in dentin using an Ultra-Micro-Indentation System. However, this cannot be applicable in the present study as the ultrasonic-excavated dentin recorded the highest Ca:P ratio (2.27± 0.05) than rotary excavation (p=0.003), but the atomic percentages of Ca and P ions were 50% lower (6.9, 3.1%, respectively) associated with lower hardness value (p<0.000).

The Scanning electron micrographs showed smooth residual dentin surfaces after the minimally caries excavation techniques free from smear layer, leaving patent dentinal tubules. This facilitates the infiltration of adhesive resin into intertubular dentin.This agreed with previous studies ^49,50 regarding the sonic abrasion (Figure 5, C-2 and C-3). For the Brix 3000, the results are consistent with Hossain et al., (2003)⁴⁷ who reported that the Carisolv treatment totally remove the smear layer leaving patent dentinal tubules. But disagreed with Hamama (2013)³⁹; and Al-Badri et al., (2023)⁵¹, who observed the presence of amorphous layer of cutting debris obliterated the dentinal tubules resulted from crushing of the excavated tissue by spoon excavator. They suggested that the mechanical action of this technique is more relevant than the chemical effect due to the high acidity of Brix (pH=3.9)⁵¹ combined with the lubricant action that modifies the hand excavation on CAD surfaces producing more debris at the excavated surfaces. This also disagrees with the study of Prabhakar et al., (2018)⁵, who observed surface irregularities with the presence of bacterial deposits, few opened dentinal tubules with minimal smear layer. However, this study⁵ showed an agreement regarding the observation of the laser-ablated surfaces that showed a wavy irregular surface with rugged appearance free from smear layer associated with patent dentinal tubules. This irregular configuration without smear layer is expected to provide a strong bonding with the composite combined with the etching behavior of laser⁵². Prabhakar et al., (2018)⁵ also found the least amount of bacterial deposits at the Er:YAG laser excavated surfaces, due to the photoacoustic and bactericidal effects of dental laser. Moreover, the presence of a water flow during laser exposure seems to prevent tissue damage allow proper cleansing of the ablated surfaces.

In contrast, the rotary excavation technique produced rough dentin surface with a noticeable smearing accompanied which might interfere with adhesion, wetting, and the penetration of the adhesive-based restorations when used without preconditioning the substrate⁵³. However, the presence of such micro-irregularities might increase the surface area for micromechanical retention. This coincides with Hamama (2013)³⁹ and Prabhakar et al., (2018)⁵ who observed the presence of well-formed smear layer with occluded dentinal tubules. However, the concept of leaving CAD following caries excavation is highly recommended due to the evidence of a high degree of collagen exposure that promotes the hybridization with adhesive resins⁵⁴ which necessitates further investigations.

Accordingly, the proposed hypotheses in this study were rejected as the use of Brix 3000, ultrasonic and laser ablation exhibited statistically significant differences in the chemomechanical properties of the residual dentine from bur excavation method and sound dentin. Despite the longer excavation time, they are capable of maintaining potentially mineralizable tissues with enhanced morphological features for subsequent bonding to restorative materials. However, further clinical studies are needed to extrapolate these findings.

The rotary technique recorded the shortest excavation time, while Brix 3000 gel showed the longest time. All techniques removed the infected caries effectively as the Diagnodent pen readings were <20 and the laser ablated surfaces recorded the lowest reading. The bur-excavated dentin showed the highest phosphate, lowest organic contents, with higher tissue hardness that were comparable to sound dentin indicating the non-selectiveness of technique in removing partially demineralized tissues that can potentially be repaired. In contrast, the implementation of MI caries removal techniques exhibited lower phosphate, higher organic contents, associated with lower microhardness especially in the deeper layers. This was associated with smooth dentin surfaces free from smear layer with patent dentinal tubules. Accordingly, this study supports the efficiency of these methods as a conservative alternative to rotary excavation, providing a promising strategy for the clinical dental practice.

Data availability. The data that support the findings of the current study are available from corresponding author upon reasonable request.

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Table 1

The mean caries excavation time (minutes), Diagnodent pen readings, and the calcium to phosphate ratios of the remaining dentin after each caries removal method and their corresponded sound dentin reference. (*) The presence of statistically significant differences among caries excavation methods within each column. Similar letters indicate that there were statistically no significant differences between methods. (^) The presence of statistically significant differences in Ca:P ratios in each method from their correspondent sound dentin within each row (alpha level of 0.05).
Groups (n = 10 per group)	Excavation time (minutes) (mean ± SD)	Diagnodent readings (mean ± SD)	Ca/P ratio (mean ± SD)
			Residual dentin	sound dentin
Rotary excavation	6.9 ± 1.2 *	17.5 ± 1.7 ^b	1.91 ± 0.05 ^c^	2.04 ± 0.04
Brix 3000	13.1 ± 1.6	16.8 ± 1.2 ^b	2.00 ± 0.06 ^c	2.00 ± 0.05
Ultrasonic	11.2 ± 1.1 ^a	15.9 ± 1.4 ^b	2.23 ± 0.04 *^	2.27 ± 0.05
Laser	10.5 ± 2.1 ^a	13.8 ± 1.6 *	1.99 ± 0.05 ^c	2.21 ± 0.06

Table 2

Raman inorganic and organic peak intensities (mean ± SD) of the remaining superficial and deep dentin layers after the caries removal techniques. Similar letters indicate that there were statistically no significant differences between groups and areas. (^) The presence of statistically significant differences in Raman peak intensities at the superficial and deep dentin layers from their correspondent sound dentin within each row. Multivariant ANOVA test and Tukey’s post-hoc tests (alpha level of 0.05)
	Raman peak intensities at 50 µm	Raman peak intensities at 100 µm	Raman peak intensities of reference points
Phosphate peak v1-PO (960 cm^− 1)
Rotary excavation	2508.8 ± 296.2 ^	2849.6 ± 272.4 ^	3188.4 ± 288.8
Brix 3000	1967.0 ± 234.1^a^	2338.2 ± 281.1^b^	2970.5 ± 258.7
Ultrasonic	2055.3 ± 280.5 ^a^	2521.5 ± 258.3 ^b^	3016.9 ± 223.4
Laser	1929.2 ± 226.8 ^a^	2451.5 ± 186.9 ^b^	2826.9 ± 214.4
Amide I (1650 cm^− 1)
Rotary excavation	25.4 ± 3.5 ^c	23.8 ± 3.1 ^c	22.1 ± 3.0 ^c
Brix 3000	37.0 ± 4.5 ^	29.3 ± 5.7 ^	19.7 ± 3.8
Ultrasonic	50.1 ± 4.9 ^d^	39.0 ± 4.9 ^e^	21.2 ± 2.7
Laser	49.9 ± 3.6 ^d^	41.1 ± 3.7 ^e^	22.1 ± 3.1
Amide III (1235 cm^− 1)
Rotary excavation	34.4 ± 4.0 ^f	33.4 ± 7.0 ^f	30.7 ± 3.5 ^f
Brix 3000	45.0 ± 4.6 ^g^	40.5 ± 4.6 ^g^	30.0 ± 8.6
Ultrasonic	69.1 ± 4.5 ^	45.3 ± 3.3 ^g^	29.1 ± 2.7
Laser	54.9 ± 5.4 ^	41.4 ± 4.7 ^g^	29.8 ± 3.2
C-H bond of pyrrolidine ring (1450 cm^− 1)
Rotary excavation	35.9 ± 2.5 ^h^	32.4 ± 6.3 ^h	30.4 ± 3.7 ^h
Brix 3000	38.7 ± 4.0 ^j^	34.8 ± 5.4 ^j^	28.7 ± 4.3
Ultrasonic	48.8 ± 5.4 ^	40.1 ± 6.0 ^j^	29.3 ± 5.8
Laser	41.3 ± 5.0 ^j^	38.3 ± 5.3 ^j^	31.7 ± 7.3

No competing interests reported.

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An in vitro assessment of the residual dentin after using three minimally invasive caries removal techniques

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