Chemical, morphological and microhardness changes of dentine after chemomechanical caries removal

Authors


Address for correspondence:

Professor Cynthia Yiu

Paediatric Dentistry and Orthodontics

Faculty of Dentistry

The University of Hong Kong

Prince Philip Dental Hospital

34 Hospital Road, Hong Kong SAR

China

Email: ckyyiu@hkucc.hku.hk

Abstract

Background

This study compared the chemical, morphological and microhardness changes in carious dentine following application of NaOCl-based (Carisolv), papain-based (Papacarie) chemomechanical caries removal agents with conventional rotary method.

Methods

Thirty-two carious and eight non-carious human permanent molars were used. The carious molars were randomly distributed into four groups: Group 1 (positive control) – molars were left without caries removal; Group 2 – caries excavated with rotary method; Group 3 – caries excavated with Carisolv; Group 4 – caries excavated with Papacarie. Group 5 (negative control) consisted of eight sound molars. After caries excavation, all specimens were prepared for chemical, morphological analysis and Vickers hardness testing.

Results

EDX analysis showed no significant difference in Ca wt%, P wt% and Ca/P ratio among Groups 2 to 5. Vickers hardness of dentine from Groups 3 and 4 was significantly lower (p < 0.05) than for Groups 2 and 5. The use of Papacarie resulted in a dentine surface without smear layer and patent tubules, while Carisolv resulted in a dentine surface exhibiting two patterns: (1) the presence of smear layer or (2) patent tubules with partial smear layer formation.

Conclusions

Papain-based (Papacarie) chemomechanical caries removal method is a reliable alternative to conventional rotary and NaOCl-based (Carisolv) caries removal.

Abbreviations and acronyms
ANOVA

analysis of variance

EDX

energy dispersive X-ray system

FESEM

field emission scanning electron microscope

MICRT

minimally invasive caries removal technique

SEM

scanning electron microscopy

Introduction

Dental caries prevention and treatment have changed significantly in the past four decades. From the aspect of excavation of diseased tissues, initially sharp metal hand drills were introduced by James Morrison in 1871, followed by low-speed rotary instruments (10 000 rpm) in 1947. The introduction of the ultra high-speed (over 250 000 rpm) air turbine handpiece in 1957 provided a great advance to rapidly access and remove caries lesions.[1] However, all rotary cutting methods cause some adverse biological reactions to the dentino-pulpal complex, mainly from pressure, heat generation and non-selective removal of both infected and sound tooth tissue.[2] One major disadvantage of rotary instrumentation in caries removal is the vibration caused by the rotation frequency of the bur, which is particularly unpleasant for patients. In the case of dental-phobic and child patients, the slow-speed bur cannot be used due to the increased anxiety that it can create in this patient group.

To overcome these problems, new caries-removal strategies have been introduced for dental treatment. These new methods provided a base for the development of minimally invasive caries removal techniques (MICRTs).[3] Minimally invasive caries removal technique is one of the most important applications of minimal intervention dentistry concepts that were established in the last decade.[4] The recent incorporation of laser ablation,[5] air abrasion,[6] sono abrasion,[7] and chemomechanical agents[2] in the removal of infected dental tissues have provided significant advancements in MICRTs. The common feature of recent MICRTs is the selective removal of caries-infected tissue, which leaves the caries-affected tissues intact. The ‘caries-affected’ dentine is characterized by demineralization of the intertubular dentine, deposition of crystals in the tubule lumen, no destruction of the collagen matrix and no bacterial penetration.[8] Conversely, the ‘caries-infected’ dentine showed distortion of the microstructure of the dentinal tubules, irreversible denaturation of the collagen fibres and marked bacterial infiltration.[8]

Chemomechanical caries removal agents are classified into either sodium hypochlorite-(NaOCl) or enzymatic-based. The first group depends on NaOCl derivatives, which chlorinate and disrupt hydrogen bonds of partially degraded collagen in carious dentine facilitating its removal.[2] The first chemomechanical caries removal method was introduced by Goldman and Kronman in 1976,[9] which consisted of a solution containing 0.05% N-monochloroglycine and NaOCl, and marketed as GK-101. It was classified as a non-specific highly reactive proteolytic agent.[9] Although this method seemed effective in caries removal, it lacked selectivity and dissolved caries-infected and caries-affected dentine, and also healthy dentine.[10]

Amino acids were later added to the NaOCl to form GK-101E [N-monochloro-DL-2 amino butyrate (NMAB)] to increase the specificity of GK-101E towards denatured protein of caries-infected dentine.[11, 12] The mechanism of action of NMAB on denaturated collagen fibrils involved the chlorination of the partially degraded collagen in the carious lesion and the conversion of hydroxylproline to pyrrole-2-carboxylic acid.[12] In addition to chlorination reaction, cleavage of the denatured collagen fibrils might occur as a result of the oxidation of glycine residues.[12] It was marketed as ‘Caridex™’ (National Patent Dental Products Inc., New Brunswick, NJ, USA) and received FDA approval in 1984.[13] The Caridex system was critically evaluated demonstrating its caries removal efficiency,[11, 14] biocompatibility[9] and pulpal safety.[15] However, Caridex also had some disadvantages, such as the complex delivery equipment[13] and the relatively long time needed for caries excavation.[2, 13] This limited its clinical usage and acceptance. The addition of urea to Caridex was a further modification to improve caries removal, especially in deciduous teeth.[14]

The latest variation of NaOCl-based chemomechanical agents came with the introduction of ‘Carisolv ™’ (Medi Team Dentalutveckling AB, Sweden) in 1998. Although Carisolv has a similar chemical structure as Caridex, it requires neither heating nor a special delivery system because of its gel form.[16] The original Carisolv consisted of two syringes containing glutamic acid, leucin, lysine, sodium chloride, erythrocin, caboxymethylcellulose, water and sodium hydroxide in one syringe and 0.25% NaOCl in the other syringe.[16] The difference between the action of Carisolv containing 0.25% NaOCl and the pure 0.25% NaOCl solution could be attributed to the addition of the amino acids to Carisolv, which reduced the organic tissue solving properties of the NaOCl in Cariosolv gel.[16] Non-cutting tip hand-instruments were designed for the Carisolv system to increase its caries removal efficiency without affecting sound dentine. The caries excavation process with the Carisolv gel was also time consuming, and this problem led to a modified Carisolv gel in 2004.[17] The modification included removal of the red colouring agent, decreasing the amino acids concentration by half and doubling the NaOCl concentration from 0.25% to 0.475%.

The second group of chemomechanical caries removal agents is the enzymatic-based agents. The only two products represented in this group are Papacarie™ (Papain-based gel, Formula & Acao, Brazil) and Biosolv™ (Experimental SFC-V gel, 3M ESPE AG, Seefeld, Germany).[18, 19] The Papacarie gel consists of papain, chloramines, toluidine blue, salts, preservatives, thickener, stabilizers and deionized water.[18] Although Papacarie contains a small amount of chloramine, the main action depends on the presence of the papain enzyme and the chloramine was added to enhance removal of denaturated tissues.[18, 20]

Enzymatic chemomechanical caries removal was introduced in 2003 by Bussadori who developed Papacarie™ (a word which means ‘caries eater’ in Portuguese). Papain is extracted from the latex of leaves and fruits of the green adult Carica Papaya tree, which is cultivated in tropical regions such as Brazil, India, South Africa, and Hawaii.[21] It is a proteolytic enzyme with bactericidal and anti-inflammatory actions. The precise mechanism of action of enzymatic-based chemomechanical caries removal agents is unknown. Bussaduri et al.[18] reported that the enzymatic caries removal method was based on the fact that infected carious tissues lost the antiprotease α-1-anti-trypsin, which inhibited protein digestion in sound collagen-based tissues.[18] However, no evidence that α-1-anti-trypsin could be identified in dentine. Recently, Bertassoni and Marshall[22] reported that the papain enzyme could partially degrade intact non-mineralized type I collagen fibrils from rat tail tendon. Furthermore, papain had been shown to reduce the mechanical properties of intact mineralized dentine as a result of degradation of proteoglycans of the matrix, suggesting that the action of papain might be non-specific.[22]

Many studies have evaluated the chemomechanical caries removal method, regarding the excavation time,[17] morphological analysis of residual dentine surface,[13, 17, 23] chemical analysis to detect any adverse effects when applied on dental tissues[24, 25] and microhardness in the residual dentine.[24-26] The majority of studies have been conducted on Carisolv; however, very few studies have evaluated the effect of enzymatic-based chemomechanical caries removal agents such as Papacarie™ on dentine surface characteristics.[22, 23, 27] Only one recent study has shown that the papain gel induced partial degradation of the collagen fibrils with no rupture of the fibrils. In addition, intact mineralized dentine had reduced mechanical properties after treatments with the papain gel.[22]

The aim of this study was to evaluate the chemical, morphological and microhardness changes in dentine following the application of NaOCl-based (Carisolv), papain-based (Papacarie) chemomechanical caries removal agents, in comparison with a standard conventional rotary method. The null hypotheses tested for this study were that there was no difference: (1) in chemical composition; (2) morphology; and (3) microhardness of dentine following the three caries excavation techniques.

Materials and Methods

Selection of teeth and caries excavation procedures

Thirty-two carious and eight non-carious human permanent molars stored in 0.5% chloramine T solution at 4 oC were used within six months following extraction. This study was approved by the Institutional Review Board (IRB Ref No: UW 11-355). The 32 carious molars included in the study exhibited frank cavitation on the occlusal surface reaching dentine. The 32 carious molars were randomly divided into four groups with eight molars per group as follows and shown in Fig. 1.

Figure 1.

Schematic diagram showing the treatment groups.

In Group (1), carious molars were left without any excavation to act as the positive control group. In Group (2), caries excavation was performed with a low-speed handpiece (NSK, Tochigi, Japan) at 35 000 rpm using a round steel bur (#014, Dentsply Ltd, Surrey, UK) after application of a caries detector dye (Batch No. 758AA, Kuraray Medical Inc., Tokyo, Japan) to define the carious lesion and verify the removal of ‘caries-infected’ dentine, leaving behind lightly stained pink ‘caries-affected’ dentine as per the manufacturer's instructions. In Group (3), the carious lesion was treated with Carisolv™ multimix gel (Batch No. 10489, Medi Team Dentalutveckling AB, Sweden), which was supplied as a twin syringe two-gel system. The gel was mixed automatically in the correct proportions in the tip (Static Mixer, Medi Team Dentalutveckling AB, Sweden) of the syringe prior to application on the carious lesion. The gel was left for 30 seconds prior to excavating the dentine using a #4 Carisolv non-cutting instrument (Medi Team Dentalutveckling AB, Sweden) (Fig. 2). Once the gel became cloudy, it was rinsed off with distilled water for 20 seconds and the process was repeated until successive application of the gel failed to become cloudy. In Group (4), the carious cavity was treated with Papacarie™ (Batch No. 8996, Formula & Acao, Brazil). The gel was left for 30 seconds prior to excavating the dentine using a #4 Carisolv non-cutting instrument. Similar to Carisolv, once the gel became cloudy, it was rinsed away and the process was repeated until successive application of the gel failed to become cloudy. Finally, Group (5) contained eight sound caries-free molars without any pre-existing cracks to act as the negative control group.

Figure 2.

#4 Carisolv non-cutting instrument tip (Medi Team Dentalutveckling AB, Sweden).

Caries excavation time and number of applications

During the caries removal process, the total excavation time for Groups 2, 3 and 4 and the number of times of gel application for Groups 3 and 4 were recorded. After caries removal, each tooth was sectioned into two halves longitudinally in the mesio-distal plane through the carious lesion using a low-speed water-cooled diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA). For the sound teeth (Group 5), the occlusal enamel was removed to expose the sound dentine, followed by a longitudinal cut mesio-distally through the dentine to separate the tooth into two sections. For all groups, one section was used for morphological examination and chemical analysis; while the remaining section was used for microhardness testing.

Chemical and morphological analysis of dentine

Each section was further cut longitudinally in the bucco-lingual direction into two sections through the centre of the excavated caries lesion. The first section was subjected to elemental analysis; while the remaining one was used in morphological observation under scanning electron microscopy (SEM) (Fig. 3). The longitudinal flat cut surface of the first section was subjected to elemental analysis under high-vacuum at 1500X magnification using energy dispersive X-ray system (EDX) (Microanalysis suites, INCA V4.11, Oxford Instrument Analytical Ltd, UK), which is attached to an environmental scanning electron microscope (Hitachi S-3400N, Hitachi High Technologies America, Inc Schaumburg, IL, USA). The chemical analysis was performed by focusing the X-ray beam on the intertubular dentine, approximately 50 μm from the margin of the deepest portion of the excavation site at three different points. The recorded values represented the average mineral weight percentages of these three points. The excavated surface of the remaining section was gold-sputter coated for morphological examination using field emission scanning electron microscope (FESEM) operated in secondary electron detection mode, 10 mm working distance, 12 KV accelerating voltage (ACCV) at X1000 and X2000 magnifications.

Figure 3.

Diagram illustrating tooth-sectioning protocol and labelling the purpose of each section.

Microhardness test

The remaining tooth sections from each group were prepared for microhardness testing. The sections were embedded in self-cured acrylic resin (ProBase Cold™, Ivoclar Vivadent AG, Liechtenstein). During the embedding process, the specimen surfaces were left exposed and covered with a damp fibreless laboratory napkin (Kimwipes™ Ex-L, Kimberly-Clark Professional, USA) to avoid dentine dehydration during setting. After setting, the surfaces of each section were wet ground with (800-, 1200-, 2400- and 4000- grit) silicon carbide papers (Microcut™, Buehler, USA) respectively to completely expose and flatten the dentine surface.

The Vickers hardness of the residual dentine was measured at 25 μm, 50 μm, 75 μm, 100 μm and 150 μm distance from the floor of the excavated lesion using a microhardness testing machine (Leitz Microhardness Tester, Ernst Leitz Wetzlar GMBH, Wetzlar, Germany) (Fig. 3). The Vickers hardness of sound dentine was similarly measured at 25 μm, 50 μm, 75 μm, 100 μm and 150 μm from the dentine surface. The hardness tester consists of pyramidal diamond indenter that was loaded to 50 g (0.49 N) for 15 seconds leaving an indentation on the polished surface. The indentations were aligned from the surface and each indentation separated from the following one by at least 50 μm. The two diagonals of each indentation were measured and the Vickers hardness was automatically calculated using Leica Qwin Lite Software (V.321, Leica Microsystems Imaging Solution Ltd, Cambridge, UK).

Dehydration of the specimens was avoided by the application of a damp fibreless laboratory napkin on top of the tested surface and removed immediately before testing. Furthermore, each individual indentation was measured immediately after its placement using the light microscopy attached to the microhardness testing machine (OrthoPlan-POL, Ernst Leitz Wetzlar GMBH, Wetzlar, Germany). A representative specimen of each group was mounted on an aluminium stub, gold-sputter coated and observed under SEM for evaluation of the indentation quality. The mean Vickers hardness of residual and sound dentine at each indentation point level was calculated using the Vickers hardness readings taken from the eight teeth.

Application of caries detector dye following caries excavation

Six additional carious and two sound molars, meeting the same criteria in the previous section, were selected. The six carious molars were randomly divided into three groups of two molars each, according to the caries excavation method: rotary, Carisolv, or Papacarie. The occlusal enamel was removed, leaving the carious and sound dentine exposed. The rotary caries excavation was performed as previously described. The initial verification of complete caries removal was performed with the conventional visual and tactile method using a blunt dental explorer, while the final verification was performed with caries detector dye (Batch No. 758AA, Kuraray Medical Inc., Tokyo, Japan). In both chemomechanical caries excavation methods, the previously described protocols were used. After completion of the caries excavation, the caries detector dye was applied for 20 seconds, then rinsed with distilled water for 10 seconds. A representative specimen of each group was selected. A photograph of the four groups was taken together for comparison.

Statistical analysis

The mean time taken for caries excavation for rotary, Carisolv and Papacarie groups were subjected to one-way ANOVA, followed by Tukey's post hoc multiple comparison tests. The number of applications for both Carisolv and Papacarie groups was subjected to an independent sample t-test. The average Ca, P weight percentages and Ca/P ratio of carious, residual and sound dentine were subjected to one-way ANOVA, followed by Tukey's post hoc multiple comparisons. The mean and standard deviation of the Vickers hardness values for the five groups were calculated and subjected to two-way ANOVA followed by Tukey's HSD post hoc multiple comparison tests. Two-way ANOVA was used to examine the effect of ‘distance from the excavated lesion’, ‘caries removal method’ and the interaction of these two factors on the Vickers hardness of dentine. The data were statistically analysed using the SPSS™ Software (V.19, IBM, NY, USA).

Results

Caries excavation time and number of applications

The mean excavation time for each group is shown in Table 1. One-way ANOVA showed that the method of caries excavation had a significant effect (p < 0.001) on excavation time. Tukey's post hoc test revealed that the excavation time was significantly longer (p < 0.01) for the Carisolv group (6.46 minutes), when compared to the rotary (4.14 minutes) and Papacarie (5.19 minutes) groups. However, no significant difference (p > 0.05) was found in the caries excavation time between the rotary and Papacarie groups. The independent sample t-test indicated that the number of gel applications was significantly higher (p < 0.05) for the Carisolv group (6 ± 1.6 times) compared with the Papacarie group (4 ± 0.6 times).

Table 1. Time of caries excavation method
Caries excavation methodExcavation time (min)a
  1. a

    Values are means ± standard deviation.

  2. Groups identified by different superscripts were significantly different at p < 0.05, n = 8.

Rotary4.14b ± 0.32
Carisolv6.46a ± 1.57
Papacarie5.19b ± 0.75

Chemical and morphological analysis of dentine

The EDX elemental analysis of Groups 1 to 5 is shown in Table 2. The major elements are represented by weight per cent, while the other trace elements such as Br, Mg, S and Cl are not represented, because of their low weight per cent when compared to Ca and P. One-way ANOVA revealed that the Ca (16.79 ± 6.88%) and P (8.5 ± 3.74%) levels of carious dentine were significantly lower (p < 0.05) than the other four groups. No significant difference (p > 0.05) in Ca/P ratio was found amongst the five groups.

Table 2. Elemental content of carious, sound and residual dentine after caries removal (wt%)
GroupCaPCa/P ratio
  1. Groups identified by different superscripts were significantly different at p < 0.05, n = 8.

Carious dentine16.79b ± 6.888.50B ± 3.742.01α ± 0.08
Rotary32.51a ± 1.916.07A ± 0.782.02α ± 0.04
Carisolv29.63a ± 3.3515.09A ± 1.251.97α ± 0.2
Papacarie30.66a ± 1.9215.86A ± 0.861.95α ± 0.08
Sound dentine31.05a ± 1.6416.04A ± 0.941.93α ± 0.04

The SEM of the dentine surface following the rotary caries excavation consistently showed the presence of a smear layer and occlusion of most of the dentinal tubules with smear plugs (Fig. 4A and 4B). The residual dentine of the Carisolv group showed two patterns; the first showed the presence of a smear layer that covered the dentinal tubules, while the other pattern showed patent dentinal tubules and the partial absence of a smear layer (Fig. 4C and 4D). The most characteristic finding was observed in the Papacarie group, in which there was nearly a total absence of a smear layer on all specimens and most of the dentinal tubules were patent (Fig. 4E and 4F). The residual dentine surfaces following chemomechanical caries removal methods by either Carisolv or Papacarie were irregular when compared to the rotary method.

Figure 4.

SEM images showing the residual dentine following caries removal. (a) and (b). SEM images in low (X1000) and high (X2000) magnification of residual dentine after rotary caries removal showing a smooth surface, occluded (O) and partially occluded (PO) dentinal tubules and the presence of smear layer. (c) and (d). SEM images in low and high magnification of residual dentine after Carisolv caries removal showing a rough surface, numerous patent (P) and partially occluded dentinal tubules and a sparse smear layer. (e) and (f). SEM images in low and high magnification of residual dentine after Papacarie caries removal showing a roughened surface and patent dentinal tubules with little or no smear layer formed.

Microhardness test

The mean Vickers hardness values of sound dentine (negative control group) and residual dentine following caries removal with rotary, Carisolv and Papacarie methods are shown in Table 3. The results of two-way ANOVA revealed a significant effect of caries excavation method (p < 0.001) and the distance from the floor of excavated lesion (p < 0.001) on the Vickers hardness of dentine. The interaction between the two factors was also highly significant (p < 0.001).

Table 3. Microhardness of sound and residual dentine after caries removal
MethodDentine Vickers hardness (Mean ± SD)
25 μm50 μm75 μm100 μm150 μm
  1. Groups identified by different superscripts were significantly different at p < 0.05, n = 8.

Rotary72.77b ± 4.2474.88b ± 6.1079.16a ± 5.7079.47a ± 2.9281.94a ± 2.32
Carisolv14.31f ± 2.3617.64e ± 2.3519..01d ± 2.5022.10d ± 3.9231.83c ± 1.60
Papacarie13.88f ± 3.0017.71e ± 2.3920.25d ± 4.8224.24d ± 4.5931.63c ± 3.58
Sound81.96a ± 4.2382.10a ± 3.1683.88a ± 2.8381.79a ± 2.3081.32a ± 3.47

Tukey's post hoc test showed that Vickers hardness of sound dentine and residual dentine following rotary caries removal was significantly higher (p < 0.001) than Vickers hardness of the residual dentine following chemomechanical caries removal by Carisolv or Papacarie. It also showed that Vickers hardness of sound dentine was significantly (p < 0.05) higher than Vickers hardness of the residual dentine following rotary caries removal at 25 μm and 50 μm indentation levels; however, no significant difference was found at 75 μm, 100 μm and 150 μm indentation levels (p > 0.05). Conversely, no significant difference (p > 0.05) in Vickers hardness of residual dentine was found between the Carisolv and Papacarie groups.

A further Tukey's post hoc test was performed to determine the effect of distance from the floor of the excavated lesion on Vickers hardness of dentine. Tukey's post hoc test showed that Vickers hardness of sound dentine group was not significantly different (p > 0.05) among all the indentation sites. However, in the rotary group Vickers hardness of dentine at both 25 μm and 50 μm indentations were significantly lower than the Vickers hardness of dentine of the remaining indentation sites (p < 0.05). In the same group, there was no significant difference in Vickers hardness of dentine between the 25 μm and 50 μm indentation sites (p > 0.05). Similarly, there was no significant difference in the Vickers hardness of dentine at the 75 μm, 100 μm and 150 μm indentation points (p > 0.05). The Vickers hardness of dentine in both Carisolv and Papacarie groups at the 25 μm and 50 μm indentations was significantly lower (p < 0.05) than at the 75 μm and 100 μm indentation sites (Table 3). The lowest Vickers hardness of dentine was recorded at the 25 μm indentation level, followed by the 50 μm indentation level. However, there was no significant difference (p > 0.05) in hardness of dentine between either 75 μm or 100 μm indentation sites. In both chemomechanical caries excavation groups, the hardness of dentine at the 150 μm indentation sites was significantly greater than all other indentation sites (p < 0.05).

Application of caries detector dye following caries excavation

Chromatic scale representing the different levels of pink colour staining of the residual and the sound dentine after the application of caries detector dye is shown in Fig. 5. Both the sound dentine and the residual dentine following rotary caries excavation were lightly pink stained. Conversely, the residual dentine following chemomechanical caries excavation was stained deeply pink after the application of the dye.

Figure 5.

Chromatic scale showed the different levels of pink colour staining of sound and residual dentine after the application of caries detector dye. (a: sound dentine; b, c, d: residual dentine following caries excavation by rotary, Carisolv, and Papacarie respectively).

Discussion

The time taken for caries excavation and the number of gel applications of the chemomechanical caries removal agents were evaluated in the present study. It was found that the time taken for Papacarie excavation was almost identical to that required for the conventional rotary technique. However, the time required for caries excavation by the rotary method in the current study exceeded that of a previous laboratory study.[28] The reason for this was due to the additional step taken to use the caries detector dye, which increased the excavation time. The caries detector dye was used to guide and limit excavation to caries-infected dentine.

This study demonstrated that chemomechanical caries removal is not necessarily a time consuming process, in comparison with the conventional rotary method as concluded in previous studies.[28-31] The result of the present study has shown that the papain-based caries removal method had shorter excavation time compared to the NaOCl-based caries removal method. The current study also supports a previous laboratory study that reported the new Carisolv gel formula reduced the excavation time in moderate carious lesions from 11.6 minutes for the original gel to 9 minutes for the new gel.[17] However, the time taken by the new Carisolv gel was still longer than the conventional rotary method.[28] The #4 Carisolv non-cutting instrument was used in both chemomechanical caries excavation methods to standardize the caries excavation method because of the similarity of its tip to the blunt back of a conventional spoon excavator, which is recommended by Papacarie manufacturer.

Although the EDX elemental analysis is conservative and simple to study mineral composition within narrow areas of tissues, it is a theoretical method based on a mathematical methodology.[32] The mineral content of normal dentine has been estimated by EDX in previous studies.[33, 34] The calcium level was reported to be 33.9 wt%[35] and 27.1 wt%,[36] while the phosphorus level was 16.7 wt%[35] and 13 wt%.[36] In the current study EDX results of the control sound dentine group were in the same range as previously reported.[35, 36] The mineral content of dentine was unaffected by either chemomechanical caries removal agents as demonstrated in previous studies.[24, 37, 38] Hence, the first hypothesis was accepted; there was no significant difference in the chemical composition of dentine following the three caries excavation techniques.

Chemical analysis using EDX method has some technical limitations. Firstly, some of the high penetration power electron probes may cause false positive results by detecting the underlying highly mineralized tissue. However, the beam used in the current study was not of a high penetration power, thus the accuracy of the analysis may reach 90–95%, depending on how flat the observed surface was. Secondly, the amount of the collected radiation is only around 1% of the total emitted X-ray.[39] To overcome this problem, the electron dose could be increased; however, this might consequently cause additional radiation damage to some sensitive biological specimens.[39] Although the EDX analysis was performed in 20 KV following the standard electron voltage range (10–20 KV), the effect of radiation damage may have had an influence on the results.

The SEM observations showed that using papain-based (Papacarie) chemomechanical caries removal agent resulted in a dentine surface characterized by absence of a smear layer, leaving patent dentinal tubules. The absence of the smear layer may be explained by the proteolytic nature of the papain gel, which removes the surface caries-affected tissue (denaturated collagen).[20, 40] This smear layer-free dentine surface may enhance bonding by facilitating the infiltration of adhesive resin into intertubular dentine and patent dentinal tubules. Moreover, the roughened dentine surface associated with chemomechanical caries removal methods may also be considered as potentially enhancing the adhesion of restorative materials due to the presence of micro-irregularities, which increase the surface area for bonding.[19, 36] The SEM observations agreed with previous studies showing that the dentine following rotary caries removal was smooth, flat and covered with a smear layer that occluded the dentinal tubules.[33, 41] Although the SEM observations of the Carisolv group agreed with the majority of studies,[19, 27, 33, 36, 41] it disagreed with that of Hossain et al.,[36] who claimed that Carisolv treatment could totally remove the smear layer leaving patent dentinal tubules. They explained the presence of an amorphous layer that obliterated the dentinal tubules resulted from the crushing and burnishing of excavated tissue caused by the Carisolv applicator tip on the surface of the dentine. However, this explanation was not supported by the current study, since the applicator tip had been used in both the Carisolv and Papacarie groups, while a smear layer was only observed in the Carisolv group. Thus, the second hypothesis was rejected as there was a significant difference in the morphology of dentine following the three caries excavation techniques.

The microhardness test showed that residual dentine following chemomechanical caries removal was softer resulting in a significant difference in hardness, when compared with residual dentine following rotary caries removal. The third hypothesis was therefore rejected. The decrease in hardness observed in the Carisolv and Papacarie groups may be explained by the selective removal of caries-infected dentine only, leaving behind the caries-affected dentine, which has lower hardness values.[26, 42] The current study agreed with the findings of Magalhaes et al.,[25] who reported that the microhardeness of residual dentine following conventional caries removal was higher than residual dentine after chemomechanical caries removal.

The results of the microhardness test showed that sound dentine hardness did not change among the different indentation points of dentine cut surface. Similarly, the hardness of the residual dentine following rotary caries removal exhibited minor changes among the different indentation points from the floor of the excavated surface. This seems to indicate that excavation with the conventional rotary method removed both caries-infected and caries-affected dentine and finished in sound dentine. This finding was supported by the chromatic scale outcome which showed that the dentine surface stained a pale pink after application of caries detector dye following rotary caries removal (Fig. 5). This also confirmed previous work, which reported that the use of caries detector dye tends to lead to over-preparation of cavities because of the non-specificity of the dye to damaged collagen fibres of the infected dentine and resulted in staining of demineralized caries-affected dentine.[34, 43, 44]

However, in both the Carisolv and Papacarie groups, dentine microhardness gradually decreased towards the cavity excavation edge (region of caries-affected dentine), indicating chemomechanical excavation preserves the caries-affected dentine.[45] Finally, the concept of leaving caries-affected dentine following caries excavation is acceptable nowadays due to the evidence of a high degree of collagen exposure and enhanced hybridization with adhesive resins,[46] but with certain concerns, that the remaining dentine must be able to support the restorative material overlying the excavated caries lesion.[47]

Conclusions

The results of the present study showed the papain-based (Papacarie) chemomechanical caries removal method to be a suitable and conservative alternative to conventional rotary excavation of carious tissue. This enzymatic chemomechanical caries removal method provided shorter excavation time and potentially enhanced morphological features of residual dentine for subsequent bonding in comparison to the sodium hypochlorite chemomechanical caries removal technique.

Acknowledgements

The authors acknowledge Formula and Acao Company, Brazil for supporting the study with Papacarie™ gel and Kuraray Medical Inc., Tokyo, Japan for providing the caries detector dye. The authors are also would like to express their gratitude to Ms Amy Wong and Mr Frankie Chan, Electron Microscope Unit, Queen Mary Hospital, University of Hong Kong for their technical support in the use of SEM.

Ancillary