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Comparative evaluation of effects of chemo-mechanical and conventional caries removal on dentinal morphology and its bonding characteristics--an SEM study.

Introduction

Dental caries is an irreversible microbial disease of the calcified tissues of the teeth, characterised by demineralisation of the inorganic portion and destruction of the organic substance of the tooth [Sivapathasundharam and Raghu, 2006]. The earliest attempt to remove caries involved the use of a hand drill, which was soon surpassed in 1871 by James Morison's treadle instrument. Since then, numerous drills have been developed to improve the efficacy of rotary instrumentation [Elkholany et al., 2002].

Despite a drill's proven efficacy in removing carious tissue, the conventional drilling technique presents negative experiences to the patient, such as tooth vibration, noise, heat and pressure on the pulp, dentine sensitivity, the possibility of overextending the cavity and healthy tissue removal. These factors trigger reaction of pain and discomfort, and the method usually requires local analgesia [Correa et al., 2007a; Carrillo et al., 2008].

Hence, a painless technique is one of the keys to avoid dentally fearful and uncooperative patients, and a skill every paediatric dentist should strive to master [Kotb et al., 2009]. With this aim, the concept of chemo-mechanical caries removal (CMCR) using 5% sodium hypochlorite (NaOCl) was introduced to remove carious tissues but was found to be unstable, toxic and aggressive to adjacent healthy tissues [Bussadori et al., 2005]. Subsequently, the Sorensen solution was developed which involved chlorination of glycine to form N-monochloroglycine (NMG) [Martins et al., 2009]. In subsequent studies, it was found that the system was more effective if glycine was replaced by amino butyric acid; the product then being N-monochloroaminobutyric acid (NMAB) also designated GK101E [Beeley et al., 2000] and commercially known as Caridex [Ganesh and Parikh, 2011]. Its efficacy in caries removal, however, required improvement as its hand-held instrument was not effective, it required a large reservoir with a pump and considerable quantities of solution. Furthermore, the product was overly expensive and had a short shelf-life [Martins et al., 2009].

However, Mediteam in Sweden continued to work on the system and the CMCR reagent known as Carisolv[TM] was developed in January 1998. Carisolv[TM] was not very popular among dental professionals mainly because it required (i) extensive training and registration of professionals and (ii) customised instruments which increased the costs.

In 2003, a Brazilian formulation for chemo-mechanical caries removal was introduced onto the market under the brand name Papacarie[R] (Formula and Acao, Sao Paulo SP, Brazil). It is a gel-like material based on papain, a proteolytic cysteine enzyme that has antibacterial and anti-inflammatory properties. In addition, it contains chloramines and toluidine blue [Bussadori et al., 2005]. In 2005, Dammaschke et al. hypothesised that calcium hydroxide [Ca[(OH).sub.2]] might be effective in chemo-mechanical caries removal due to its high pH and anti-microbial activity thus making calcium hydroxide (CH) a possible chemo-mechanical caries removal agent [Dammaschke et al.,2005].

Any method used for caries removal will yield a different pattern of dentine substrate [Correa et al., 2007b]. It has been reported that chemo-mechanically treated dentine has a higher surface energy than conventionally treated dentine. This implies that the chemo-mechanically treated dentine may have greater affinity for adhesive materials and better bonding than conventionally treated dentine [Hosoya et al., 2001]. A stable adhesion between composite resin and dental structure is fundamental to the clinical success of restorations because an adhesion failure allows infiltration of bacteria and oral fluids that may lead to the development of secondary carious lesions [Correa et al., 2007b].

Considering the factors noted above, and the scarcity and significance of literature on newer chemo-mechanical agents, the present study was undertaken. The objectives were to evaluate the surface morphology of the remaining dentine after caries removal with the conventional method of using drills and a chemo-mechanical method using Papacarie[R] and CH and further comparing the resin tags formed at the resin-dentine interface.

Materials and methods

Extracted human permanent molars (45), with large and visible carious cavities involving the occlusal surface and extending into dentine, were used as samples in this institutionally-approved study. All the teeth were cleaned with prophylactic instruments and stored in distilled water until use. For the study the samples were divided into two basic groups:-

GROUP 1: consisted of 15 samples prepared for ultramorphological evaluation of residual dentine after caries removal.

GROUP 2: consisted of 30 samples prepared for evaluating the bonding characteristics of residual dentine by analysing the resin tags formed at the resin-dentine interface.

Both the groups were further sub-divided into three subgroups containing an equal number of specimens. In subgroup A, carious tissue removal was performed under coolant with a round, diamond bur (BR-31, Adenta), having a size of diamond grit ranging between 106-125 [micro] and attached to a high speed handpiece. In subgroup B, carious tissue removal was performed using freshly mixed CH and saline. The paste was allowed to remain in the cavity for 30-40 seconds and the softened carious tissue was removed with a blunt excavator. The procedure was repeated 4-5 times on average until no further softened carious tissue could be obtained. The cavity was then wiped with a cotton-pellet moistened with water and rinsed. The complete procedure of caries excavation required a mean time of 6-8 minutes. In subgroup C, carious tissue removal was performed using Papacarie[R]. The product was applied and left in the cavity for 30-40 seconds. The softened carious dentine was then scraped using a blunt excavator. The procedure was repeated 3-4 times on average until there was no clouding of the gel. The gel was then removed and the cavity was wiped with a moistened cotton pellet and rinsed. The complete procedure of caries excavation required a mean time of 6-8 minutes. The caries-free appearance of the cavities was further confirmed with an exploratory probe in all the samples.

After caries removal in the specimens of Group 1, the crowns were separated from the roots and split in the centre of the cavity to obtain the treated dentinal surfaces and embedded into acrylic resin with the dentinal surface facing upwards. After caries removal in the specimens of Group 2, the cavities of teeth were etched with 34% phosphoric acid for 15 seconds, followed by rinsing, drying, application of an adhesive (Prime & Bond NT, Dentsply) following the manufacturer's instructions and filling with composite resin (Ceram X, Nanoceramic, Dentsply) using increments that were light cured for 40 seconds each.

The crowns were separated from the roots and sectioned vertically through the resin fillings and dentine with the help of a diamond disc under running water into two halves (mesial and distal) to expose the resin-dentine interface. They were then embedded in self-cure acrylic resin and polished with 600, 1,000, 2,000 grit polishing paper and Soflex finishing and polishing systems and further immersed in 4% sodium hypochlorite for 20 mins, followed by 20% hydrochloric acid for 30 seconds. The specimens of both groups were rinsed with distilled water and sequentially dehydrated in 60%,70%, 80%, 90% alcohol for 20 mins each and in 100% alcohol for 1hr.

Sample preparation for SEM viewing

The samples to be observed for surface morphology and dentine-resin interface were dried, mounted on aluminium stubs, placed in a vacuum chamber, sputter-coated and observed under a scanning electron microscope. A series of micro-photographs were taken at a magnification of 2,000x and 5000x for viewing the surface morphology and 1,000x for viewing the resin-dentine interface. Resin tags were evaluated quantitatively by measuring the length of the resin tags according to a scale given on the photographs. Five different measurements were performed on each photograph and the mean was taken as the representative value for that specimen.

For qualitative evaluation of the tags, a four- step (0-3) scale method according to Ferrari et al. [Ferrari et al., 2002] was used for evaluation of resin-dentine interfaces.

Score 0--no resin tag formation

Score 1--few and short resin tags

Score 2--when long resin tags were visible

Score 3--dense resin tags with numerous lateral branches.

The evaluation was performed and the results subjected to statistical analysis using SPSS software version 15.

Results

The dentine surfaces of samples in subgroup I showed a well formed smear layer partially or completely occluding the dentinal tubules. Tracks formed due to use of burs were evident (Figure 1). The resin-dentine interface revealed a thick hybridised complex with long tags (43.14-68.28 um) which were entangled in some places (Figure 2).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

The dentine surfaces of a few samples in subgroup B showed a uniform, well-formed smear layer while those of other teeth showed an amorphous layer with accumulation of debris. The dentinal tubules were almost completely occluded or narrowed in diameter due to the smear layer and accumulated debris (Figure 3). At most locations on the resin-dentine inter-diffusion zone, the tags were short or even absent with the mean length ranging from 4.00-38.86 [micro]m. At a few locations, the tags were greatly entangled but not in continuity with the dentinal tubules. No microtags were evident (Figure 4).

The dentine surfaces of few samples in subgroup C exhibited different patterns of residual dentine. At some places, a regular and cracked surface with little smear layer was observed with presence of bacteria while at other places a rough and irregular surface was observed. The dentinal tubules were open in most locations due to a minimal smear layer (Figure 5). At the resin-dentine interface, treatment with Papacarie[R] yielded a thick hybridised complex with very long and thick tags extending almost through the entire resin-dentine inter-diffusion zone and mean tag length ranging from 49.4 to 112.0 [micro]m. Short microtags or lateral branches were evident (Figure 6).

Comparison of mean tag length in the groups (Table 1) revealed the mean tag length to be a minimum in subgroup B (19.90 [+ or -] 13.49 [micro]m) and maximum in subgroup C (68.20 [+ or -] 18.11 [micro]m).

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

In subgroup A, the mean tag length was 53.88 [+ or -] 8.60 [micro]m. Between group comparisons of tag length (Tables 1 and 3) showed that subgroup A and C had significantly higher tag lengths as compared with subgroup B while the mean value of subgroup C was observed to be significantly higher compared to subgroup A. Qualitatively, using the scoring criteria of Ferrari et al. [2002] between-group comparisons showed that subgroup C had significantly higher scores as compared with subgroups A and B respectively. However, no significant difference was observed between subgroups A and B (Tables 2 and 3).

Discussion

With the development of new dental restorative materials, the management of dental caries has drastically evolved from GV Black's "Extension for prevention" to "Construction with conservation". This concept includes early detection of lesions, non-surgical interventions and a modified surgical approach that includes smaller tooth preparations, adhesive dental materials and repair rather than replacement of failing restorations. CMCR holds a lot of promise in achieving this objective [Ganesh and Parikh, 2011].

The smear layer produced in dentine affected by caries possesses acid-resistant crystals that may hinder the diffusion of primer into the intact underlying dentine. This layer acts as a barrier, decreasing the dentine's permeability and is also considered to be an impediment to the establishment of intimate contact between tooth and resin. Thus, the treatment of the dentinal surface and the resulting characteristics of dentinal substrates will be important for adhesion and will affect the performance of composite resin restorations. Adhesion of resins to dentine is considered to be mainly related upon micro-mechanical retention based on the formation of resin tags inside dentinal tubules, branching or microtags, and the formation of a hybrid layer or resin-dentine interdiffusion zone. Obtaining a hybrid layer involves applying an acid etch to dentine to remove the smear layer and smear plugs, opening up the dentinal tubules and increasing dentinal permeability as well as exposing the collagen network of inter-tubular dentine due to mineral removal [Correa et al., 2007b]. The results of this present study, in context with the morphology obtained after treatment with rotary instruments, are in accordance with earlier studies [Banerjee et al., 2000; Correa et al., 2007b; Peric and Markovic, 2007].

Papain has been suggested to act by exclusively by breaking down the partially degraded collagen molecules and contributing to the degradation and elimination of the fibrin "mantle" formed by the carious process without damaging intact collagen fibrils [Bussadori et al., 2005]. Since its introduction in 1920 by Hermann, CH has been widely used. It is a strong alkaline substance which dissociates into calcium and hydroxyl ions in aqueous medium. Various biological properties have been attributed to this substance, such as antimicrobial activity [Bystrom et al., 1985], tissue-dissolving ability [Hasselgren et al., 1988, Andersen et al. 1992], inhibition of tooth resorption [Tronstad, 1988], and induction of repair by hard tissue formation [Foreman and Barnes, 1990].

In 1980, Bergenholtz and Reit reported by Pashley et al., [1986] suggested that the topical application of CH reduced dentine permeability due to:

a) Physical blockage of the openings of the tubules by Ca[(OH).sub.2]

b) Production of intra-tubular mineralisations or precipitates

Dammaschke et al. [2005] observed that chemo-mechanical caries removal with CH showed only 50-60% caries-free specimens. The limitation of CH to dissolve soft tissue has been explained as follows: the solubility of Ca[(OH).sub.2] powder to water is very low (0.16g/100g of water at 20[degrees]C), and the number of dissociated hydroxyl ions is little, although the mixture of Ca[(OH).sub.2] to water exhibits a pH of 12 or higher. As soon as the Ca[(OH).sub.2] dissolves the soft tissues, the hydroxyl ions are consumed during the reaction.

The presence of more residual carious dentine or bacteria after treatment with chemo-mechanical methods has been observed in previous studies [Hosoya et al., 2001; Spleith et al., 2001; Yazici et al., 2003; Peric and Markovic, 2007]. This finding could be explained by less extensive preparation compared with the rotary instruments. The chemo-mechanical method removes caries, preserves sound dentine, but residual dentine is not completely bacteria-free. Yazici et al. [2003] explained the presence of bacteria in chemo-mechanically treated cavities, where the absence of a smear layer enabled direct pushing of bacteria into dentinal tubules with hand instruments [Spleith et al., 2001; Peric and Markovic, 2007].

The micro-cracks observed in the present study with Papacarie[R] were also observed in earlier studies [Banerjee et al., 2000; Correa et al, 2007a, b]. Banerjee suggested that these cracks may be the result of the hydrophilic nature of the gel causing dehydration of the dentine surface and their appearance might be accentuated by lack of a smear layer.

No study has yet been undertaken to evaluate the effects of CH for caries removal on residual dentine. However, its effectiveness in caries removal cannot be denied. One of the disadvantages of chemo-mechanical caries removal, that is the presence of bacteria in the remaining dentine, can be easily overcome by the high antimicrobial activity of CH. Apparently, there were no bacteria found in dentine surfaces treated with CH in our study. CH used in the present study was also mixed in an aqueous medium of normal saline and may not have been able to remove carious tissue completely. It is probable that mixing CH with a viscous water soluble substance or non-water soluble substance can help increase the duration of action of the paste as well as enhance its time of contact with vital tissues [Fava and Saunders, 1999].

The presence of open tubules in chemo-mechanical caries removal with other chemo-mechanical agents is attributed to the initial high pH of the gel and the mechanical preparation technique. The study by Tonami et al. [2003] revealed that treatment with chloramines, a component of Papacarie[R], resulted in the opening of dentinal tubules in the outer layer of carious dentine. Thus, it is suggested that several factors such as incomplete caries removal, inability to enter the deeper dentinal layers along with the absence of additives like chloramine are responsible for the obtained surface morphology of residual dentine treated with CH [Tonami et al., 2003].

Piva et al. [2008] observed similar micro-tensile bond strength results in mechanically excavated samples and papain-based gel treated samples when Prime & Bond NT was used after the different excavation methods while after use of a self-etch system reduced microtensile bond strengths to carious dentine were obtained. After the acid-etching and water rinsing steps for the conventional system, complete removal of the papain-based product from the tooth surface can be expected. On the other hand, as the self-etching system lacks the rinsing step and requires the use of a weak acid, the smear layer is not completely removed but only partially demineralised. Thus remnants of the gel could stagnate on dentine surfaces and hybrid layers may not be completely formed which potentially interferes with the bonding mechanism [Piva et al., 2008]. Therefore, considering the above mentioned studies, the type of adhesive used in the present study was Prime & Bond NT which is a 3-step adhesive.

In the present study, tags obtained after using Papacarie[R] were the longest and densest, as compared with the other two techniques. The probable reason for the observation is the presence of a minimal smear layer, open dentinal tubules and also the complete removal of the gel after acid etching and water rinsing. Pashley et al. [1986] observed that the treatment of acid-etched dentine surfaces with CH produced a statistically significant (p < 0.001) reduction in dentine permeability. In a macroscopic sense CH treatment did not provide a physical barrier that might interfere with dentine permeation but microscopically, the CH treatment did produce a slight reduction in the dimensions of the tubule orifices of acid-etched dentine. Thus, the relatively poor quality of tags obtained with CH can be attributed to the above discussed physical properties of CH and its effect on dentinal permeability. It is probable that etching the dentinal surface with a more aggressive solution, increasing the etching time or even adding a suitable additive to CH could have yielded better results.

The study by Correa et al. [2007b] revealed innumerable elongated tags and a small amount of microtags for all experimental groups after using rotary instruments, Carisolv[TM] and Papacarie[R] for caries removal. However, no definitive conclusion could be gained by their study as they did not use any quantitative or qualitative criteria for their assessment. However, the results of the present study are based on both, quantitative as well as qualitative criteria and thus show that chemo-mechanical caries removal using Papacarie[R] is better than the other two methods of use of rotary instruments and CH.

Conclusions

Chemo-mechanical caries removal using Papacarie[R] resulted in minimal smear layer formation with open dentinal tubules, yielded denser and more numerous tags than CH and rotary instruments and is an effective alternative to conventional techniques of caries removal. Though the use of CH resulted in occluded dentinal tubules, with suitable additives it may prove as a promising CMCR agent for the future.

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R. Arora, M. Goswami, S. Chaudhary, T.R. Chaitra, A. Kishor, M. Rallan Department of Paedodontics and Preventive Dentistry, Kothiwal Dental College and Research Centre, Mora Mustaqueem, Moradabad-244001, India.

Corresponding author: : Dr R. Arora, Department of Paedodontics and Preventive Dentistry, Kothiwal Dental College and Research Centre, Mora Mustaqueem, Moradabad-244001, Uttar Pradesh, India.

Email: rachita.arora1@gmail.com
Table 1. Comparison of mean tag length in different groups

                                         Mean              Mean
S.No.   Subgroup                 n    ([micro]m)    SD     Rank

1.      A [Rotary instruments]   10     53.88      8.60    17.75
2.      B [Calcium hydroxide]    10     19.90      13.49   5.50
3.      C [Papacarie[R]]         10     68.20      18.11   23.25

[chi square]=21.316 (df=2); p<0.001 (Kruskal-Wallis test)

Table 2. Frequency distribution of grade scores in
different groups (according to Ferrari et al., 2002)

        Subgroup I (n=10)     Subgroup II (n=10)  Subgroup III (n=10)
        [Rotary instruments]  [Ca[(OH).sub.2]]    [Papacarie[R]]
Grade
Score   No.        %          No.       %         No.        %

0       0          0          1         10.0      0          0
1       3          30.0       6         60.0      0          0
2       7          70.0       3         30.0      4          40.0
3       0          0          0         0         6          60.0

Table 3. Between group comparison of tag length and objective scoring

                                                         Objective
S.No   Comparison                      Tag length         Scoring

                                       "z"     "p"      "z"     "p"

1      Subgroup A vs B [Rotary         3.780   <0.001   1.834   0.105
       instruments vs.
       Ca[(OH).sub.2]]

2      Subgroup A vs C [Rotary         2.080   0.035    3.033   0.005
       instruments vs. Papacarie[R]]

3      Subgroup B vs C                 3.781   <0.001   3.496   <0.001
       [Ca[(OH).sub.2] vs.
       Papacarie[R]]

Mann-Whitney U test
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Title Annotation:scanning electron microscopy
Author:Arora, R.; Goswami, M.; Chaudhary, S.; Chaitra, T.R.; Kishor, A.; Rallan, M.
Publication:European Archives of Paediatric Dentistry
Article Type:Report
Geographic Code:9INDI
Date:Aug 1, 2012
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