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Year : 2016  |  Volume : 19  |  Issue : 3  |  Page : 274-279
Histological response of human pulps capped with calcium hydroxide and a self-etch adhesive containing an antibacterial component

1 Department of Conservative Dentistry and Endodontics, MES Dental College and Hospital, Perinthalmanna, Kerala, India
2 Department of Conservative Dentistry and Endodontics, Bharati Vidyapeeth Dental College and Hospital, Sangli, Maharashtra, India
3 Department of Conservative Dentistry and Endodontics, P.M. Nadagouda Memorial Dental College, Bagalkot, Karnataka, India

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Date of Submission29-Dec-2015
Date of Decision14-Feb-2016
Date of Acceptance19-Apr-2016
Date of Web Publication9-May-2016


Aim: To compare human pulp tissue response following direct pulp capping with calcium hydroxide and a self-etch adhesive containing antibacterial component.
Materials and Methods: Sixty-six erupted sound premolars scheduled to be extracted for orthodontic reasons were selected from 17 human subjects. Pulp exposures were made. Direct pulp capping was then performed using calcium hydroxide and a self-etch adhesive containing antibacterial component in its primer. The teeth were then restored with composite resin. Two teeth were maintained intact as a control group. After 7 and 30 days, teeth were extracted and processed for light microscopic examination using a histological scoring system. The teeth were divided into four groups (N = 16) according to the pulp capping materials used and their days of extraction. The results were then statistically analyzed by Mann-Whitney U-test.
Results: After the 7-day observation period, the inflammatory reaction to the self-etch adhesive containing antibacterial component group was significantly less severe than that in the calcium hydroxide group (P < 0.05). After the 30-day observation period, the inflammatory reaction was slight in both groups, but specimens with dentin bridge formation in the self-etch adhesive group were significantly less common than those in the calcium hydroxide group (P < 0.05).

Keywords: Calcium hydroxide; CLEARFIL PROTECT BOND; direct pulp capping; human pulp

How to cite this article:
Parthasarathy A, Kamat SB, Kamat M, Kidiyoor KH. Histological response of human pulps capped with calcium hydroxide and a self-etch adhesive containing an antibacterial component. J Conserv Dent 2016;19:274-9

How to cite this URL:
Parthasarathy A, Kamat SB, Kamat M, Kidiyoor KH. Histological response of human pulps capped with calcium hydroxide and a self-etch adhesive containing an antibacterial component. J Conserv Dent [serial online] 2016 [cited 2022 Dec 9];19:274-9. Available from:

   Introduction Top

Direct pulp capping is a form of vital pulp therapy indicated in selected cases to preserve and protect the vitality of the pulp. [1]

Pulp alterations under different restorative materials were related to the bacterial microleakage. [2] The failure of the pulp-capping treatment has been attributed to the bacterial contamination through microleakage rather than to the irritant properties of the capping materials. [3] The formation of dentin bridge is actually an intrinsic response of the exposed the pulp in the absence of bacteria. [4] Hence, pulp healing could be expected if the material seals the cavity against the microleakage thereby preventing the entry of bacteria. This prevention of microleakage thereby aids in the formation of a healthy dentinal bridge.

Calcium hydroxide has been the most widely used pulp-capping agent as a result of its ability to induce dentinal bridge and its bactericidal effect. But owing to its disadvantages, [5] a variety of alternatives have been proposed for direct pulp capping and one among them is the self-etch adhesive. Self-etching products have shown good performance in inhibiting gap formation and preventing microleakage. [6] Moreover, according to the hydrodynamic theory, there is a possibility that no pulp irritation will result in as there will be no movement of dentinal fluid. [7] The recent addition to it is the incorporation of an antibacterial component [12-methacryloyloxydodecylpyridinium bromide (12-MDPB)] in its primer that has considerable antibacterial effects, which make them promising as a direct pulp-capping agent. The inhibitory effects of MDPB on the proliferation and mineralization were lower than those of bisphenol A-glycidyl methacrylate (Bis GMA). MDPB has superior biocompatibility compared to BIS-GMA in terms of hard tissue formation by odontoblastic cells, suggesting its possible less negative influences on dentinogenesis. [8]

This study was designed to evaluate the histological features of human pulp tissue following the application of a self-etch adhesive containing antibacterial component and calcium hydroxide on exposed pulp of sound human teeth and also to assess the feasibility of this self-etch adhesive as a direct pulp-capping agent.

   Materials and methods Top

Sixty-six fully erupted sound premolars scheduled to be extracted for orthodontic reasons were selected from 17 healthy subjects in the age range of 15-25 years. The research protocol was designed and the permission for conducting the study was obtained from the ethical committee and the head of the institution. The subjects were thoroughly explained about the experimental rationale, clinical procedures, and possible risks, and then the informed consent was obtained.

Each tooth was examined radiographically for proximal caries and periapical lesion. Pulp sensibility testing was performed before the experimental procedure and again just before the extraction. After adequate local anesthesia and cotton roll isolation class I cavity preparations, approximately 3-mm long, 3-mm wide, and 3.5-mm deep, were prepared in the occlusal surface using a straight fissure bur (FG-57; SSW, Lakewood, CO, USA) at high speed under copious water/spray coolant. The cavity walls were vigorously washed and cleaned with sterile distilled water. Then, pulp exposure approximately 1 mm in diameter was performed gently in the middle of the cavity using diamond (FG-801) at a high speed without penetrating into the pulp space. Hemorrhage was arrested with physiological saline in sterile cotton pellets.

Thirty-two teeth from eight subjects were then capped with a self-etch adhesive containing antibacterial component (12-MDPB) in its primer (CLEARFIL PROTECT BOND, Kuraray, Japan) and thirty-two teeth from another eight subjects were then pulp capped with calcium hydroxide (DYCAL, Dentsply, USA). Two teeth from the 17 subjects were treated as control.

Pulp-capping procedures

Self etch adhesive group {CPB (CLEARFIL PROTECT BOND)}

The self-etch adhesive's primer containing the antibacterial component (12-MDPB) was brushed to the pulpal wound, dentin, and enamel for 20 s and gently dried with air. Then the bonding agent was applied to the exposed pulp and cavity walls, and light-cured for 10 s.

Calcium hydroxide group {CH (DYCAL)}

The base and catalyst of calcium hydroxide were mixed according to the manufacturer's instructions and then applied directly to the exposed pulps leaving the cavity walls uncovered as much as possible.

All the cavities were restored with composite resin in 2 mm increments (Clearfil AP-X, Kuraray, Japan) using exponential ramped up curing technique and the restorations were finished and polished.

On the 7 th and 30 th day, after direct pulp-capping teeth were extracted under local anesthesia.

The grouping of teeth was done, according to the pulp-capping material used and the days of extraction, with a sample size of 16 in each group (N = 16) which is as follows:

Immediately after the extraction, a small hole was made near the cementoenamel junction (CEJ) to facilitate the fixative to easily enter and fix the pulp tissue. The extracted teeth were then fixed in a buffered 10% formalin solution for 24 h, decalcified in a 2% mixture of hydrochloric acid and nitric acid for 6 days, and dehydrated in aqueous ethanol. Six-micron thick serial buccolingual sections were cut longitudinally through the center of the exposure site. The sections were then subjected to hematoxylin and eosin staining and gram-staining techniques. For all the sections, four histological features were evaluated according to the criteria listed in [Table 1] [Table 2] [Table 3] [Table 4], [8],[9] and the results were tabulated as shown in [Table 5]. Histological scoring criteria were based on COX et al.[9] and Akimoto et al. [10]
Table 1: Inflammatory cell response

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Table 2: Tissue damage Grade Characterization

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Table 3: Hard-tissue formation

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Table 4: Stained bacteria

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Table 5: Results

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   Results Top

Seven-day observation period

CPB 7 - group 1

Fourteen specimens (87%) of this group showed only slight inflammation. Only two specimens (13%) had moderate inflammation with neutrophils infiltration at the exposure site. Dilated blood vessels were seen with no signs of bacteria or reparative dentin [Figure 1].
Figure 1: (Group 1) Black arrow - Minimal inflammatory reaction seen below the exposed area. Beneath this inflammatory cell infiltration and hemorrhagic area, pulpal tissue is normally composed of mesenchymal cells and blood capillaries. Blue arrow - Normal pulp tissue

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CH 7 - group 2

Ten specimens (62%) showed moderate inflammation characterized by neutrophils and mononuclear cell infiltration at the exposure site while only four specimens (25%) showed slight inflammation [Figure 2]. Two specific specimens (13%) showed severe inflammatory reaction with necrosis. No signs of reparative dentin were seen. Some disorganization of pulpal tissue along with some kind of response was found in almost all the specimens and there were no signs of bacteria.
Figure 2: (Group 2) Red arrow - Black, discolored foreign material seen superficially (calcium hydroxide). Black arrow - Inflammatory reaction with necrosis

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After 7 days, group 1 showed significantly lesser inflammation than group 2 (P < 0.05).

Thirty-day observation period

CPB 30 - group 3

Fourteen specimens (87%) were devoid of any signs of reparative dentin or any kind of barrier. But two specimens (13%) showed a hard tissue deposition that was not very clear to be differentiated as reparative dentin [Figure 3]. Two particular specimens showed a moderate amount of inflammation with some kind of foreign material into the pulp space while others showed only a slight inflammation. There were no traces of bacteria.
Figure 3: (Group 3) Black arrow - No indication of any hard-tissue bridge formation below the exposed area. Green arrow-pulp tissue with very minimal inflammatory cells and a few engorged vessels

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CH 30 - group 4

Ten specimens (63%) showed a calcified dentinal matrix below the exposure site and two specimens showed a proper hard-tissue deposition below the exposure site [Figure 4]. All the specimens showed a slight inflammation with edematous vessels. Four of the specimens exhibited bacteria [Figure 5].

Group 3 exhibited significantly lesser reparative dentin formation than group 4 (P < 0.05). It is also important to note that group 4 exhibited bacteria in four of their specimens.
Figure 4: (Group 4) Black arrow - Restorative material (calcium hydroxide). Blue arrow - Arrow mark showing below the black, discolored material appears as dentin. Below this dentin bridge pulpal tissue is normal and free of inflammatory cell infiltration

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Figure 5: Gram-stained section indicates the presence of bacteria

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   Discussion Top

The study evaluated and compared the efficacy of a bonding agent containing 12-MDPB as a pulp-capping agent.

The failure of pulp capping was believed to be due to the toxic effects from the dental materials placed over it. [11] However, studies later proved that restorative materials previously reported as toxic do not cause pulp inflammation or pulp necrosis when placed directly on the exposed pulp, if bacteria were sealed off at the margins. [12]

Cytotoxicity evaluation of the antibacterial adhesive (CLEARFIL PROTECT BOND) on established cell lines also proved that the cell survival (fibroblast) percentage ranged between 66% and 97% and the toxicity of this particular antibacterial adhesive with 12-MDPB is comparatively lesser than the other generations of bonding agents. [13]

MDPB has superior biocompatibility than BIS-GMA in terms of hard-tissue formation by odontoblastic cells, and possible less negative influences on dentinogenesis. [8]

In our study, most of the pulps of the self-etch adhesive group demonstrated only slight inflammation, thus demonstrating the fact that the toxic effect of this antibacterial adhesive is moderate and acceptable that correlates with the findings of Nishida et al. [8] Calcium hydroxide, on the other hand, demonstrated necrosis initially (7 days), which has been shown to be related to its high pH. [14]

The success of direct pulp-capping material depends upon two main factors: Tissue repair and marginal sealing effect. The tissue repair representing odontoblast-fibroblast differentiation and a hard-tissue deposition were actually dependent on a healthy pulp tissue and not on the pulp-capping material. [15] Impaired tissue repair has been a concern with any other generation of bonding agent as the unpolymerized resin particles close to the pulp gets displaced into them triggering a foreign body response characterized by inflammatory cell infiltration as well as the appearance of giant cells. In our study, only two specimens of the self-etch adhesive containing antibacterial component group exhibited such a resin displaced inflammatory reaction while others exhibited slight inflammation. It indicates that this self-etch adhesive did not release many particles into the pulp and thereby the pulp was not adversely affected. The observed pulpal histological features could be attributed to the adhesion characteristics of the self-etch adhesive systems. [15] In these systems, the acidic primer infiltrates the collagen fibers as it simultaneously decalcifies the inorganic component to the same depth, and the acidic primers are neutralized by inorganic components of dentin, such as calcium or phosphate, during mineralization. This self-limiting mechanism leads to limited depth dissolution, so the innermost part of the hybrid layer is completely polymerized, leaving a limited amount of unpolymerized monomer. Moreover, the pH of the self-etch adhesive is generally above 2, which may also be the reason for lesser irritation to the pulp than the other generations of adhesive systems. Also a good seal is achieved because the smear plugs are left intact.

Marginal sealing also plays an important role in determining the final outcome of direct pulp capping. [16] The pulp could form some hard-tissue barrier if a proper biological seal was provided so that bacteria did not gain access to the wound. [17] Many studies in the recent past have proved the satisfactory biological seal and the antibacterial property of the adhesive system containing 12-MDPB. [18] In our study, none of the specimens of the self-etch adhesive group demonstrated bacteria in the bacterial staining, which substantiated the sealing efficacy and the antibacterial property. In the calcium hydroxide group, bacteria were observed in two specimens, which may cause its poor sealing efficacy. [19] Even though some of the bacteria would have been lost during the histological procedures, the bacterial staining method adopted in our study has been accepted as a valuable tool for histological assessment of the bacteria. [20]

Only two specimens of the antibacterial self-etch adhesive group demonstrated a hard tissue like formation while almost all the specimens of calcium hydroxide group demonstrated a hard tissue like formation. Considering the fact that the resin-dentin bonds undergo degradation in vivo over time, [21] the risk of pulp infection may be greater without a hard-tissue barrier than in the case of hard-tissue bridge.

In case of self-etch adhesives, the formation of a properly hybridized dentin-adhesive interface has been considered to seal both dentin and pulp effectively, allowing complete tissue healing and tertiary dentin formation. [17],[22],[23] Nevertheless, other contradictory experimental data [24],[25] showed that dentin adhesives interrupt the potential of pulpal cells to express their dentinogenic activity. The results of the present study are in accordance with the study by Tziafas et al., [26] which proved that although the adhesive system containing 12-MDPB has acceptable biocompatibility they interrupt the dentinogenesis process.

Our research has therefore opened up a new frontier in studies regarding direct pulp-capping materials by including an antibacterial component that is the most essential component of any pulp-capping therapy. Hence, to determine whether the self-etch adhesive containing antibacterial component can be used for direct pulp capping requires further studies with a longer observation period and larger sample size including carious tooth.

   Conclusions Top

Within the limitations of the study, we can conclude that although CLEARFIL PROTECT BOND produced minimal inflammation, their ability to induce reparative dentin is lesser than that of calcium hydroxide.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

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Correspondence Address:
Ambalavanan Parthasarathy
MES Dental College and Hospital, Malaparamba, Perinthalamanna, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-0707.181947

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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