Editorial Type:
Article Category: Research Article
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Online Publication Date: 01 Jul 2019

Clinical and Histological Study on Direct Pulp Capping With CO2 Laser Irradiation in Human Teeth

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Page Range: 336 – 347
DOI: 10.2341/18-030-C
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SUMMARY

The study aimed to histologically evaluate wound healing of exposed human pulp on direct pulp capping using super-pulsed CO2 laser preirradiation. In this single-blind clinical trial, 28 third molar teeth of 17 volunteers were randomly capped with either CO2 laser irradiation (n=14) or Dycal (calcium hydroxide cement; n=14) and restored using resin composite. The laser was operated in super-pulsed mode (pulse duration, 0.2 ms; interval, 5.8 ms; 0.003 J/pulse). The irradiation conditions were a power output of 0.5 W, an irradiation time of 15 seconds, repeat mode (10-ms irradiation and 10-ms intervals, for a total beam exposure time of 7.5 seconds), total applied energy of 3.75 J, and an activated air-cooling system. Each tooth was extracted at six or 12 months posttreatment and prepared for histological evaluation. We evaluated the parameters of pulp tissue disorganization, inflammatory cell infiltration, reparative dentin formation (RDF), and bacterial penetration. There were no significant differences between groups for all parameters at each postoperative period (Mann-Whitney U-test, p>0.05). CO2 laser irradiation completely controlled bleeding and exudate from the exposed pulp. The CO2 laser group had a tendency to delay RDF compared with the Dycal group, but 4 of 7 teeth from the CO2 laser group showed a complete dentin bridge at 12 months posttreatment.

INTRODUCTION

Preventing pulp devitalization helps extend the longevity of the tooth, especially with an immature tooth. Therefore, dentists try to avoid removing dental pulp. Direct pulp capping (DPC) is an effective method for treating accidentally exposed pulp that may occur during cavity preparation, traumatic injury, or removal of deep carious lesions in clinical practice. Numerous materials for DPC, including calcium hydroxide (CH) and mineral trioxide aggregate (MTA), have been developed, and their effectiveness and biocompatibility have been reported.1-3

On the other hand, managing exposed pulp surfaces is a common issue and one of the most important factors in the clinical success of DPC or pulpotomy notwithstanding the type of pulp-capping materials used.2,3 Accordingly, some studies have focused on the method of hemorrhage control and disinfection at the site of exposed pulp before a capping material is dressed.4-7 Sodium hypochlorite (NaClO) at different concentrations is an effective and well-known surface-acting solvent for vital pulp tissue.4,8-11 However, dentists sometimes encounter rebleeding and exudate from exposed pulp tissue while placing capping material or applying restorative materials, even after achieving hemostasis with NaClO. Hafez and others10 reported that application of 3% NaClO was effective for hemorrhage control, but renewed bleeding sometimes occurred at the margin between the exposed pulp and dentin after etching or priming. They suggested that the renewed bleeding may be responsible for the initial inflammation. Kitasako and others12 histologically observed pulp tissue protrusion into the cavity at the periphery of the exposed area via exudate from pulp tissue after chemical lavage despite a well-controlled procedure, small mechanical exposure, healthy pulp, and local anesthesia. If bleeding reoccurs at the exposed area of the pulp after applying the DPC material, it is clinically complicated to remove the capping material and then stem the bleeding again.

A number of studies in dentistry have been conducted using a laser in pulp-capping experiments.13-17 Wavelength is a highly influential factor for obtaining adequate results from laser treatment because of the different characteristics of laser beams (ie, a laser beam is selected according to its intended purpose). A CO2 laser emits infrared light with a wavelength of 10.6 μm, which is efficiently absorbed by water in vital tissue. CO2 laser energy causes the water to vaporize and certain morphological changes to occur, including carbonization, hemocoagulation, and protein denaturation in the target tissue.18 CO2 laser is often used as a substitute for a steel scalpel when cutting soft tissue, as it achieves excellent hemostasis as a result of its thermal action.19 Several studies showed increased success of DPC and pulpotomy assisted by CO2 laser irradiation compared with the conventional method.20-23 Our laboratory examined the rat pulp response to irradiation with a super-pulsed CO2 laser to achieve hemostasis and disinfection of the exposed pulp prior to applying a capping material.24,25 CO2 laser irradiation was effective for field control and controlling hemorrhage from the exposed pulp without severe inflammation. But the specimen from the CO2 laser group demonstrated an irregular fibrous dentin matrix near the denatured and carbonized tissue and a tendency to delay dentin bridge formation compared with applying CH cement or an adhesive resin system without laser irradiation.

The present study aimed to histologically evaluate wound healing of exposed human pulp after DPC using a self-etch adhesive system and CO2 laser preirradiation compared with a commercially available CH cement (Dycal, Dentsply Caulk, Milford, DE, USA). The null hypothesis of this study was that CO2 laser irradiation would not affect pulpal healing and reparative dentin formation (RDF) in exposed human pulp.

METHODS AND MATERIALS

This single-blind clinical trial was evaluated and approved by the Ethics Committee of the Nippon Dental University School of Life Dentistry at Niigata (receipt and permission number: ECNG-H-102). Prior to enrollment in the trial, all volunteers were informed of the benefits and risks and clearly instructed on reporting their pain history as well as the influence of the treatment that they would receive. Thereafter, informed consent forms were signed by all volunteers. One operator was responsible for all of the procedures in the dental clinic of the Nippon Dental University Niigata Hospital. This study was conducted in full accordance with the World Medical Association Declaration of Helsinki.

One or two teeth per volunteer were included in the study. Teeth were randomly assigned to the CO2 laser group or the Dycal group. A simple randomization method was used for selection of volunteers who provided two teeth. Namely, the first tooth was assigned to be treated with CO2 laser irradiation and the second tooth was to be treated with Dycal. A total of 28 mature permanent third molar teeth scheduled to be extracted from 17 volunteers were randomly capped with either CO2 laser irradiation (n=14) or Dycal (n=14) and restored using resin composite (Figure 1). None of the tested teeth had any caries or history of pulpitis. The volunteers included 15 women and two men ranging in age from 18 to 33 years. Mean age ± standard deviation at the time of treatment was 21.8 ± 3.45 years.

Figure 1. . Diagram showing the flow of volunteers.Figure 1. . Diagram showing the flow of volunteers.Figure 1. . Diagram showing the flow of volunteers.
Figure 1 Diagram showing the flow of volunteers.

Citation: Operative Dentistry 44, 4; 10.2341/18-030-C

CO2 Laser

An Opelaser PRO (Lot No. PG-049, Yoshida Dental Mfg Co, Tokyo, Japan) was used for CO2 laser irradiation. The specifications of this laser were wavelength of 10.6 μm, power output of 0.5 to 5 W (changeable at 0.1-W increments), focus beam diameter of 0.15 mm (at a distance of 10 mm from the tip of the handpiece), and green guide beam with a wavelength of 532 nm. The laser was operated in super-pulsed mode (pulse duration: 0.2 ms; interval: 5.8 ms; 0.003 J/pulse). Irradiation conditions included a power output of 0.5 W, an irradiation time of 15 seconds, repeat mode (10-ms irradiation and 10-ms intervals, for a total beam exposure time of 7.5 seconds), a beam diameter of 0.15 to 1.09 mm (at approximate distances of 10-20 mm from the exposed pulp surface), energy density of 0.32 to 16.99 J/cm2 per pulse, total applied energy of 3.75 J, and an activated air-cooling system. The laser handpiece was kept moving in a small circular motion during laser irradiation to avoid heat concentration in one area.

Clinical Procedure

All teeth were examined by preoperative clinical tests using a pulp vitality tester (Vitality scanner model 2006, Lot No. 26-10027, Kerr Co, Danbury, CT, USA) and radiographs to confirm the normal condition of pulp and periodontal and periapical tissues. The teeth were polished with a cleaning brush and isolated with a rubber dam or cotton rolls after local anesthesia with 2% lidocaine containing epinephrine bitartrate (ORA Injection Dental Cartridge, Lot No. 24730, Showa Yakuhin Kako Co, Tokyo, Japan). The operating field was cleaned with 0.025% benzalkonium chloride, rinsed with water spray, and disinfected with diluted iodine tincture. Class 1 cavities were prepared on the occlusal surface using an FG No. 440 regular cut diamond point (ISO 015, Shofu Inc, Kyoto, Japan) in a high-speed handpiece under copious amounts of water spray. The pulp was then exposed with a CA No. 3 steel round bur (ISO 012, Shofu Inc) in a low-speed handpiece under distilled water. A new bur was used for each tooth. Alternate irrigation with 6% NaClO (Purelox, Lot No. 4053, Oyalox Co, Tokyo, Japan) and 3% hydrogen peroxide (H2O2, Oxydol, Lot No. FUA0033, Daiichi Sankyo Co, Tokyo, Japan) were performed several times to remove operative debris, including dentin chips, and achieve hemostasis. The cavity was then rinsed with normal saline. The excess water was removed using sterilized, small cotton pellets, and the cavity was gently air dried. In the CO2 laser group (n=14), the exposed pulp was irradiated with the CO2 laser under the specified conditions and directly capped with a two-step self-etching adhesive system, Clearfil SE Bond (SE, Kuraray Noritake Dental Inc, Tokyo, Japan; Figure 2). In the Dycal group (n=14), the exposed pulp was directly capped with Dycal, and SE was applied to the cavity. SE comprised two components, whereby the primer was applied, left for 20 seconds, and gently air dried, and the bonding agent was applied and then photopolymerized for 10 seconds at 1000 mW/cm2 with a light-emitting diode light-curing unit (Pencure 2000, Lot No. AF0051, J. Morita Mfg Co, Kyoto, Japan). Light intensity was measured using a curing radiometer (Optilux radiometer model 100, Lot No. 144060, KaVo Dental Systems Japan Co, Tokyo, Japan). Clearfil Majesty LV, a flowable resin composite (Kuraray Noritake Dental Inc), was filled into the cavity floor and photopolymerized for 40 seconds following direct capping and bonding procedures. Clearfil Majesty, a hybrid restorative resin composite (Kuraray Noritake Dental Inc), was added to the cavity using a three- or four-increment filling technique and photopolymerized for 40 seconds for each filling. The composition of the materials used in this study is shown in Table 1. All materials were applied according to the manufacturers' instructions. Excess material and occlusal contact were removed using a superfine diamond point in a high-speed handpiece under copious water spray. Restorations were polished using a silicon point for resin composite one week postoperatively, and all teeth were clinically examined at one week and one-, three-, six-, and 12-month intervals. The volunteers were asked about their experiences regarding symptoms and signs including postoperative sensitivity and pain, and pulp and periapical responses to the electric pulp test, percussion, palpation, and mobility were examined.

Figure 2. . Clinical photographs of the CO2 laser group (specimen 6). (a): Bleeding from exposed pulp. (b): Pulp surface irradiated with CO2 laser.Figure 2. . Clinical photographs of the CO2 laser group (specimen 6). (a): Bleeding from exposed pulp. (b): Pulp surface irradiated with CO2 laser.Figure 2. . Clinical photographs of the CO2 laser group (specimen 6). (a): Bleeding from exposed pulp. (b): Pulp surface irradiated with CO2 laser.
Figure 2 Clinical photographs of the CO2 laser group (specimen 6). (a): Bleeding from exposed pulp. (b): Pulp surface irradiated with CO2 laser.

Citation: Operative Dentistry 44, 4; 10.2341/18-030-C

Table 1 Composition of the Different Materials Used in This Study
Table 1

Specimen Preparation

The teeth were extracted after either the six or 12 month observation (Table 2) under local anesthesia, and the apical thirds of the roots were removed for penetration of the fixative solution. They were immediately immersed in 4% paraformaldehyde phosphate buffer solution (pH 7.4; Wako Pure Chemical Industries Co, Tokyo, Japan) at 4°C for one week. The excess tissue was trimmed off the teeth, which were decalcified with 10% EDTA decalcifying solution (pH 7.4, Wako Pure Chemical Industries Co) at room temperature for three months. After the resin composites in the decalcified teeth were carefully removed, the teeth were rinsed with running water for six hours and then dehydrated in ascending grades of ethanol, dealcoholized in xylene, and embedded in paraffin. Serial sections of 5-μm thickness were cut using a sliding microtome (Jung HistoSlide 2000R, Leica Microsystems, Wetzlar, Germany) and stained with Mayer hematoxylin and eosin (H&E) and gram bacterial staining. The stained sections were observed under a light microscope (Eclipse E1000, Nikon Co, Tokyo, Japan), and pulp tissue disorganization (PTD), inflammatory cell infiltration (ICI), RDF, and bacterial penetration (BP) were evaluated according to the following criteria,26 which are modified versions of the ISO 10993 and 7405 standards, to allow detailed evaluation of the changes in pulp tissue following DPC.

Table 2 Duration of Actual Observation (Days)
Table 2

PTD

Score 1: Normal or almost normal tissue morphology (none)

Score 2: Odontoblast layer disorganization but the deep part of the pulp appears normal (mild)

Score 3: Loss of general tissue morphology (moderate)

Score 4: Necrosis in the coronal one-third or more of the pulp (severe)

ICI

Score 1: Absence or presence of a few scattered inflammatory cells in the pulp (none)

Score 2: Mild acute/chronic cell lesions (mild)

Score 3: Moderate inflammatory cell lesions seen as abscesses or densely stained infiltrates of polymorphonuclear leukocytes, histiocytes, and lymphocytes in one-third or more of the coronal pulp and/or the mid-pulp (moderate)

Score 4: Pulp necrosis due to severe degree of infection or lack of tissue in one-half or more of the pulp (severe)

RDF

Score 1: No dentin bridge formation (none)

Score 2: Initial dentin bridge formation extending to not more than one-half of the exposure site (initial)

Score 3: Partial/incomplete dentin bridge formation extending to more than one-half of the exposure site but not completely closing the exposure site (partial)

Score 4: Complete dentin bridge formation (complete)

BP

Score 1: Absence of stained bacterial profiles in any part of the section (none)

Score 2: Presence of stained bacterial profiles along the mesial or distal walls of the cavity (mild)

Score 3: Presence of stained bacterial profiles within cut dentinal tubules or pulpal walls of the cavity (moderate)

Score 4: Presence of stained bacterial profiles within the dental pulp (severe)

In addition, the following histologic features were recorded: hemorrhaging, dentin chips (location, size, and number), and irritation (reactionary) dentin formation.

Measurement of the Diameter of Exposed Pulp Area

The diameter of the exposed pulp area was measured using a stereomicroscope (Measuring Microscope MM-40, Nikon Co), and the widest dimension was recorded as the size of the pulp exposure in the H&E-stained section of the specimen.

Statistical Analysis

The results of the histopathological evaluation were statistically analyzed using the Mann-Whitney U test for between-group differences during each observation period. The exposure size data were analyzed using two-way analysis of variance, testing for capping methods (two levels) and observation periods (two levels). Statistical procedures were performed at a significance level of 0.05 using SPSS statistical software (version 14.0J, Base System SC, IBM SPSS Japan, Tokyo, Japan).

RESULTS

Clinical Assessment

From the results of the examination and medical history obtained, slight and temporary pain induced by cold water was present in specimen 1 treated with the CO2 laser at five months posttreatment, specimen 1 treated with Dycal at three months posttreatment, specimen 4 treated with Dycal at five months posttreatment, and specimen 11 treated with Dycal at two months posttreatment; this condition disappeared spontaneously within a few weeks. The other teeth showed no symptoms, and all the tested teeth had a vital response to the electric pulp test performed immediately prior to tooth extraction.

Diameter of Exposed Pulp Area

The diameter (mm, mean ± SD) of exposed pulp area in each group and observation period was 1.18 ± 0.25 for the CO2 laser group at six months, 1.28 ± 0.34 for the CO2 laser group at 12 months, 1.36 ± 0.14 for the Dycal group at six months, and 1.12 ± 0.14 for the Dycal group at 12 months. There were no significant differences between the groups in pulp exposure size (p>0.05).

Histopathological Evaluation

The results of the histopathological evaluation are summarized in Figure 3. Representative histopathological images of each group are shown in Figures 4 to 9. The results of the Mann-Whitney U test for histopathological evaluation showed no significant differences between the CO2 laser group and the Dycal group for all parameters at each observation period (p>0.05).

Figure 3. . Overview of the results of histopathological evaluation. PTD, pulp tissue disorganization; ICI, inflammatory cell infiltration; RDR, reparative dentin formation; BP, bacterial penetration.Figure 3. . Overview of the results of histopathological evaluation. PTD, pulp tissue disorganization; ICI, inflammatory cell infiltration; RDR, reparative dentin formation; BP, bacterial penetration.Figure 3. . Overview of the results of histopathological evaluation. PTD, pulp tissue disorganization; ICI, inflammatory cell infiltration; RDR, reparative dentin formation; BP, bacterial penetration.
Figure 3 Overview of the results of histopathological evaluation. PTD, pulp tissue disorganization; ICI, inflammatory cell infiltration; RDR, reparative dentin formation; BP, bacterial penetration.

Citation: Operative Dentistry 44, 4; 10.2341/18-030-C

Figure 4. . Representative histologic image of the CO2 laser group (six months, specimen 7). Irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls connecting the cavity floor with the dentinal tubules. Newly formed and initial reparative dentin (RD) can be seen at the exposed pulp surface. Pulpal morphology is normal. (H&E staining, original magnification: a = 40× and b =100×). / Figure 5 Representative histologic image of the CO2 laser group (12 months, specimen 9). A complete dentin bridge has been formed in this specimen. Newly reorganized odontoblast-like cells (arrowheads) are seen beneath the dentin bridge and are connected to the primary odontoblasts at both ends of the exposure. The space (*) between those layers and the dentin bridge is a sectioning artifact. Pulpal morphology is normal. (H&E staining, original magnification = 40×). / Figure 6 Representative histologic image of the Dycal group (six months, specimen 2). A thin and complete dentin bridge has formed adjacent to the black material (Dycal, DY). The dentin bridge is continuous with irritation dentins (ID) at both ends of the exposure. Denticle-like reparative dentin (arrow) has formed around the Ca(OH)2 agent particles beneath the dentin bridge. The space (*) is a sectioning artifact. Pulpal morphology is normal. (H&E staining, original magnification: a = 40× and b = 100×). / Figure 7 Representative histologic image of the Dycal group (12 months, specimen 8). The pulp exposure site was completely closed with a relatively thick dentin bridge adjacent to Dycal (DY). Newly reorganized odontoblast-like cells (arrowheads) can be seen beneath the dentin bridge. This is an excellent example of pulpal healing and repair. (H&E staining, original magnification = 40×). / Figure 8 Representative histologic image of the CO2 laser group (six months, specimen 5). A collection of inflammatory cell infiltrations including mononuclear leukocytes and macrophages near the pulp exposure site. The mid-pulp has a normal morphology. A small amount of irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls, but reparative dentin formation and reorganization of the odontoblastic layer have not occurred. (H&E staining, original magnification = 40×). / Figure 9 Representative histologic image of the Dycal group (12 months, specimen 13). Severe tissue disorganization (necrosis) extends to the coronal one-third or more of the pulp. A small amount of irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls, but no reparative dentin is evident. Some particles of Ca(OH)2 agent (arrowheads) are scattered among the fibroblasts at the mid-pulp. (H&E staining, original magnification = 40×)Figure 4. . Representative histologic image of the CO2 laser group (six months, specimen 7). Irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls connecting the cavity floor with the dentinal tubules. Newly formed and initial reparative dentin (RD) can be seen at the exposed pulp surface. Pulpal morphology is normal. (H&E staining, original magnification: a = 40× and b =100×). / Figure 5 Representative histologic image of the CO2 laser group (12 months, specimen 9). A complete dentin bridge has been formed in this specimen. Newly reorganized odontoblast-like cells (arrowheads) are seen beneath the dentin bridge and are connected to the primary odontoblasts at both ends of the exposure. The space (*) between those layers and the dentin bridge is a sectioning artifact. Pulpal morphology is normal. (H&E staining, original magnification = 40×). / Figure 6 Representative histologic image of the Dycal group (six months, specimen 2). A thin and complete dentin bridge has formed adjacent to the black material (Dycal, DY). The dentin bridge is continuous with irritation dentins (ID) at both ends of the exposure. Denticle-like reparative dentin (arrow) has formed around the Ca(OH)2 agent particles beneath the dentin bridge. The space (*) is a sectioning artifact. Pulpal morphology is normal. (H&E staining, original magnification: a = 40× and b = 100×). / Figure 7 Representative histologic image of the Dycal group (12 months, specimen 8). The pulp exposure site was completely closed with a relatively thick dentin bridge adjacent to Dycal (DY). Newly reorganized odontoblast-like cells (arrowheads) can be seen beneath the dentin bridge. This is an excellent example of pulpal healing and repair. (H&E staining, original magnification = 40×). / Figure 8 Representative histologic image of the CO2 laser group (six months, specimen 5). A collection of inflammatory cell infiltrations including mononuclear leukocytes and macrophages near the pulp exposure site. The mid-pulp has a normal morphology. A small amount of irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls, but reparative dentin formation and reorganization of the odontoblastic layer have not occurred. (H&E staining, original magnification = 40×). / Figure 9 Representative histologic image of the Dycal group (12 months, specimen 13). Severe tissue disorganization (necrosis) extends to the coronal one-third or more of the pulp. A small amount of irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls, but no reparative dentin is evident. Some particles of Ca(OH)2 agent (arrowheads) are scattered among the fibroblasts at the mid-pulp. (H&E staining, original magnification = 40×)Figure 4. . Representative histologic image of the CO2 laser group (six months, specimen 7). Irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls connecting the cavity floor with the dentinal tubules. Newly formed and initial reparative dentin (RD) can be seen at the exposed pulp surface. Pulpal morphology is normal. (H&E staining, original magnification: a = 40× and b =100×). / Figure 5 Representative histologic image of the CO2 laser group (12 months, specimen 9). A complete dentin bridge has been formed in this specimen. Newly reorganized odontoblast-like cells (arrowheads) are seen beneath the dentin bridge and are connected to the primary odontoblasts at both ends of the exposure. The space (*) between those layers and the dentin bridge is a sectioning artifact. Pulpal morphology is normal. (H&E staining, original magnification = 40×). / Figure 6 Representative histologic image of the Dycal group (six months, specimen 2). A thin and complete dentin bridge has formed adjacent to the black material (Dycal, DY). The dentin bridge is continuous with irritation dentins (ID) at both ends of the exposure. Denticle-like reparative dentin (arrow) has formed around the Ca(OH)2 agent particles beneath the dentin bridge. The space (*) is a sectioning artifact. Pulpal morphology is normal. (H&E staining, original magnification: a = 40× and b = 100×). / Figure 7 Representative histologic image of the Dycal group (12 months, specimen 8). The pulp exposure site was completely closed with a relatively thick dentin bridge adjacent to Dycal (DY). Newly reorganized odontoblast-like cells (arrowheads) can be seen beneath the dentin bridge. This is an excellent example of pulpal healing and repair. (H&E staining, original magnification = 40×). / Figure 8 Representative histologic image of the CO2 laser group (six months, specimen 5). A collection of inflammatory cell infiltrations including mononuclear leukocytes and macrophages near the pulp exposure site. The mid-pulp has a normal morphology. A small amount of irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls, but reparative dentin formation and reorganization of the odontoblastic layer have not occurred. (H&E staining, original magnification = 40×). / Figure 9 Representative histologic image of the Dycal group (12 months, specimen 13). Severe tissue disorganization (necrosis) extends to the coronal one-third or more of the pulp. A small amount of irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls, but no reparative dentin is evident. Some particles of Ca(OH)2 agent (arrowheads) are scattered among the fibroblasts at the mid-pulp. (H&E staining, original magnification = 40×)
Figure 4 Representative histologic image of the CO2 laser group (six months, specimen 7). Irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls connecting the cavity floor with the dentinal tubules. Newly formed and initial reparative dentin (RD) can be seen at the exposed pulp surface. Pulpal morphology is normal. (H&E staining, original magnification: a = 40× and b =100×) Figure 5 Representative histologic image of the CO2 laser group (12 months, specimen 9). A complete dentin bridge has been formed in this specimen. Newly reorganized odontoblast-like cells (arrowheads) are seen beneath the dentin bridge and are connected to the primary odontoblasts at both ends of the exposure. The space (*) between those layers and the dentin bridge is a sectioning artifact. Pulpal morphology is normal. (H&E staining, original magnification = 40×) Figure 6 Representative histologic image of the Dycal group (six months, specimen 2). A thin and complete dentin bridge has formed adjacent to the black material (Dycal, DY). The dentin bridge is continuous with irritation dentins (ID) at both ends of the exposure. Denticle-like reparative dentin (arrow) has formed around the Ca(OH)2 agent particles beneath the dentin bridge. The space (*) is a sectioning artifact. Pulpal morphology is normal. (H&E staining, original magnification: a = 40× and b = 100×) Figure 7 Representative histologic image of the Dycal group (12 months, specimen 8). The pulp exposure site was completely closed with a relatively thick dentin bridge adjacent to Dycal (DY). Newly reorganized odontoblast-like cells (arrowheads) can be seen beneath the dentin bridge. This is an excellent example of pulpal healing and repair. (H&E staining, original magnification = 40×) Figure 8 Representative histologic image of the CO2 laser group (six months, specimen 5). A collection of inflammatory cell infiltrations including mononuclear leukocytes and macrophages near the pulp exposure site. The mid-pulp has a normal morphology. A small amount of irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls, but reparative dentin formation and reorganization of the odontoblastic layer have not occurred. (H&E staining, original magnification = 40×) Figure 9 Representative histologic image of the Dycal group (12 months, specimen 13). Severe tissue disorganization (necrosis) extends to the coronal one-third or more of the pulp. A small amount of irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls, but no reparative dentin is evident. Some particles of Ca(OH)2 agent (arrowheads) are scattered among the fibroblasts at the mid-pulp. (H&E staining, original magnification = 40×)

Citation: Operative Dentistry 44, 4; 10.2341/18-030-C

After Six Months

One tooth from the CO2 laser group and three from the Dycal group showed complete RDF and exhibited non-PTD and non-ICI. In teeth showing dentin bridge formation, the reorganized layers of newly formed odontoblast-like cells were seen just beneath the reparative dentin. Two teeth from the CO2 laser group and three from the Dycal group showed no RDF but exhibited PTD and ICI. None of the specimens stained positively for bacteria.

After 12 Months

All of the teeth from the CO2 laser group showed RDF, including three teeth with partial RDF and four with complete RDF. A small amount of heat denaturation and carbonized dentin remained at the pulp-exposed area in a few teeth. In contrast, the Dycal group showed one tooth with partial RDF, three with complete RDF, and three with no RDF. Severe PTD and moderate ICI were observed in the teeth with no RDF. The reparative dentin formed by the Dycal group in the early stage tended to include the osteodentin type, while that formed by the CO2 laser group exhibited the tubular type. Irritation (reactionary) dentin was observed at the pulpal dentin wall around the pulp-exposed area in both groups. These dentins were induced by stimulation of cavity preparation that came through the dentinal tubules. The reparative dentin and irritation dentin had varied thicknesses, which did not depend on the capping method. None of the specimens stained positively for bacteria.

DISCUSSION

Bacterial microleakage was identified as an important factor in pulpal inflammation. Several studies have described that the response of the dentin pulp complex does not depend on the dental materials used for pulp capping but on the materials' ability to prevent bacterial microleakage.12,26-31 In this study, there was no bacterial invasion to the cavity or the pulp chamber, and none of the volunteers, except for four cases, complained of any pain throughout the observation periods. Nevertheless, some teeth from both groups showed an inflammatory response when evaluated using light microscopy. In particular, severe inflammation was observed in six teeth from the Dycal group (three each at six and 12 months posttreatment). These findings indicate that bacterial leakage is not the only cause of failure in DPC. We identified two major reasons for the presence of severe inflammation.

The first reason was trauma to the pulp associated with the deep cavity preparation using a diamond point in a high-speed handpiece and the mechanical pulp exposure using a steel round bar in a low-speed handpiece. Generally, dentin caries in vital teeth indicate certain protective responses. Acids produced by bacteria in carious lesions dissolve the hydroxyapatite of the tooth substance into calcium and phosphate ions. These ions and saliva form carious crystals in dentinal tubules that are formed by precipitating calcium phosphate, such as brushite (dicalcium phosphate dihydrate), whitrokite (beta-tricalcium phosphate), and octacalcium phosphate, which is termed sclerotic dentin.32,33 In addition, carious dentin gradually produces tertiary (reactionary or reparative) dentin along the pulpal wall in response to irritation due to BP. The barrier protection of tertiary dentin is superior because there is no continuity between the affected permeable tubules of the regular primary dentin and those within the tertiary dentin.34,35 Sclerotic and tertiary dentins also occur with aging, and it is believed that they reduce exogenous stimuli and tooth pain due to the existence of impermeable dentin.36,37 We selected only intact teeth, and the volunteers were relatively young and were not expected to have the above-mentioned protective barrier. Therefore, fast and furious treatment stimuli, such as deep cavity preparation, might highly affect pulp tissue and cause inflammation.

The second reason was biocompatibility and stimulation of the pulp tissue with the capping materials. CH and similar cement, such as Dycal, have been used as the gold standard material for DPC for many decades because of their antibacterial effect and ability to stimulate repair and induce RDF.1,26,38-40 In addition, Dycal has the disadvantage of forming a necrotic layer at the pulp exposure site because of its high alkaline pH (11-13), which should play a protective role in deep pulp but reduces the volume of pulp tissue simultaneously, thereby increasing the risk of pulp morbidity.1,40 The current Dycal formula used in this study has been improved to a lower pH (9-11)41; however, some studies have indicated the cytotoxicity of Dycal.42,43 Moreover, Dycal specimens with inflammation of the pulp tissue in this study showed black particles of CH preparation diffused into the deep part of the pulp caused by the dissolution of Dycal (Figure 9). Recently, many clinicians have used MTA as their first choice of capping material in substitution for CH because of its higher biocompatibility with pulp cells.42-44 Several in vivo studies have reported that MTA forms dentin bridges earlier, more frequently, and of a greater thickness than those observed with CH.45,46 However, we used Dycal as the control in this study because we wanted to compare the results of this experiment with those of our previous studies, which also used Dycal as the control.

On the other hand, we selected the adhesive system as the capping material for the CO2 laser group to evaluate the individual efficiency of CO2 laser irradiation without any agents to facilitate pulpal repair and RDF. There are arguments for and against using adhesive resins as the DPC agent. Some studies on DPC with adhesive resins have shown favorable results due to good adhesive bonding that eliminates microleakage in DPC treatment.10,12,29,30,47,48 In contrast, some studies concluded that adhesive resins should not be applied to exposed pulp because they do not allow histopathological repair of pulp tissue with moderate or severe inflammation due to a poor biocompatibility and cytotoxic effect regardless of bacterial invasion.49-54 Pameijer and Stanley reported that adhesive systems using the “total etch” technique have been shown to have disastrous effects during DPC in primates.49 Common adhesive components including bisphenol A-glycidyl methacrylate, urethane dimethacrylate, 2-hydroxyethyl methacrylate, triethylene glycol dimethacrylate, and others display toxic effects in pulp cells such as fibroblasts and odontoblasts and might cause an inflammatory response to the dental pulp and hinder pulpal healing.55-58 Several studies, including previous studies of our laboratory, revealed that teeth capped with adhesive resins showed delayed RDF compared with those capped with Dycal or MTA but almost normal tissue morphology without severe reactions was seen throughout the observation period.25,26,59-62 Some studies reported that SE was a reliable adhesive agent to both enamel and dentin,63 which provides sufficient marginal sealing to prevent BP,64,65 and that the cytotoxicity of SE was relatively lower than that of other adhesive systems.57,58 Lu and others59,60 indicated that SE had good biocompatibility to pulp tissue when used for DPC. The results of this study showed that SE did not allow BP. Fewer teeth showed pulp inflammation in the CO2 laser group compared with the Dycal group, although the difference was not statistically significant. We suppose that an adhesive resin may not be the most suitable material for DPC, but SE may be available for DPC. In addition, the denaturation tissue at the exposed surface coagulated by the CO2 laser might prevent resin monomer from diffusing the pulp tissue.

The results of statistical analysis for the histopathological evaluation revealed no significant differences between the groups for RDF, but three of seven teeth in the Dycal group showed complete RDF at six months posttreatment, whereas the CO2 laser group had only one tooth that exhibited complete RDF. These findings indicated a tendency similar to our previous study using rat pulp in that initiation of dentin bridge formation in teeth capped with Dycal appears earlier than in teeth capped with CO2 laser irradiation.25 It was suggested that the total laser energy was too strong for the rat pulp to form a dentin bridge, as the volume of rat pulp is very small. As a result, CO2 laser irradiation caused thermal damage and created relatively thick denaturation tissue around the pulp-exposed area, which might inhibit pulp tissue from forming a dentin bridge. In another study, we reported that delayed pulp tissue healing following CO2 laser irradiation might be related to the thickness of the heat-denatured layer, which increases in proportion to the intensity of laser irradiation.66 Thus, the reaction of the pulp is largely affected by the irradiation settings and the device as well as the wavelength.13-17,20-25,67,68 In this study, the heat-denatured tissue formed by CO2 laser irradiation was scarcely observed around the exposed pulp area histopathologically. The exposed pulp irradiated with the CO2 laser was closed by the complete dentin bridge in four cases at 12 months posttreatment despite the absence of any pharmacological agents. The exposed pulp surface showed a chalky white or light brown discoloration after irradiation with the CO2 laser, which might have been due to coagulation and protein denaturation by the photothermal effect. It is known that CO2 laser irradiation with high energy creates dark brown or black carbonized tissue. Therefore, it is speculated that the total energy of CO2 laser irradiation performed in this study might be relatively low in human pulp and might not completely interfere with dentin bridge formation. Lasers also yield a photoactive effect (low reactive-level laser/light therapy [LLLT]) that demonstrates a reversible reaction, which contributes to providing the biostimulation effect followed by an accelerated wound-healing process.69,70 It is not clear whether the effect of LLLT contributed to the results of this study. Generally, lower energy and longer irradiation time compared with those of this study are needed to obtain the effect of LLLT in the clinic. Therefore, it was suggested that there was little effect of LLLT under the irradiation conditions in this study.

Alternate irrigation with 6% NaClO and 3% H2O2 was performed to control bleeding and remove dentin chips, debris, and coagulum in both groups. Hemostasis was achieved with alternate irrigation in all the teeth; however, teeth without laser irradiation gradually accumulated exudate around the exposed site following chemical lavage, while teeth irradiated with the CO2 laser exhibited neither secondary bleeding nor exudate until the filling of the resin composite into the cavity was completed because of artificial scab formation at the irradiated area because of the photothermal effect. It should provide smooth and accurate capping and restoration regardless of the type of capping materials used. One of the great advantages of CO2 laser is its ability to work without making contact with the pulp tissue, which helps avoid bacterial infection. Moritz and others reported that the most important effects of CO2 laser irradiation are sterilization and scar formation as well as minimal hematoma formation in the exposed pulp area due to the thermal effects of laser irradiation.22 Laser irradiation should minimize hematoma formation between the pulp tissue and the capping material and allow close contact between them.

Based on the findings of this study, we accepted the null hypothesis that CO2 laser irradiation would not affect pulpal healing and RDF in the exposed human pulp. However, further studies using various irradiation conditions and combining the use of capping materials and CO2 laser irradiation are required to determine the most suitable irradiation conditions and procedure to increase the success rate in DPC with CO2 laser.

CONCLUSIONS

Under the experimental conditions of this study, CO2 laser irradiation completely controlled exudate as well as bleeding of the mechanically injured human pulp. DPC with the self-etching adhesive system following CO2 laser irradiation without carbonization of the exposed pulp demonstrated favorable pulpal healing and dentin bridge formation that was comparable to that with Dycal.

Acknowledgments

This study was supported by a Grant-in-Aid for Young Scientists (B) (grants JP18791412 and JP24792040) from the Japan Society for the Promotion of Science (JSPS). The authors would like to thank Enago (www.enago.jp) for the English language review.

Regulatory Statement

This study was conducted in accordance with all the provisions of the local human subjects oversight committee guidelines and policies of the Research Ethics Committees Northern Ireland. The approval code for this study is ORECNI, 16/NI/0101.

Conflict of Interest

The authors of this article certify that they have no proprietary, financial, or other personal interest of any nature or kind in any product, service, and/or company that is presented in this article.

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Copyright: Operative Dentistry, 2019 2019
Figure 1
Figure 1

Diagram showing the flow of volunteers.

Figure 2
Figure 2

Clinical photographs of the CO2 laser group (specimen 6). (a): Bleeding from exposed pulp. (b): Pulp surface irradiated with CO2 laser.

Figure 3
Figure 3

Overview of the results of histopathological evaluation. PTD, pulp tissue disorganization; ICI, inflammatory cell infiltration; RDR, reparative dentin formation; BP, bacterial penetration.

Figure 4
Figure 4

Representative histologic image of the CO2 laser group (six months, specimen 7). Irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls connecting the cavity floor with the dentinal tubules. Newly formed and initial reparative dentin (RD) can be seen at the exposed pulp surface. Pulpal morphology is normal. (H&E staining, original magnification: a = 40× and b =100×)

Figure 5 Representative histologic image of the CO2 laser group (12 months, specimen 9). A complete dentin bridge has been formed in this specimen. Newly reorganized odontoblast-like cells (arrowheads) are seen beneath the dentin bridge and are connected to the primary odontoblasts at both ends of the exposure. The space (*) between those layers and the dentin bridge is a sectioning artifact. Pulpal morphology is normal. (H&E staining, original magnification = 40×)

Figure 6 Representative histologic image of the Dycal group (six months, specimen 2). A thin and complete dentin bridge has formed adjacent to the black material (Dycal, DY). The dentin bridge is continuous with irritation dentins (ID) at both ends of the exposure. Denticle-like reparative dentin (arrow) has formed around the Ca(OH)2 agent particles beneath the dentin bridge. The space (*) is a sectioning artifact. Pulpal morphology is normal. (H&E staining, original magnification: a = 40× and b = 100×)

Figure 7 Representative histologic image of the Dycal group (12 months, specimen 8). The pulp exposure site was completely closed with a relatively thick dentin bridge adjacent to Dycal (DY). Newly reorganized odontoblast-like cells (arrowheads) can be seen beneath the dentin bridge. This is an excellent example of pulpal healing and repair. (H&E staining, original magnification = 40×)

Figure 8 Representative histologic image of the CO2 laser group (six months, specimen 5). A collection of inflammatory cell infiltrations including mononuclear leukocytes and macrophages near the pulp exposure site. The mid-pulp has a normal morphology. A small amount of irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls, but reparative dentin formation and reorganization of the odontoblastic layer have not occurred. (H&E staining, original magnification = 40×)

Figure 9 Representative histologic image of the Dycal group (12 months, specimen 13). Severe tissue disorganization (necrosis) extends to the coronal one-third or more of the pulp. A small amount of irritation (reactionary) dentin (ID) formation is observed at the pulpal dentin walls, but no reparative dentin is evident. Some particles of Ca(OH)2 agent (arrowheads) are scattered among the fibroblasts at the mid-pulp. (H&E staining, original magnification = 40×)

Contributor Notes

*Corresponding author: 1-8 Hamaura-cho, Chuo-ku, Niigata, 951-8580, Japan; e-mail: collagen@ngt.ndu.ac.jp
Accepted: 30 May 2018
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