Histopathological study of direct and indirect pulp capping with dentin bonding agents in human teeth

Dr. Roberto Espinosa Fernandez
Professor, Graduate Prosthodontics, Faculty of Dentistry, University Center of Health Sciences, University of Guadalajara. Mexico.
e-mail: robertoesp@avantel.net

Dr. Alvaro Cruz González
Professor of Endodontics, Faculty of Dentistry, University Center of Health Sciences, University of Guadalajara. Mexico.

Dr. Diego Espinosa Sanchez
Graduate School of Dentistry, University Center of Health Sciences, University of Guadalajara. Mexico

Dr. Eduardo Flores Herrera
A graduate of the Graduate Prosthodontics, University Center of Health Sciences, University of Guadalajara. Mexico.

Dr. Israel Ceja Andrade
Master of Environmental Health Sciences. Researcher, Center for Engineering Sciences, University of Guadalajara, Mexico.

Summary

Introduction-The modern adhesive techniques have included acid etching and adhesives as direct and indirect pulp capping therapy. The objective of this in vivo study was to evaluate the possible histological changes of the dental pulp as a result of the direct and indirect pulp capping in human teeth with adhesives.

Materials and Methods-60 teeth that required extraction due to periodontal problems were used; class V cavities were prepared in all of them. The teeth were divided into 4 groups: Group # 1, indirect pulp capping with total etch adhesive systems. Group # 2, indirect pulp capping with calcium hydroxide and a glass ionomer liner. Group # 3, direct pulp capping with total etch adhesive systems. Group # 4, direct pulp capping with calcium hydroxide and a glass ionomer liner. After 6 months all teeth were extracted and prepared for scanning electron microscope and for histopathologic analysis.

Results: Pulps of groups # 1 and # 3 showed similar results: severe chronic, large areas of necrosis, and presence of macrophages wrapped around the adhesive particles. The pulps of groups # 2 and # 4 showed similar results: no inflammation, tissue reorganization, formation of a dentinal bridge and reparative dentin.

Conclusions and Recommendations: According to the obtained results, we can conclude that in the direct and indirect pulp capping with adhesives, they penetrate the pulp causing chronic inflammatory reaction and cellular toxicity. Meanwhile, when a liner is used, pulpal tissue is reorganized and dentinal bridge and reparative dentin are formed. In deep zones of the dentin, it is recommendable to apply calcium hydroxide and a glass ionomer liner in order to avoid toxicity of the adhesives to the pulp and pulp Therefore avoid pathology.

Keywords: Adhesives, citotoxicity, Calcium Hydroxide, Pulp Capping.


Introduction
The current bonding systems have opened a myriad of possibilities for the implementation of successful new treatments, through hybridization, which forms an excellent mechanical bond between resin and dentin, so that opens new expectations in restorative dentistry.

It is often forgotten that care must be taken not to cause pulpal irritation as a result of clinical procedures. Pulp reactions caused by restorative materials depend on the structure and amount of remaining dentin between preparation and the pulp, often called the remaining dentin thickness. The remaining dentin than 2 mm is considered adequate to perform restorative procedures, even if used adhesive techniques (Christensen, 1994; Major, 2002).

The current adhesive techniques have included acid etching and adhesive coating therapy directly and indirectly. Some authors argue that the deep dentinal etching does not cause pulp damage or toxic effects caused by materials used in adhesive techniques (Bränström, 1978; Fusayama, 1987), but other authors claim that acid etching causes irritation and subsequent restoration, pathology Histopathologic phenomena pulp and internal resorption, when applied in deep cavities with a remaining dentin thickness of 0.5 to 1 mm (HEBLING, 1999, Stanley HR. 1975). Clinical research studies have shown that due to the use of dentinal etching systems and adhesives, have significantly increased adverse pulpal effects (Gerurtsen, W. 2000, Cox CF. Et al.1998; Camps, J. et al. 2000 ), thereby casting doubt on the biocompatibility of the components of adhesives.

Intertubular Diffusion is the ability of a material to spread through the dentinal tubule, this is proportional to the length and diameter of the tubules and the molecular weight of substances (Pashley, DH. Et al. 1984; Bränström, M. 1966) . The modern adhesives contain a large amount of HEMA (2-hydroxyethyl methacrylate) with a molecular weight of 130 (Craig R. 1998). This component allows for excellent interdigitation between the adhesive and dentin amended. However, in areas near the pulp chamber HEMA has the ability to diffuse through the tubules to reach the pulp chamber and cause pulpal pathology (Camps, 2000, Pashley et al 2000).

The aim of this study was to evaluate in vivo the possible histologic changes of the dental pulp as a result of indirect and direct coating with adhesives in human teeth.


Materials and Methods
60 pieces were used dental extraction requiring the periodontal indications. The sample consisted of 10 patients, whose average age was 54 years, the youngest of 47 and over 61. This study was conducted according to the guidelines established in the code of bioethics for the Department of Dental Health, the Official Mexican and regulations in force for Bioethics at the University of Guadalajara. Patients who agreed to participate in the study authorized in writing treatments and procedures for the same.

Healthy teeth were selected, that although some of them had restorations and wear, had no carious lesions or fractures. In the initial diagnosis of patients reported no symptoms. No tooth proposed for the study had periapical pathology, confirmed radiographically, and showed positive response to pulp vitality tests.

Class V cavities were prepared in all samples. Patients fillings are made with both techniques. Of the 60 specimens were prepared close to 40 without communication pulp pulp pulp and 20 with open communication. The samples were divided into 4 groups: Group # 1 of 20 teeth indirect conducted in the coating with a total-etch adhesive system and resin, the group # 2 of 20 teeth, which are indirect coating made with calcium hydroxide ( Ca (OH) 2) covered by a layer of glass ionomer liner (DIV), the process of implementing the total-etch adhesive system and resin. The group # 3 of 10 teeth, which were made preparations pulp communication and direct capping with total-etch adhesive system and resin y. The group # 4 with pulp making preparations communication direct pulp capping with Ca (OH) 2, DIV adhesive system prior to it (table 1).

Clinical Procedures:
The cavity preparation of the teeth of the first two groups were made according to the following steps: After the application of local anesthetic and the total isolation of the operative field with rubber dam, became cavities near the pulp chamber with a diamond bur coarse # 6856 (Brasseler, USA) using a high speed and irrigation with water and air. The indirect coating of each sample group # 1 was performed with a bonding system, used the Singlebond and resin Filtek Z250 (3M), following the manufacturer's instructions: He wiped each cavity with air, was recorded immediately after enamel dentin with 37% phosphoric acid (Scotchbond, 3M) for 15 seconds. After washing profuse withdrew the excess moisture inside the cavity with a sterile cotton swab, making sure not to dehydrate the dentin. Was 2 layers of dentin bonding agent (Singlebond, 3M) and evaporated the solvent for 15 seconds with a gentle air blast, polymerizing the adhesive for 20 seconds by making a second application of adhesive as described above, and was carried out restoration using resin (Filtek Z250, 3M) using the technique of incremental layers obliquely polymerizing each for 20 seconds. In the 20 samples of group # 2 was implemented in the area closest to the pulp a point of Ca (OH) 2 mixed with pure sterile water covering it with a DIV (Vitremer, 3M), the restoration was conducted following the same procedures as restorative described above.

In the 20 samples of the groups # 3 and # 4, you are deep preparations made following the same procedure of the previous groups, and performed these steps use a carbide bur No. 330 with which communication was carried out pulp, proceeding to profuse washing the cavity with saline until bleeding relented. The direct coating performed in 10 samples of group # 3 was conducted with the same adhesive that group # 1 and following the same steps. The 10 samples of group # 4, the direct coating was carried out with Ca (OH) 2 following the same steps of group # 2.

Patients included in the study received at least two different treatments, although it was not possible at all, it was intended that each were conducted on the four treatments described above.

Patients were evaluated bimonthly cited, it was made by clinical inspection prior examination of the possible symptoms. Pulp vitality tests were made in each tooth included in the study, looking for any difference in both hypersensitivity or possible necrosis. Finally periapical radiographs were taken. This evaluation procedure was carried out from the pre-selection assessment study and every two months over the six month period of this study. These results were reflected in the general history of the study (Table No. 3).

As the specimens reached 180 days, extractions were performed until all the groups.

Preparation of samples for electron microscopy:

10 samples were used for each of the groups # 1 and # 2, to be analyzed in the scanning electron microscope. After the removal of these 20 samples were stored immersed in saline. 24 hours after he cut the root of the tooth with a diamond disk, keeping only the anatomic crowns. To remove all organic material housed in the pulp cavity, the samples were submerged in sodium hypochlorite 6% for 20 minutes. Then washed with water and dried. We proceeded to develop a diamond cutting disc exclusively buccal lingual enamel. Subsequently the teeth were sectioned with a chisel blow # 3G-0090 (American Dental) and hammer, in order to preserve the internal structures no wear or presence of foreign elements.

Samples were prepared for observation using scanning electron microscopy (SEM) (JEOL JSM-5400 LB). Were mounted on a slide and were coated with 24 carat gold by sputtering (JEOL) with the objective to be tested at high vacuum each in the electron microscope.


Preparation of samples for histological sections:

At the end of 180 days extractions were performed mainly using elevators, when necessary, use of forceps was applied on the apical root as possible to avoid causing damage to the area of restoration. Immediately after extraction is short root in half with a carbide bur No. 701-L (Jet, Beavers Dental) with cooling water and air, thus promotes easy and fast penetration of fixative. The samples were immersed in 10% formaldehyde (pH 7.4) for 72 hours. To decalcified groups were immersed in acid ethylene diamine tetra-acetic acid trisodium 17% (EDTA) under continuous stirring for 5 months, renewing the acid weekly. At the end of this time proceeded to include the samples in paraffin for making cuts on a microtome and mounting on slides. 20 serial sections were processed for each piece of the area of the cavity. We used hematoxylin and eosin staining proceeding to microscopic analysis of each of magnification 100X, 400X and 1000X.


Results

The bimonthly evaluation results are shown in table No. 3, which shows that in both the pulp vitality tests, and radiographic studies throughout the duration of the study did not show symptoms or signs or evidence of pulpal pathology radiographic periapical lesions.

Electron microscopy:

The only electron microscopic evaluation was performed in all 20 samples, 10 of group # 1 and 10 of group # 2, samples of the groups # 3 and # 4, not analyzed through this system because when making the communication dentin pulp is removed which is the subject of this review.

By analyzing the electron microscope was observed and measured the remaining dentin thickness, resulting in the remaining dentin thickness of all samples of the two groups was less than 1 mm. The results of the 10 samples of group # 1 (indirect coating resins), it was noted that within the dentinal tubules present throughout its length dentin bonding agent, from the floor of the cavity to the pulp chamber.

The adhesive penetrated the dentin tubules to the pulp chamber flowing fully impregnating the predentin, so we formed a mixture of adhesive and predentin. It was also found very large irregular adhesive residue, forming extensions within the pulp chamber (figure 1).

Test results from 10 samples of group # 2 (indirect coating with Ca (OH) 2 and DIV, are the remaining dentin thickness is similar to that of group # 1. Dentinal tubules was observed throughout its length free of adhesive . tubules in the end are completely empty pulp without adhesive residue and dentin of the pulp chamber roof is free of foreign elements (Figure No. 2).

Histopathologic findings:
The results of histological sections from 10 samples of group # 1, which made indirect coating and etching adhesives were similar: The original remaining dentin thickness remained the same with no evidence of tertiary dentin formation or repair. In the pulp tissue was observed near the coating cell lysis including the odontoblast layer, was struck by the lack of the predentin layer. Both the acellular zone, such as cell-rich zone are shown with total disarray with few scattered fibroblasts, few glasses and a larger diameter and some macrophages (Figure No. 3). We found several adhesive cells scattered in the pulp tissue, surrounded by macrophages (Figures No. 4 and 5). The histopathologic features observed in the samples of this group showed a chronic inflammatory state in the process of necrosis, which extends even to remote areas of the coating area.

The 10 samples of group # 2 in the coating was made indirectly with Ca (OH) 2 and DIV unlike previous samples, we observed formation of tertiary dentin in both the coating area and around the perimeter of the pulp chamber clearly defines the line calciotraumatic where he initiated the formation of reparative dentin with the following histological features: a lesser dentinal tubules diameter and quantity of dentin areas atubular, amorphous and hypercalcified (Figure No. 6 and 7). The predentin is clearly distinguishable accompanied by the odontoblast layer in position and normal amount, just below the space they can see where the acellular nerve plexus delimited by the cell-rich layer, and finally the connective tissue in normal . The presence of vessels and amount shown in normal diameter. Cellular organization can be seen clearly and predentin formation (Figure No. 8).

In none of the samples of group # 3 was formed dentin bridge persisted pulp tissue contact with the coating material (Figure No. 9). There are areas of necrotic tissue areas coexisting with hyperemic vessels showing, with considerable disruption of the tissue and the absence of collagen fibers and fibroblasts, with an apparent coagulation of the extracellular matrix. In pulp tissue near the area of the coating shows large amount of inflammatory exudate, cellular disorganization generalized reduction in the number of cells, showing characteristics of a necrotic degenerative process. Samples from this group were characterized by the presence of many particles polymerized adhesive within the pulp chamber enveloped by macrophages (Figure No. 10).

All samples of group # 4 showed dentin bridge formation, with a thickness variable reparative dentin, predentin and odontoblast layer, as well as the characteristics of the repair process cellular physiological reorganization comparable with a normal pulp (Figures No. 11 and 12 ).

Discussion
The total etching technique in deep areas and the application of adhesive bonding as direct and indirect pulp capping, has been adopted by many dentists to its application to daily clinical benefit based on supposedly provides this treatment, for greater retention and sealing of the cavity. However, several authors, while recognizing that this system favors the sealing and retention, have shown that in the deepest potential of injuring the pulp is high (Costa, et al 1999). Many factors are related to the implementation of engraving in deep dentin adhesive application. Importantly, not only the aggressor is the element recorded the pulp, but also components that are toxic adhesive to pulp cells (Mondelli, 1998).

10 of the 20 samples of group # 1 to the indirect coating was made were analyzed by scanning electron microscope, demonstrated the ease of dissemination with dentin adhesives in coming to penetrate deep into the pulp chamber, ratified the histologic presence the adhesive in the resulting pulp with pulp tissue toxicity; been demonstrated that direct and indirect pulp-capping with acid etching and bonding does not promote biological repair mechanisms.

Normally predentin inside the pulp chamber consists of a large number of interwoven collagen fibers form the matrix of the formation of the following layers of dentin. The adhesive penetrated the dentin tubules to the pulp chamber flowing fully impregnating the predentin, a mixture of adhesive and predentin forming a bloc similar to what is known as hybridization in adhesion, penetration of the adhesive was wrapped such that large number of odontoblasts at the time of the photopolymerization were included in the resinous mass. It was also found very large irregular adhesive residue, forming extensions within the pulp chamber (figure 1). These adhesive residue in the pulp chamber dentin matrix involve zero causing the formation of tertiary dentin.

Several in vivo studies have been carried out concluding that the traditional method of applying a base Ca (OH) 2 in the deep areas, prior to etching and adhesive pulp offers the best treatment for proper repair and maintain pulp-free conditions. (Costa, CA.et to 1999; Rakich, DR. 1999; Shuster, GS. Et al 2000).

The results of this research study confirmed the results obtained by other authors (Camps, J. et al 2000; Costa, CA. HEBLING, J. 2002), Costa CA. 2003) which concluded that the presence of adhesives on pulp causes a chronic inflammatory reaction with vascular dilatation, areas of necrosis, the phenomena of internal resorption and no dentin bridge formation. This process is asymptomatic and no periapical pathology. While in cases where a base was applied previously observed protective cellular reorganization and formation of reparative dentin (Mjor, I. 2002).

Unlike studies that are made solely on premolars which are to be removed for orthodontic indications, this study was conducted in middle-aged patients. This aspect is particularly important because the samples have been exposed over time with the natural consequence of different types of deterioration such as gingival recession, gingival dentine chronic irritation, occlusion and occlusal wear, some types of restorations, and so on. Studies in young teeth show an overview of the pulpal response, but this study in mature parts show the results of clinical work that we make daily in elderly patients and actual situation.

It has been shown that there is great difference between the concept of vitality and tenderness (Stanley, H. Pameijer C. 1997), so that the pulp tissue can be in the process of necrosis and positive response to stimuli such as temperature changes response of vitality, but it is well known that nerve tissue in a necrotic process is the last to die, so in these cases can be detected without pulp vitality pulp sensitivity confusing clinical diagnosis. Draws attention to the results of this study that no patients had pain or discomfort, in clinical diagnosis bimonthly any of the samples presented increased sensitivity or loss thereof. The radiographic diagnosis was not presented bimonthly perceptible change in the X-rays compared with the initial diagnosis or between subsequent shots. Importantly, these same pieces in histologic sections showed a degenerative process caused by necrotic pulp and adhesives in the pulp. Thus, we conclude that the absence of symptoms does not necessarily represent the health status of the pulp.
            
Conclusions and Recommendations
According to the results of this research study, we conclude that in deep cavities where the remaining dentin thickness between cavity and pulp chamber is very thin (1.0 mm), the adhesive flows through the dentinal tubules to penetrate pulp causing a chronic inflammatory reaction with vascular dilatation, areas of necrosis, and no dentin bridge formation. This process is asymptomatic with no radiographic symptoms and pathological signs. While in the cases where we applied a protective base reorganization was observed cellular reparative dentin formation.

In areas of deep dentin is recommended, prior to etching dentin, calcium hydroxide application covered by a glass ionomer liner to avoid the toxicity that the substances may create the pulp and thus does not cause pulpal pathology.

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