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Brazilian Journal of Oral Sciences
Piracicaba Dental School - UNICAMP
EISSN: 1677-3225
Vol. 9, Num. 3, 2010, pp. 337-344

Braz J Oral Sci, Vol. 9, No. 3, July-September, 2010, pp. 337-344

Response of human dental pulp to calcium hydroxide paste preceded by a corticosteroid/antibiotic dressing agent

Elisa Maria Aparecida Giro1, Juliana Oliveira Gondim2, Josimeri Hebling1, Carlos Alberto de Souza Costa3

1 DDS, MSc, PhD, Professor, Department of Orthodontics and Pediatric Dentistry, School of Dentistry of Araraquara, Universidade Estadual Paulista - UNESP, Araraquara, SP, Brazil
2 DDS, MSc, Assistant Professor, Department of Dentistry, School of Dentistry, Federal University of Ceará, Sobral, Fortaleza, CE, Brazil; Graduate Student, Department of Orthodontics and Pediatric Dentistry, School of Dentistry of Araraquara, Universidade Estadual Paulista - UNESP, Araraquara, SP, Brazil
3 DDS, MSc, PhD, Professor, Department of Physiology and Pathology, School of Dentistry of Araraquara, Universidade Estadual Paulista - UNESP, Araraquara, SP, Brazil

Correspondence to: Elisa Maria Aparecida Giro Departamento de Clínica Infantil, Faculdade de Odontologia de Araraquara UNESP Rua Humaitá, 1680 Centro. CEP:14801-903 CP: 331, Araraquara, São Paulo, Brasil Phone:+ 55 16 33016336; fax:+55 16 33016329 E-mail: egiro@foar.unesp.br

Received for publication: June 18, 2009
Accepted: June 21, 2010

Code Number: os10032

Abstract

Aim: To evaluate the treatment with corticosteroid/antibiotic dressing in pulpotomy with calcium hydroxide.
Methods:
Forty-six premolars were pulpotomized and randomly assigned into 3 groups. In Group I pulpal wound was directly capped with calcium hydroxide, and Group II and Group III received corticosteroid/antibiotic dressing for 10 min or 48 h, respectively, before pulp capping. Teeth were processed for histological analysis after 7, 30 or 60 days to determine inflammatory cell response, tissue disorganization, dentin bridge formation and presence of bacteria. Attributed scores were analyzed by Kruskal-Wallis and Mann-Whitney tests (α =0.05).
Results:
On the 7th day, all groups exhibited dilated and congested blood vessels in the tissue adjacent to pulpal wound. The inflammatory cell response was significantly greater in Group III (p<0.05). On the 30th day, in all groups, a thin dentin matrix layer was deposited adjacent to the pulpal wound and a continuous odontoblast-like cell layer underlying the dentin matrix was observed. On the 60th day, all groups presented a thick hard barrier characterized by an outer zone of dystrophic calcification and an inner zone of tubular dentin matrix underlined by a defined odontoblast-like cell layer.
Conclusions:
Within the limitations of present study, considering that the treatment was performed in healthy teeth, it may be concluded that the use of a corticosteroid/antibiotic dressing before remaining tissue protection with calcium hydroxide had no influence on pulp tissue healing.

Keywords: calcium hydroxide, corticosteroid, dental pulp capping, permanent dentition, pulpotomy.

Introduction

The application of antiinflammatory agents on exposed pulp tissue in an attempt to prevent or minimize inflammatory reaction and to favor healing has been investigated for a long time1-7. Corticosteroid can be used as a dressing agent for deep cavities and exposed pulp tissue in order to control the inflammatory pulp response and reduce postoperative pain1,3,8-10. However, there is no consensus in the dental literature concerning the use of this medicament1-5,8.

Topic corticosteroid application for 5 min on the cavity floor did not significantly change intrapulpal pressure when compared to healthy teeth2. The therapeutic effect of a corticosteroid agent seems to depend upon its potency, concentration and ability to diffuse into connective tissue. Otosporin®, one of the commercial denominations of hydrocortisone/antibiotic association, presents high displacement capacity in dentin in comparison to other corticosteroid agents available to different therapies6. This product has been reported to prevent intense inflammatory reaction, cause no pulp damage when applied as a dressing agent in exposed pulps and preserve pulp vitality, showing a mild inflammatory reaction restricted to the superficial pulp zone underneath the pulp capping site3. On the other hand, the effects of corticosteroid agents have been questioned when they are applied for a long period because inhibition of collagen synthesis and interference with pulp recovery may occur in this situation11.

Calcium hydroxide has been indicated as the material of choice to treat exposed pulp tissue because it presents biocompatibility, antibacterial activity and is able to induce mineralized barrier formation7,12-15. Studies have shown that the high pH values of this material when in contact with pulp tissue causes coagulation necrosis, which stimulates mineralized tissue formation7,12-13,15-17.

The use of calcium hydroxide for direct pulp capping is supported by several studies3,7. However, considering that the mechanical trauma produced during pulpotomy therapy results in mild inflammatory pulp response, few clinical and histopathologic studies have investigated the use of corticosteroid before direct pulp capping3-4.

The aim of this in vivo study was to evaluate pulp healing in teeth subjected to corticosteroid/antibiotic dressing agent application prior to direct pulp capping with calcium hydroxide paste.

Material and methods

For this study, 46 intact, caries-free and periodontally healthy premolars scheduled to be extracted for orthodontic reasons (28 maxillary and 18 mandibular) were obtained from 23 patients aged between 12 and 16 years. All patients enrolled in the trial were attending the Pediatric Dentistry Clinic of the Dental School of Araraquara, Brazil, and none of them were using medicines or have any systemic disease.

After receiving full explanation about the experimental rationale, clinical procedures and possible risks, parents of all volunteers were asked to carefully read the research protocol before signing the informed consent form. Both the consent form and the research protocol were previously submitted and approved by the Human Research Ethics Committee of the São Paulo State University UNESP, Brazil (Protocol # 52/99).

The teeth were examined clinically and radiographically for the presence of caries or periapical pathology, and were polished with rubber cup and prophylactic paste at low speed. Local anesthesia was administrated (2% mepivacaine, DFL Ind. e Comércio Ltda, Rio de Janeiro, RJ, Brazil) and rubber dam isolation was provided followed by antisepsis with 70% alcohol.

Pulp chambers were opened under aseptic conditions with #4 round bur (Beavers Dental Div. of Sybron Canada, Ontario, Canada) at high speed and under irrigation with sterile cooled water. After pulp exposure, the coronary pulp tissue was carefully cut with a sharp dentin spoon. Saline solution and sterile cotton pellets were used to control hemorrhage. Teeth were randomly assigned to 3 groups: Group I (control), Group II (10-min Otosporin® application) and Group III (48-h Otosporin® application).

Group I (n=14 teeth; 7-day period = 6 teeth, 30-day period = 4 teeth and 60-day period = 4 teeth):

In this group, calcium hydroxide paste was directly applied onto the pulp wound with a sterile instrument and gently compressed with sterile cotton pellets. This pulp-capping agent was prepared by mixing 120 mg of calcium hydroxide powder (calcium hydroxide p.a.; Merck KGaA, Darmstadt, Germany) with 60 µL of sterile distilled water. No dressing agent was applied before pulp capping. Then, a hard-setting calcium hydroxide cement (Dycal; Dentsply, Milford, DE, USA) hand-mixed according to the manufacturer's instruction was applied, followed by placement of a reinforced zinc-oxide and eugenol hard-setting cement base (IRM; Dentsply Indústria e Comécio Ltda., Petrópolis, RJ, Brazil), and cavity restoration with amalgam (Permite; SDI, Southern Dental Industries, Australia).

Group II (n=16 teeth; 7-day period = 6 teeth, 30-day period = 5 teeth and 60-day period = 5 teeth):

In this group, a sterile cotton pellet embedded with corticosteroid/antibiotic dressing agent (Otosporin®; Glaxo Wellcome S.A., Rio de Janeiro, RJ, Brazil) was applied for 10 min on the pulp wound, and then removed with sterile cotton pliers. The pulp chamber was irrigated with sterile saline and gently dried with a sterile cotton pellet. Calcium hydroxide paste was applied onto the pulp wound with a sterile instrument and gently compressed with sterile cotton pellets. Then, a hard-setting calcium hydroxide cement was applied, followed by placement of a reinforced zinc-oxide and eugenol hard-setting cement base, and cavity restoration with amalgam, as described in Group I.

Group III (n=16 teeth; 7-day period = 5 teeth, 30-day period = 6 teeth and 60-day period = 5 teeth):

In this group, a sterile cotton pellet embedded with Otosporin® dressing agent was applied onto the pulpal wound and the cavity was temporarily restored with reinforced zinc-oxide and eugenol hard-setting cement. After 48 h, the temporary restorative material and the cotton pellet embedded with the dressing agent were removed as previously described. Then, the pulpal wound was capped with calcium hydroxide paste and the cavity restored following the clinical sequence described for Groups I and II.

In all 3 groups, the pulpotomy procedures were performed by the same experienced operator.

After the experimental periods of 7, 30 or 60 days, teeth were extracted under local anesthesia and had their roots sectioned halfway between the cementoenamel junction (CEJ) and the root tip with a high-speed handpiece under copious water spray cooling. Teeth were immersed for 48 h in 10% buffered formalin, decalcified in Morse's solution, dehydrated, and vacuum-embedded in wax paraffin. Six-micron-thick serial sections were cut (`820' Spencer Microtome, Carson, CA, USA) parallel to the long axis of the tooth. The sections were mounted on glass slides and stained with hematoxylin and eosin, Masson's Trichrome and Brown and Brenn technique for bacterial assessment.

All histological sections were evaluated by a previously standard-trained pathologist and the analyses conducted in a blind fashion protocol under light microscope (Carl Zeiss 62774, Oberkachen, West Germany). Each tooth was independently examined in serial sections and specific scores were attributed according to the established criteria presented in Tables 1, 2, 3, 4. The attributed scores for the histological events were analyzed statistically by Kruskal Wallis and Mann-Whitney tests to determine any significant differences (p<0.05) among the ranked groups and periods.

Results

The patients did not report pain or any kind of discomfort during the postoperative periods in any of the groups. The observed scores for the histological events in the different experimental groups and periods are shown in Table 5.

7-Day Period

On the 7th day after the procedure, 6 teeth of Group I, 6 of Group II, and 5 of Group III were analyzed.

In all groups, a thick coagulation necrosis layer was observed adjacent to the pulp-capping agent, with pulp cells remaining within this necrotic tissue. In most specimens, the subjacent pulp tissue exhibited few polymorphonuclear neutrophils and few mononuclear cells. Small congested and dilated blood vessels were observed close to the coagulation necrosis or getting into this necrotic tissue (Figure 1).

In Group I (control), pulp response was characterized by a mild inflammatory reaction associated with mild deposition of dentin matrix in all 6 specimens. Stained bacteria along the outer third of lateral cavity walls were observed in 1 specimen of this group.

Group II presented histological characteristics of mild tissue disorganization. Moderate tissue disorganization was seen in only one specimen in which bacteria was evidenced along the outer third of the lateral cavity walls. In 2 specimens, elongated pulp cells (odontoblast-like cells) associated with mild deposition of dentin matrix underneath the coagulation necrosis zone was observed. In the 4 specimens that showed disorganized elongated cells no dentin matrix deposition was seen.

In Group III, pulp response was similar to that of Group II. However, mild inflammatory response was observed in 4 specimens (Figure 2) and moderate inflammatory reaction was seen in 1 specimen. It should be emphasized that no microleakage was evidenced in all evaluated specimens.

30-Day Period

After 30 days of the procedure 4 teeth of Group I, 5 teeth of Group II, and 6 of Group III were analyzed.

In Group I (control), all specimens showed a monolayer of elongated cells associated with moderate deposition of hard tissue subjacent to pulp wound (Figure 3). Mild inflammatory reaction associated with mild tissue disorganization was seen in 2 out of 4 specimens.

In Group II, all specimens presented dystrophic calcification in the deepest zone of the coagulation necrosis layer. Presence of a monolayer of elongated cells (odontoblast- like cells) underlying the dystrophic calcification zone was noted. These differentiated elongated pulp cells were responsible for the deposition of a thin dentin matrix layer which, associated to the dystrophic calcification, characterized the moderate hard tissue barrier formation. The subjacent pulp tissue exhibited normal histological characteristics (Figure 4). Delay in the pulp repair occurred in one specimen in which calcium hydroxide remnants were displaced into pulpal space. In this specimen, heterogeneous deposition of dentin matrix associated to mild tissue disorganization was observed. No bacterial contamination in the lateral cavity walls or in the pulp tissue was observed in any of the specimens.

In Group III, stained bacteria were seen along the outer third of the lateral cavity walls in 2 specimens, in which mild dentin matrix deposition was observed. In one of these specimens, mild inflammatory response occurred associated with tissue disorganization. However, moderate hard tissue barrier formation was observed in 4 out of 5 specimens in which no inflammatory response was determined (Figure 5).

60-Day Period

After 60 days of the procedure 4 teeth of Group I, 5 teeth of Group II, and 5 of Group III were analyzed.

At this period, an intense deposition of partially calcified dentin matrix underneath the thick layer of dystrophic calcification was observed in all groups. As a result, the dystrophic calcification associated with the dentin matrix gave rise to a noticeable hard barrier that remained between the capping material and the vital pulp tissue. It could also be seen a homogeneous monolayer of odontoblast-like cells underlying the tubular dentin matrix which was synthesized and deposited by these elongated pulp cells. Subjacent pulp tissue exhibited normal histological characteristics (Figure 6).

One specimen of group I (control) and 1 specimen of group III presented mild persistent inflammatory pulp response associated with mild tissue disorganization and in these specimens stained bacteria along the outer third of the lateral cavity wall were observed.

The Kruskal Wallis statistical test applied to the attributed scores for the histological parameters showed that the period of evaluation had a significant influence (p<0.05) on tissue disorganization and on hard tissue barrier formation in Group II. In the other groups (I and III) this test indicated that tissue disorganization, and hard tissue barrier formation were also influenced significantly (p<0.05) by the evaluation period and the inflammatory cell response.

There was greater inflammatory cell response (p<0.05) in Groups I and III than in Group II on the 7th postoperative day. However, no significant difference was observed among the three groups on the 30th and on the 60th postoperative days (p>0.05).

Discussion

Pulpotomy consists in cutting inflamed or infected coronary pulp and capping the remaining vital pulp tissue with a material that maintains its vitality18. This pulpal therapy has been strongly recommended for young permanent teeth, especially in clinical situations when a large pulp exposure is caused by decay or trauma or when the root is not completely formed7,12,14,16-18. In the present study, the antiinflammatory effect of Otosporin® application before (10 min or 48 h) pulp capping with calcium hydroxide was investigated.

Calcium hydroxide has been the material of choice recommended for pulpotomy7,12,14,16-20. The application of this material on pulpal wound causes superficial coagulation necrosis, which seems to participate in the pulpal healing process in association with hard barrier formation, although the calcium hydroxide mechanism of action is not yet well understood12-13,16-17,19-22.

The dentin bridge may be a sign of healing or mild irritation, and the histological reaction to direct calcium hydroxide application onto pulpal tissue has been shown in several studies12,16-17,20,22. In the present study, 7 days after application of this material, it could be seen the presence of odontoblast-like cells underlying the thick area of coagulation necrosis subjacent to pulp-capping agent, and only a mild inflammatory cell infiltrate. After 30 days, necrotic tissue in a dystrophic calcification process could be noted as well as the start of pulp repair with dentin bridge formation close to this tissue. Dentin bridge formation was apparently complete after 60 days of calcium hydroxide application, and the remaining pulp tissue presented normal histological characteristics.

The efficacy of calcium hydroxide as a pulp capping agent was reported to depend on its ability in releasing calcium and hydroxyl ions into the subjacent connective pulp tissue. The degree and speed of calcium and hydroxyl ion dissociation as well as the change in tissue pH values which the calcium hydroxide paste was applied to depend on the vehicle used to prepare the paste23 (distilled water, in the present study). At the postoperative period of 7 days, a large zone of coagulation necrosis was observed in contact with pulp-capping material. This was probably due to the vehicle utilized, which allowed a highly alkaline pH since the moment that it was put in contact with the tissue. A previous study on the effects of pH on enzymatic activity in pulp tissue24, have shown that the alkaline phosphatase activity, which is related to mineralization process, is increased by alkaline pH.

On the 30th postoperative day, the coagulation necrosis area presented calcification in its inner layer. A new odontoblastic layer was present, and newly formed dentin matrix was deposited nearby the pulpotomy area. This hard tissue barrier was larger on the 60th postoperative day. In all groups, it was characterized by the presence of dystrophic calcified tissue in the superficial layer and dentin deposition in the inner layer. These reactions are similar to those observed on healthy pulp tissue when direct calcium hydroxide application was made12,16-17,20,22.

In a previous study13, on the other hand, an irregular dentin bridge formation was seen and no calcification or odontoblastic layer formation were observed on the 90th postoperative day. In a longer postoperative period (6 month), calcification and necrosis underneath the dentin bridge, mild hyperemia and mild chronic inflammation dominated by lymphocytes were observed, but no odontoblastic layer formation was noted13.

Franz et al.25 in a study in humans and Yoshiba et al.26 in monkeys have also observed that the mineralized tissue barrier formed by pulps treated with calcium hydroxide consisted of a superficial osteodentin layer and a tubular dentin layer subjacent to that one, next to the pulp tissue. The thickness of this mineralized barrier increased in direct relationship to the length of the postoperative period25 and its production tended to be made far from the capping material26.

Corticosteroid can be used as a dressing agent in order to minimize pulp inflammation and consequently to provide relief of postoperative pain and sensisitivity1,4-5,8. However, there is no consensus regarding the benefits of the use of this agent. The results of studies that employ corticosteroids as a cavity liner support that these medications are effective in reducing or preventing postoperative thermal sensitivity1,8,27. On the other hand, when applied alone as direct pulp capping the results are poor3-5. Many investigators believe that the treatments are more successful when the products are used in combination with calcium hydroxide or when previous topical application is made before direct pulp capping3,28.

Researchers have shown that application of corticosteroid/antibiotic association for short period of time was effective to control inflammation in the pulp tissue without determining changes in the healing process3,18. Kakehashi et al.27 have reported that 1-min application of predinisolone onto pulp tissue appeared not to alter the normal healing process in the absence of microorganisms, whereas appeared not to retard the pulpal degeneration process in the presence of bacterial infection. In the present study, the histological analysis showed no inhibition on blood vessels proliferation or on collagen deposition processes after corticosteroid/antibiotic dressing application for 10 min or 48 h.

The absence of significant difference among control and treated groups II and III (10 min or 48 h Otosporin® dressing, respectively) on different postoperative periods is in agreement with the observation that the corticosteroid/antibiotic association has no effect when applied on healthy dental pulp11. Therefore, it can be suggested that previous pulp-capping application of a corticosteroid/antibiotic agent may be unnecessary in routine clinical situations of accidental pulp exposure without inflammation like complicated crown fractures or complicated crown-root fractures in which the search for dental treatment is immediate or in cases of pulpotomies.

When pulp tissue is inflamed, Souza and Holland3 have reported that dressing with corticosteroid/antibiotic association for 48 h produced best results, presenting complete hard barrier deposition with absence of dispersion of inflammatory cells in the remaining tissue. Also in inflamed pulp tissue, Santini18 has observed that corticosteroid/antibiotic association maintained vitality for a longer period without interfering in the healing produced by the use of calcium hydroxide in pulpotomy made in young permanent human teeth.

In the present study, significant differences were observed for the scores of tissue disorganization and reparative dentine formation in the postoperative periods in all groups. However, significant difference was detected only between Group I (control) and Group III (48-h Otosporin® dressing) for inflammatory cell response. In these groups, the intensity of the inflammatory cell response was greater at 7 days than in the other periods. In Group II (10-min Otosporin® dressing), no significant difference was found in the inflammatory cell response scores during the course of the experiment. Sazak et al.28 have also observed greater inflammation in Ledermix and calcium hydroxide association treated group when compared to calcium hydroxide alone on the 7th day.

Bacterial contamination is an important factor that may influence the response of injured pulp tissue and could be considered the primary cause of pulp death5,12,29. After direct pulp-capping with calcium hydroxide, tissue healing and dentin bridge formation will be promoted by the pulp as long as bacterial microleakage is prevented12. Bacteria can arise from the original carious lesion, from saliva, from the margins of the restoration by microleakage, and from the operator and instruments during the treatment5,12,29. In the present study, presence of bacteria in the most superficial third of the lateral cavity walls was detected in only 6 specimens. From these, mild inflammatory response and tissue disorganization were observed in only1 specimen of Group III on the 30th postoperative day. Since only noncarious teeth were used and the clinical procedures were performed under aseptic conditions, it may be assumed that bacteria probably reached the tooth-restoration interface through microleakage.

Within the limitations of present study, considering that the pulpotomy was performed in healthy teeth, it may be concluded that the use of a corticosteroid/antibiotic dressing before remaining tissue protection with calcium hydroxide had no influence on pulp tissue healing.

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