Periapical tissue repair of dog teeth after intracanal administration of capsaicin 0 . 075 %

Objetivo: O objetivo deste estudo foi realizar uma avaliação descritiva sobre a reparação tecidual da região periapical de dentes de cães. Material e Métodos: Foram utilizados trinta e seis canais radiculares, sendo dezoito preenchidos com capsaicina 0,075% e hidróxido de cálcio (pasta teste) e os outros dezoito canais preenchidos com hidróxido de cálcio (grupo controle). As análises histológicas foram realizadas para classificar a inflamação como ausente, leve, moderada ou grave. As análises também classificaram a presença ou ausência da reparação tecidual nos períodos de 7, 14 e 30 dias. Resultados: O uso de capsaicina 0,075% não alterou a resposta inflamatória e reparação quando comparado ao grupo controle. Conclusão: Nossos dados sugerem que o grupo teste, o qual utilizou capsaicina 0,075% como pasta intracanal, pode ser indicado para reparação tecidual. Além disso, outros experimentos devem ser realizados para avaliar a possível utilização clínica da capsaicina para odontalgias atípicas. ABSTRACT


INTRODUCTION
C apsaicin is a compound derived from the chili pepper extract [1].Repeated applications cause a prolonged period of hypoalgesia, which is generally referred to as desensitization.Therefore, Capsaicin has been recommended as treatment for a variety of neuropathic pain syndromes [2].
The atypical odontalgia is a neuropathic pain characterized by discomfort in a tooth or in a site where extraction has been performed, with no clinical or radiographic signs related to disease [3].Periapical tissue repair of dog teeth after intracanal administration of capsaicin 0.075%

Pavan NNO et al.
There is no agreement on the most appropriate therapeutic protocol for treating neuropathic pain, once this condition is difficult to diagnose and well elaborated clinical control studies to assess the effectiveness of drugs for atypical odontalgias are lacking [4].
Several drugs have been used in the treatment of atypical odontalgias and tricyclic antidepressants stand out among them [5].Besides,there is also an association with oral anticonvulsants [6], analgesics [7] and the use of topical capsaicin in mucosa [8].However, capsaicin topically applied in mucosa promotes intense irritation, characterized by the burning sensation, which leads to treatment interruption.The intracanal route could be an alternative use of capsaicin without causing adverse effects.
There are few studies [9] showing inflammatory and repair effect of capsaicin in living tissue by intracanal via, thus knowledge of the irritating potential of any dental material for living tissue is essential, because any constituents present in these materials could induce degeneration, irritation or tissue necrosis [10].
Capsaicin, in this experiment, is in close contact with the periapical tissues, and the principal features of the effects of capsaicin on primary afferent neurons are excitation, desensitization, neurotoxicity and selectivity [2].Our working hypothesis was that, although capsaicin takes an initial excitation, there is no exacerbate inflammatory response resulting from this technique.
Therefore, the aim of the present study was to histopathologically evaluate the periapical region of dogs teeth, following the application of 0.075% capsaicin paste.overfilled via canal.We performed a descriptive analysis of the inflammatory changes and subsequent repair, using the hematoxylin and eosin staining technique.

Animals
All surgical and experimental procedures were approved by the Ethics in Animal Research Committee of the State University of Maringá and were in compliance with the Ethical Guidelines of Animal Experimentation.
For the experiment, 36 teeth of three beagle dogs were used.The experiment included central and lateral incisors, second and third upper premolars, and second, third, and fourth lower premolars.

Anesthesia
The animals were anesthetized intravenously with 0.03 mg/kg of 0.2% acepromazine and 3 μg/kg fentanyl citrate, and Ringer's lactate solution was administered to maintain venous access.Anesthesia was induced with intravenous 3 mg/kg propofol and 2 mg ketamine to 10% (8 mg/kg ketamine; Agener Union Chemical Pharmaceutical National, Brazil).Anesthesia was maintained with isoflurane, and 0.5% bupivacaine was used for local anesthetic block.

Endodontic procedure and Experimental groups
After the dogs had been anesthetized, and teeth received rubber dam isolation, pulp chamber was accessed with #1090 cylindrical diamond bur.The pulp tissue was extirpated and the instrumentation up to #50 K-files (Dentsply Maillefer Instruments SA, Ballaigues, Switzerland) using the crown-down technique was performed.Irrigation was made with 2.5% sodium hypochlorite.After that, the apical foramen was cross-sectioned with Gates Glidden n°1 (Dentsply Maillefer Instruments SA, Ballaigues, Switzerland).The confirmation was performed by means of radiography.
Smear layer was removed with EDTA for 3 minutes and after that, root canal was finally irrigated with 2.5% sodium hypochlorite.Teeth were then separated into two groups.
The analyses were made at 7, 14 and 29 days after the material was introduced to root canals.Therefore, there were six teeth filled with calcium hydroxide + propyleneglycol + topical anesthetics and the remaining six teeth were filled with calcium hydroxide + propyleneglycol + topical anesthetics + 0.075% capsaicin for each evaluation period.Finally, the dogs were euthanized by overdose of anesthetics.

Material collected and Histology
The maxillary bones were cut, fixed in 10% formalin, and decalcified in sodium citrate and formic acid.Each tooth with sustention tissue was then processed for embedding in paraffin.Sections (6 μm) were cut and stained with hematoxylin and eosin.

Evaluation Methodology
Descriptive histological analysis was performed to classify inflammation as absent, mild, moderate, or severe [11].It also classified the presence or absence of repair.

days
Analyzing the control group, in the root canals, a moderate inflammatory infiltrate in the periapical region, with predominance of neutrophils and macrophages (Figure 1A), was observed at 7 days.The surrounding area of root apex, which was previously occupied by the alveolar bone, has been filled by richly vascularized loose connective tissue, providing blood supply responsible for phagocytic cell formation and transportion.Tissue repair began just after the elimination of aggressor agents and the rest of cell tissues [12].Granules of calcium hydroxide were visualized inside macrophages and were dispersed within the loose connective tissue.The alveolar bone indicates a significant resorption, leaving only few bone trabeculae, where there were many osteoclasts (Figure 2A).Cementum resorption was also observed (Figure 3).
At the capsaicin 0.075% group (test paste), there were no significant differences in the findings.Alveolar bone has undergone significant resorption (Figure 2B) and this reabsorbed region was filled with loose connective tissue, with inflammatory infiltration.In areas closer to overfilled paste, there was moderate inflammation.In more distant areas, the inflammation was classified as intense with predominance of macrophages (Figure 1B).Roots showed superficial cementum resorption, similarly in the control group.

days
At 14 days, the positive control morphology of periradicular region was similar to that observed at 7 days, except for a reduced number of osteoclasts (Figure 1C).
For the test paste, the morphology of periradicular region was not different, although at distant regions from the defect, blood vessels and fibroblasts with active morphology and irregular deposition of collagen are visualized.At this point in time, the tissue repair is beginning and a the presence of fibrosis tissue is visualized (Figure 4).The inflammatory response was moderate to intense (Figure 1D).

days
At 30 days, the control group presented inflammatory process, which was classified as moderate.It was circumscribed by a fibrous tissue that partially replaced the loose connective tissue (Figure 1E).Hydroxyapatite granules could be detected inside the vessels and macrophages (Figure 5).During this period, we observed the presence of cementum resorption and a significant reduction in number of osteoclasts at the trabecular bone surface (Figure 6).The test paste has shown that at the region immediately after the overfilled material, that loose connective tissue was replaced by dense connective tissue, which circumscribed the inflammatory infiltrate and was classified as mild (Figure 1F).Cementum resorption remained superficial (Figure 7).

DIscussIoN
In subchronic inflammation, the exudate is gradually resorbed and the neutrophilic infiltrate is replaced by mononuclear leukocytes with macrophages predominance, and these histological events could be observed in this experiment at 7 and 14 days.
During these periods, in the 0.075% capsaicin group, the inflammatory response was classified as moderate with predominance of polymorphonuclear cells, and at 30 days the inflammatory infiltrate was regarded as mild, indicating the evolution of the inflammatory process to its resolution.(Figure 1E) The inflammation of the test group has demonstrated similarity to the control group, evidencing that capsaicin does not intensify the damaged tissue.This is probably due to desensibilization induced by capsaicin which led to changes in the release of substance P, neurokinin A, Calcitonin gene-related peptide (CGRP) and other neurotransmitters [13].
Therefore, the inflammatory response seen in the three periods was due to overinstrumentation of the apex, procedure that was performed to promote contact of the material with periapical region, and also to promote contact with calcium hydroxide [14], present in both groups.This tissue damage induces cytokines release, growth factors and arachidonic acid products [12], which also participated in the initiation and maintenance of vascular phenomena, especially as the aggression found in our study that has been classified as mild to moderate.
The central mechanism for connective tissue repair is inflammation, and repair can be considered the last event of inflammatory response.
Repair of the 0.075% capsaicin group was similar to the control group.At 14 days, morphologically active fibroblasts could be noticed in areas more distant from defects (Figure 4), demonstrating the beginning of repairing process, and also the presence of blood vessels (Figure 4) important for the repair, since neovascularization resulting from the surgical site is an essential event [15].
At 30 days, at the periapical region immediately around the overflowed material, a wide area of dense connective tissue circumscribing the mild inflammatory infiltrate is observed (Figure 1E and 1F).
Capsaicin in this experiment has not affected the speed or quality of repair when compared to control group, therefore it is not a modifying factor in the repair process.
However, there are studies indicating reduction in the healing degree of the peripheral tissue when in contact with capsaicin, besides the increasing rate of the dental pulp necrosis [16].Perhaps, these effects occur because capsaicin depends on the exposure time, its formulation and certainly its concentration, because capsaicin is dose-dependent [17].
Results obtained in this experiment are in agreement with the findings of another study [9], where the intracanal route has also been used in dog teeth, although capsaicin was used with concentration at 0.025% and 0.05%.
Intracanal administration of 0.075% capsaicin in dog teeth did not show histological changes in inflammatory events and repair at the doi: 10.14295/bds.2015.v18i1.1070 Periapical tissue repair of dog teeth after intracanal administration of capsaicin 0.075% Pavan NNO et al.

Figure 5 -
Figure 5 -After 30 days of root canal filling, showing hydroxyapatite granules inside the vessels and macrophages, respectively (A and C) (100x magnification, H-E staining) and 0.075% capsaicin (B and D) (40x and 100 magnification respectively, H-E staining).
Periapical tissue repair of dog teeth after intracanal administration of capsaicin 0.075% Pavan NNO et al.