Comparative evaluation of the efficacy of the cyclooxygenase pathway inhibitor and nitric oxide synthase inhibitor in the reduction of alveolar bone loss in ligature induced periodontitis in rats: An experimental study.

BACKGROUND: Alveolar bone loss is the most striking feature of periodontal disease. The aim of this study was to investigate the effect of a cyclooxygenase (COX) pathway inhibitor and nitric oxide synthase (NOS) inhibitor in the reduction of alveolar bone loss in an experimental periodontal disease (EPD) model.
MATERIALS AND METHODS: The study was conducted on 60 Wistar rats divided into three groups of 20 rats each and then subjected to a ligature placement around the left maxillary second molars. Group 1 rats were treated with COX inhibitor (diclofenac sodium 10 mg/kg/d), group 2 with NOS inhibitor (aminoguanidine hydrochloride 10 mg/kg/d) and group 3 served as controls, receiving only saline, intraperitoneally 1h before EPD induction and daily until the sacrifice on the 11(th) day. Leukogram was performed before ligation, at 6 h and at the first, seventh and 11(th) days after EPD induction. After sacrifice, all the excised maxillae were subjected to morphometric and histometric analysis to measure the alveolar bone loss. Histopathological analysis was carried out to estimate cell influx, alveolar bone and cementum integrity.
RESULTS: Induction of experimental periodontitis in the rat model produced pronounced leucocytosis, which was significantly reduced by the administration of diclofenac sodium and aminoguanidine on the 11(th) day. In morphometric and histometric examinations, both the test drugs significantly (P < 0.05) inhibited the alveolar bone loss as compared with the control group.
CONCLUSION: Both COX inhibitor and NOS inhibitor are equally effective in inhibiting the inflammatory bone resorption in an experimental periodontitis model.

INTRODUCTION

The most frequent cause of tooth loss in adults is periodontitis. It represents a complex host-parasite interaction in which the local release of mediators such as prostanoids and cytokines leads to the destruction of tooth supporting connective tissues and alveolar bone.[12] The bacterial toxins, enzymes and metabolites present in the dental plaque play a key role in the initiation of this inflammatory process.[3]

Administrations of non-steroid anti-inflammatory drugs (NSAIDs) which inhibit prostaglandin synthesis by inhibiting the cyclooxygenase (COX) pathway, have proven beneficial in treating periodontitis in humans.[45] Also, retrospective human studies have also shown less periodontal destruction in subjects chronically ingesting various anti-inflammatory drugs in comparison with controls.[67] Diclofenac sodium is a one such NSAID that is most commonly used in the management of acute and post-operative pain. The majority of anti-inflammatory effect of diclofenac is due to inhibition of prostaglandin synthesis by COX pathway inhibition.[89]

Another molecule involved during inflammation is nitric oxide (NO). It is a free radical produced from L-Arginine through the action of isoenzymes globally termed as nitric oxide synthases (NOS). The nitric oxide synthase exists in three different isoforms: Two consecutive endothelial NOS (eNOS), brain or neuronal NOS (bNOS) and inducible NOS (iNOS). The eNOS and bNOS produce low NO concentrations for a small time period: Conversely, iNOS, identified in several cell types such as macrophages and polymorphonuclear cells, is expressed in response to inflammatory stimuli such as IL-1, TNF-α, IFN-g and LPS, preferably in a synergistic manner, yielding high amounts of NO for a long time period, which results in tissue destruction,[10] It is a ubiquitous internal messenger in vertebrates, modulating blood flow,[11] regulating platelet aggregation[12] and neurotransmission.[13] The production of NO is also important for non-specific host defense, helping to kill tumors and intercellular pathogens.[11]

Biopsy specimens of periodontitis affected tissue have shown to have a greater level of iNOS expression than the healthy tissue, suggesting the definite role of NO in the inflammatory response of periodontal tissues.[14] Furthermore, the NO synthase inhibitor, aminoguanidine hydrochloride (AG), has shown significantly inhibited alveolar bone loss as compared with the control group in an experimental disease model.[1516]

Considering the lack of studies that assess the effect of diclofenac on alveolar bone loss, the present investigation was undertaken to assess and compare the efficacy of the COX pathway inhibitor (diclofenac) and NO synthase inhibitor (aminoguanidine) on alveolar bone loss in an experimental periodontal disease (EPD) model in rats.

MATERIALS AND METHODS

Animals

Thirty male Wistar rats were procured from Venkateshwara Enterprises, Bangalore, for the purpose of conducting this experimental study. The selected rats were 6 weeks old and weighed 150-200g. The rats were then transferred to the animal house, Bapuji Pharmacy College, Davangere, and were housed in plastic cages using husk as the bedding material. The animals were subjected to normal atmospheric conditions at 21°C, and subjected to the same regimen of lighting (12 h of light and 12 h of dark) and the same time of feeding and handling. Animals received rodent diet in the form of pellets and water ad libitum.

Ethical approbation

Ethical clearance for the experimental study was obtained from the Ethical Committee, Bapuji Dental College and Hospital, after a review of the experimental protocol.

Induction of EPD

A sterilized black 2-0 braided silk thread (ligature*) was placed around the cervical portion of the upper left second molar tooth of the rats under ketamine† anesthesia (0.08 mL/100 g).[17] The ligature was knotted on the buccal side so that it remained subgingivally in the palatal side and supragingivally on the buccal (vestibular) side. The contralateral side was used as the unligated control.

A blood sample (1 mL) was taken from the orbital sinus of the rat eye and added to a tube containing EDTA‡ anticoagulant immediately before and 6 hr and 1, 7 and 11 days after the induction of periodontitis. Total WBC count was performed using a Neubauer chamber, whereas the differential count was performed using smears stained with Leishman's stain.§

Study design

Thirty Wistar rats were randomly divided into three groups of 10 rats each. Group 1 rats received diclofenac sodiumk 10 mg/kg (i.p.) in 1% Tween’ 80 solution¶, whereas group 2 received aminoguanidine hydrochloride# 10 mg/kg (i.p.) in 1% Tween’ 80 solution; group 3 , the control group, was injected with saline (i.p.) 1 h before the induction of EPD and daily for 11 days. The rats were sacrificed on the 11th day by cervical dislocation. Their maxillae were excised and fixed in 10% neutral formalin for 24-48 h. After defleshing the excised maxillae, the jaws were subjected to morphometric, histometric and histopathological analysis.

Morphometric analysis

In order to measure the bone loss morphometrically, images were captured using a three- chip CCD camera** (Preview, Media Cybernetics, Bethesda MD, USA) attached to a stereomicroscope†† (Olympus, Tokyo, Japan) with a 7X objective. The actual measurement was done using Image Pro Plus software‡‡ V- 4.1.0.0 (Media Cybernetics). Measurements were made along the long axis of the vestibular root surfaces of all the molar teeth and the distance from the cusp tip to the alveolar bone crest was measured based on the method described by Leito.[15] Three readings for the first molar and two readings for the second and third molars were made. Alveolar bone loss was obtained by subtracting the values of the unligated side from the ligated side. The sum of these seven values was used as a measure of the total alveolar bone loss on the vestibular surface for each animal.

Histometric analysis

After the measurement of bone loss, same specimens were decalcified using 5% nitric acid and 5% formic acid for 4-5 weeks. The specimens were then dehydrated, embedded in paraffin and sectioned along the molars in a mesio-distal plane for hematoxylin and eosin staining. Three sections of 5 μm each, which included two roots of the first and two roots of the second molars, were used. Images of the second molar furcation area were captured using a three- chip CCD camera (Media Cybernetics) attached to a research microscope (Olympus) with 4X objective. Measurements were made using Image Pro Plus software V-4.1.0.0(Media Cybernetics). Bone loss in the furcation region, i.e., the area between the bone crest and the cementum surface was measured histometrically (in microns) in the decalcified specimens based on the method described by Nociti et al.[18]

Histopathological analysis

After histometric analysis in the same histological section, the areas between the first and second molars, where the ligature was placed, were analyzed under a research microscope§§ with a 2.5X objective (Leica DMRB®, Erst-Leitz Straβe, Wetzlar, Germany). Considering the inflammatory cell influx and alveolar bone and cementum integrity, 0 to 3 score was given as described previously.[15] Score 0: Absence of or only discrete cellular infiltration (inflammatory cellular infiltration is sparse and restricted to the marginal gingival region), preserved alveolar process and cementum. Score 1: Moderate cellular infiltration (inflammatory cellular infiltration present all over insert gingival), some but minor alveolar process resorption and intact cementum. Score 2: Accentuated cellular infiltration (inflammatory cellular infiltration present in both gingival and periodontal ligament), accentuated degradation of the alveolar process and partial destruction of cementum. Score 3: Accentuated cellular infiltration, complete resorption of the alveolar process and severe destruction of cementum.

Statistical analysis

Statistical analysis was carried out by one-way analysis of variance (ANOVA; single factor) followed by Bonferroni's test to compare means and Kruskal - Wallis and Mann-Whitney tests to compare medians. A P < 0.05 was considered statistically significant.

RESULTS

Both diclofenac sodium and aminoguanidine highly significantly (P < 0.001) reduced the leucocytosis and neutrophilia at 6 h and 1 day after ligation when compared with the control group and also diclofenac sodium and aminoguanidine significantly (P < 0.05) reduced the lymphocytosis observed at the 11th day when compared with the control group.

Effect of diclofenac sodium and aminoguanidine on alveolar bone loss

Both diclofenac sodium and aminoguanidine hydrochloride at the dosage of 10 mg/kg significantly (P < 0.05) reduced the alveolar bone loss in an EPD as measured morphometrically [Figure 1] and also histometrically when compared with the control group [Figure 2]. Figure 3a shows the macroscopic aspects of the contralateral side (unligated side) with no resorption of the alveolar bone, severe bone resorption in the control group [Figure 3b] and reduced bone resorption in the groups treated with diclofenac sodium [Figure 3c] and aminoguanidine hydrochloride [Figure 3d]. The histopathological analysis of the region between the first and second molars on the contralateral side not subjected to EPD, showing the structure of the normal periodontium where periodontal ligament, alveolar bone, cementum and dentin can be observed [Figure 4a]. The histopathology of the periodontium of the animals subjected to EPD and received no treatment revealed severe cementum destruction and complete alveolar process resorption [Table 1 and Figure 4b], receiving a median score of 3, whereas histopathology of the periodontium of animals subjected to EPD and received diclofenac sodium and aminoguanidine hydrochloride revealed minor alveolar bone resorption and intact cementum, receiving a score of 1 [Table 1, Figure 4c and d]. These valves were statistically highly significant (P < 0.001) when compared with the control group.

Figure 1

Effect of diclofenac sodium and aminoguanidine hydrochloride on experimental periodontal disease. Data represent the mean of 20 rats in each group. FNx01P < 0.05 was considered statistically significant compared with the rats subjected to periodontitis, which received only saline

Figure 2

Photomicrograph: (Hematoxylin and eosin, ×4) showing the furcation area in relation to the second molar tooth. (a) No bone resorption in the group treated with diclofenac sodium. (b) No bone resorption in the group treated with aminoguanidine hydrochloride. (c) Severe bone resorption in the control group

Figure 3

Macroscopic aspects of the periodontium. (a) Contralateral side (unligated side) with no resorption of the alveolar bone. (b) Severe bone resorption in the control group. (c) Reduced bone resorption in the group treated with diclofenac sodium. (d) Reduced bone resorption in the group treated with aminoguanidine hydrochloride

Figure 4

Histopathology of the periodontium of rats subjected to experimental periodontal disease. Photomicrograph: (Hematoxylin and eosin, ×2.5) showing the first and second molar interdental regions. (a) Normal periodontium where periodontal ligament, alveolar bone, cementum and dentin are intact (unligated side). (b) Severe cementum destruction and complete alveolar process resorption (control group). (c) Minor alveolar bone resorption and intact cementum (group treated with diclofenac sodium). (d) Minor alveolar bone resorption and intact cementum (group treated with aminoguanidine hydrochloride)

Table 1

Histopathological analysis (scores) of rat maxillae with experimental periodontitis

DISCUSSION

Periodontitis is a local inflammatory reaction that results in the destruction of the dental attachment apparatus thus provoking tooth loss. The pathogenesis of periodontitis involves the release of various inflammatory mediators. Among these mediators, prostaglandins following COX pathway activation and cytokines such as IL-1 and TNF-α seem to be important mediators in periodontitis.[192021] These proinflammatory cytokines induce the NO production, which may act as a stimulator of bone resorption.[2223]

There is overwhelming evidence that correlates PGE2 levels within the periodontal tissues and within the crevicular fluid (CF) to the clinical expression of periodontal disease.[2]

Gomes et al.[24] and Heijl et al.,[25] have suggested that prostaglandins produced during an acute inflammatory response may be an important mediator for the generation of bone resorption. Retrospective human studies have also shown less periodontal destruction in subjects chronically ingesting various anti-inflammatory drugs in comparison with controls.[2627] Diclofenac sodium is one such commonly used NSAID derived from phenyacetic acid which inhibits prostaglandin synthesis and also reduces neutrophil chemotaxis and superoxide production at the inflammatory site. It is a potent inhibitor of COX, both in vivo and in vitro. Hence, it is indicated in the management of acute and chronic painful conditions and post-operative and post-traumatic inflammation.[89]

Another important inflammatory mediator is NO, a short-lived, reactive-free radical and a ubiquitous intercellular messenger molecule with important cardiovascular, neurological and immune functions. NO plays a multifaceted role in the inflammatory and immune responses depending on the source of NO produced. Low concentrations of basally produced NO maintain normal homeostasis and are protective under physiological conditions in circumdental tissues. However, NO may be detrimental when produced in excess in inflammation and may destroy the host tissues as well not only invading microorganisms.[28]

Matejka et al. have shown that the gingiva contains increased L-arginine and citrulline levels, which are the precursor and the by-product of NO synthesis, respectively, in human periodontal disease[29] and Lappin et al. also have shown a greater level of iNOS expression in periodontitis - affected tissue than the healthy tissue. The source of iNOS in the periodontal tissues, as determined by monoclonal antibody, is the macrophage, with the endothelial cells.[14] The resident bacteria of the gingivomucosal tissue induce an increase in reactive nitrogen species, which is greatly enhanced by plaque formation in periodontitis.[30]

The present study supported the observations made by other authors by showing that ligatures placed subgingivally around the second molar teeth stimulated periodontal breakdown and alveolar bone resorption probably due to microbial plaque, the associated microorganisms and their toxins.[31] Although dogs, non-human primates and ferrets are the promising models for studying periodontal disease, in our study, rats were used because of their ease of availability, handling and maintenance and because they are less expensive.

Aminoguanidine 10 mg/kg body weight as described by Leitao et al.[15] and diclofenac sodium 10 mg/kg body weight was given.[8] Both the drugs were given by dissolving them in 1% Tween 80’ solution, which has an added advantage of reducing gastric irritation, other than just increasing the solubility. The administration of aminoguanidine decreased the neutrophilia observed at 6 h and lymphocytosis observed at Day 11 after ligation compared with the control group, similar to the observations made by Leitao et al.[15] Likewise, diclofenac sodium therapy also reduced the neutrophilia observed at 6 h and the lymphocytosis observed at Day 11 after ligation when compared with the control group. Similar effects were observed with other NSAIDS like meloxicam and indomethacin.[32]

Administration of aminoguanidine reduced the alveolar bone loss when compared with the control group, similar to observations made by Leitao et al.[15] Diclofenac sodium also reduced the alveolar bone loss when compared with the control group. This observation is also in accordance with the findings of a previous study conducted by Bezerra et al.[32]

Histometric measurement of bone loss was carried out in the furcation area, i.e., the area between the bone crest and the cementum surface, as described by Nociti et al.[16] Nociti et al. also showed that both diclofenac and aminoguanidine were equally potent in reducing the alveolar bone loss compared with the control group.

The histopathology of the periodontium in the experimental groups treated with diclofenac sodium and aminoguanidine revealed minor alveolar bone resorption and intact cementum, whereas the control group revealed severe destruction of alveolar bone and cementum This shows that the administration of diclofenac sodium reduced degradation of alveolar bone and destruction of cementum compared with the control group. Similar observations were made by Bezerra et al. while studying the effect of other NSAIDs like indomethacin on bone resorption.[32] Similarly, aminoguanidine also reduced the degradation of alveolar bone and destruction of cementum compared with the control group. These findings are in accordance with the observation made by Leitao et al.[15]

Unlike hemogram, the histopathology and the measurement of bone loss were not sequentially determined. Hence, the actual amount of bone loss at different time periods could not be estimated. Therefore, further studies with an increased sample size are required.

In the past few years, chemotherapeutic approaches for the management of periodontal diseases have largely focused on controlling the bacterial etiology using antibiotics, antiseptics and antimicrobial agents. Relatively, very few pharmacological approaches have been considered for uncoupling the host-mediated periodontal destruction. Certainly, one could not rationalize using such a chemotherapeutic approach without also addressing the etiological component. However, NSAIDs like diclofenac sodium and NOS inhibitors like aminoguanidine are effective in preventing tissue destruction, even in the absence of a well-defined etiology.

This study was conducted in an animal model and the results can serve only as indirect evidence taking into consideration the difference between animals and humans. Further studies are needed in this regard as no evidence exists in the dental literature that focuses on the effect of diclofenac sodium on the alveolar bone. In the coming era, the use of drugs such as diclofenac sodium, which inhibits prostaglandin synthesis, and aminoguanidine hydrochloride which inhibits NO synthesis, may prove to be a highly successful adjunctive chemotherapeutic agent in the management of periodontal diseases in humans.