Evaluation of optical density of rats tibiae bone repair treated with rofecoxib

The aim of this study was to evaluate the influence of rofecoxib on bone repair process in tibiae of rats by means of analysis of gray levels acquired by digital optical density. Ten Wistar rats were assigned to two groups: an untreated control group and a group treated with rofecoxib. The experimental procedures comprised the following stages: general anesthesia, preparation of unicortical bone defect on the left tibiae of each rat, medication with rofecoxib and radiographic examination. Digital radiographic images were taken using Visualix GX-S-HDI digital sensor, an X-ray equipment. Radiographics were taken at baseline, 7, 14, 21 and 30 days postoperatively and the gray levels were evaluated using the Vix win 1.4 system of the region of interest (ROI). The mean values of ROI readings were analyzed statistically by repeated measures ANOVA with significance level of 5%. There were no statistically significant differences among gray levels between rofecoxib and control groups (p=0.2429), but there were statistically significant differences between times baseline, 7, 14, 21 and 30 in both groups (p=0001). The findings of this study suggest that no influence was found when rofecoxib was used in bone tibiae repair after 30 days post-treatment.

NSAIDs that inhibit only COX-2, but not COX-1, scientists hypothesized they could offer the same pain relief as traditional NSAIDs, but with greatly reduced risk of fatal or debilitating peptic ulcers 9,12,14 .
The nonsteroidal anti-inflammatory drugs have been investigated to influence the process of bone repair.Martins et al. 10 suggested that Ketophofen influenced bone repair process by observing delayed new bone formation after 21 days and de lima et al. 5 associated the intake of NAIDs with bone repair process and have demonstrated that bone is one of the few tissues with remodeling capacity, being able to recover its structure and function after a trauma.
Digital radiographs allow evaluating radiographic bone repair analyzing the regions of interest (ROI) by using the scale of gray levels that correspond to the optical density of regions.The direct digital radiographs can be acquired utilizing CCD system (Charge Couple Device) that uses a silicio chip to capture the images that can be manipulated using a software that assist in image visualization which can improve the diagnostic accuracy 15,8,4 .
Based on these considerations, the purpose of this study was to evaluate the influence of rofecoxib, a selective COX-2 inhibitor (cyclooxygenase-2 inhiBitors), on the bone repair of unicortical defects created in tibiae of rats by analysis of the digital optical density (OD) at different periods of time.

mAteriAl And methods
Ten adult male rats from the same line (Rattus norvegicus albinus, Wistar) aged 120 days and weighing from 350 to 400g were obtained from the animal laboratory of the faculty of Dentistry of São José dos Campos (UNESP).The animals were housed in proper cages and environment and were maintained on laboratory chow (labina) and water ad libitium.The study protocol was approved (protocol number, 031/2003) by the Ethical Committee of Research of the School of Dentistry of São José dos Campos-UNESP.
The skin over the left tibiae was shaved and antisepsis was performed with iodized alcohol made on the proximal area of the tibiae with an interchangeable #5 blade mounted on a Bard-Parker handle and a #7 spatula was used to retract the soft tissues and periosteum.A perforation was made on the bone with a 3mm diameter trephine bur coupled to an electric engine (Kavo do Brasil SA, Joinville, SC, Brazil) at 1,100 rpm speed and under constant irrigation with saline (fig 1A and B).Skin was sutured with silk suture number 4 (Ethicon-Johnson & Johnson, Somerville, NJ, USA) and antisepsis was done with iodized alcohol.
for the radiographic examinations, the rats in the control and rofecoxib groups were initially submitted to general anesthesia by application of the same anesthetics, syringes and needles used for surgery, figure 1 -A although the anesthetic dose in this stage range from 0.08 to 0.1ml.Thereafter, each rat was positioned in dorsal recumbecy on a supporting table.Before the baseline direct digital radiographs were obtained, an impression was taken from the left tibiae with low fusion impression compound (Godibar; lysanda Produtos Odontológicos, SP, Brazil), in a way that the initial position could be reproduced in the further stages.Impression was taken on the lower portion of an individual metallic clamping device shaped as a retangular box, with 3 free ends and a groove for fixation on the supporting table (fig.2).Visualix GX-S-HDI TM digital sensor (Gendex Dental System, Dentisply International, Chicago, Il, USA) was placed on the lower portion (free impression material) of each device.
Because it is a CCD (Charge-Couple Device) sensor, the image could be immediately transmitted to a computer screen.Thereafter, the tibiae was introduced in the clamping device, positioned on upper portion of the sensor and fixed to the device and impression mould with #9 utility wax (Epoxiglass Ind. Com.De Produtos Quimicos ltda, Diadema, SP, Brazil).In this way, the bone defect was also turned upwards and the initial position could be reproduced in all evaluation periods (fig.2.).Afterwards, the left tibiae of each rat was radiographed with an X-ray equipment (Spectro-70X TM ; Dabi Atlante, São Paulo, SP, Brazil) at 10mA and 65kVp; with 0.10 s exposure time and 25cm focus-object distance (fig.2).Radiographs were taken from rats in both groups at baseline and 7, 14, 21 and 30days after unicortical bone defects were created on the left tibiae of the animals.Optical density was evaluated using Vix win TM 1.4 system (Gendex Dental System) and are expressed by gray levels.The gray levels were analyzed statistically by repeated measures -Analysis of Variance -ANOVA and Tukey test (Statitix for windows (version 8.0, 2003, Analytical Software Inc. Tallahassee, florida, USA) at 5% significance level.Statistical assumptions were evaluated before statistical analysis.The results indicated that the residuals were normally distribute and, by plotting against predicted values, the uniformity was checked, then none of the ANOVA assumptions were violated.

resUlts
Repeated measures ANOVA showed no significant differences between control and rofecoxib groups (p=0.2429), and interactions between treatment and periods (0.5678), while there were statistically significance differences (p=000.1)among the elevation periods to optical density expressed by the gray levels (Table 1).The Tukey test revealed that the rofecoxib and control groups presented the height levels of gray in 7 days, after which, the values decreased until the 30 th day (Table .2).

discUssion
This study evaluated the tibiae bone repair of rats treated with rofecoxib, a selective COX-2 inhibitor, and control groups using direct digital radiographic techniques evaluations of tibiae bone repair.The findings of the present study show that NSAIDs did not interfere with bone remodeling process in NSAIDs groups after 30 days.The NSAIDs were selected to this study because they were prescribed for the treatment of various conditions that require analgesia and anti-inflammatory activity.People also have access to NSAIDs for relief mild aches, pain, and headaches.The NSAIDs are specific inhibitors for COX-2 and inhibited while sparing the activity of COX-1.NSAIDs were available (celecoxib, rofecoxib, valdecoxib (in order of US market release), however rofecoxib was withdrawn from the market in September 2004 due to clinical responses.white 14 and Kondro et al. 7 report that rofecoxib doubled the risk of heart attack or stroke if it was taken for longer than 18 months.The risk was thought to be due to blood clots.Even though, others COX-2 inhibitors have still been commercially available on the market 13,14 .
Clinical studies have been conducted not only to analyze vascular injuries but also the effect of NSAIDs on bone repair.de lima et al. 5 suggested that these drugs have a potential to stimulate bone healing.Although Allen et al. 2, yazdi et al. 16 , Altman et al. 1 and Ho et al. 6 have reported that NSAIDs may inhibit bone repair depending on type of drug, period of intake and dosage administered.An human study conducted by Brägger et al. 3 showed that no influence on bone repair was observed in human with periodontal disease treated with NSAIDs.The findings of the present study did not show differences between bone remodeling process among the two groups analyzed after 30 days.
Other aspect of these evaluations was the use of digital radiographs to evaluated bone repair.Digital radiographic images were acquired using Visualix GX-HDI TM digital sensor.This CCD (Charge couple device) is composed by a silicio sensor smaller than the conventional periapical film that has bidimensional transistor arrangements.Each arrangement corresponds to a pixel, which represents the smallest image formation unit responsible for radiographic contrast (each pixel can vary in a scale from 250 shades of gray), and the set of lines and columns corresponds to the digital matrix of the photon receptor.The great advantage in utilizing this system was that it allows image manipulation, great detail richness in addition to the possibility of optical density evaluation and measurement 4,11,15 .
Martins et al. 10 used the digital radiographic method to evaluate the bone density gain of rats' tibiae -like our study -that had Ketoprofen administrated for 30 days compared to control group.Although the authors' results found difference in optical density repair after 21 days, our study could not find the same results when rofecoxib was administrated.However, both studies agree that the evaluation method of optical density was efficient in detecting changes during the studied evaluation periods.
The results of the present study must be analyzed with caution, since they regard a pilot study with a medication already withdrawn from the market, however different NSAIDs that inhibit COX-2 are still available in the market.Therefore, we suggest that other studies are conducted with different medication that present the same action mechanism which can influence bone tissue repair response in order to observe weather the results are maintained with a lager number of subjects and longer evaluation time.conclUsion within the limits of this study these findings indicate no influence of rofecoxib on bone repair process when it was administered until the 30 th day.
figure 1 -A.Perforation being made on rat left tibiae; B. Unicortical bone defect.