LINEAR DENSITY ANALYSIS OF BONE REPAIR IN RATS USING DIGITAL DIRECT RADIOGRAPH

1PhD Student Department of Stomatology, Division of Dentistry Clinics, São Paulo Dental School, FOUSP. Professor Department of Dentistry Clinics, University Center Hermínio Ometto, UNIARARAS. 2Professor Department of Stomatology, Division of Dentistry Clinics, São Paulo Dental School, FOUSP. 3Professor Department of Radiology, Piracicaba Dental School, FOP-UNICAMP. 4Assistant Professor, Department of Radiology, University Center Hermínio Ometto, UNIARARAS. 5Professor Department of Stomatology, São Paulo Dental School, FOUSP Adjunct Professor Department of Radiology, College of Medicine, The University of Iowa, USA.


INTRODUCTION
Direct digital radiographic image has appeared with the aim of achieving improvement in interpretation with a reduction in the radiation dose, allied furthermore to greater facility in obtaining radiographies, with consequent reduction in working time 6,7,12,13,20 .
This imaging modality offers a great potential to facilitate the manner of diagnosing and treating dental pathologies 12,19   .The accuracy of direct digital dental radiography and the assessment of alveolar bone structures for detection of bone defects were described in literature by some authors 2,10,11,14,15   .However, advances in densitometry analysis using direct dental digital radiograph tools, with a rigorous control of experiment, were not described in the literature.
Thus, the purpose of this study was to assess the usefulness of direct digital radiographic analysis tools, for measurement of bone density for analysis of repairing fractures in rats under Dexamethasone effects.

MATERIAL AND METHODS
The study population consisted of 30 young male adult Wistar rats with an average weight of 220 grams, provided by the by Central Vivarium of the Biomedical Institute of University of São Paulo, following approval by the institutional review committee.
General anesthetics was done by inhalation of ethyl ether, each animal being placed in a closed glass vat containing cotton wool soaked with ethyl ether * .After anesthesia had been induced, it was maintained by placing a test tube containing ethyl ether close to the animal's nose, so that it would be breathed in during the surgical procedure.
The surgical area was decontaminated by application of 4.0% chlorhexidine digluconate, and it was isolated by surgical fields so that there would be no contamination from neighboring areas.An incision of approximately 3.0cm was made in the anterior lateral part of the animal's back using a scalpel with handle number 03 and blade number 15.The tissue was severed by using blunt-nosed scissors, until the animal's rib was exposed.Once exposed the rib was fractured with surgical scissors.
Suturing was done plane by plane with mono nylon 5.0* mounted thread, in order to assure adequate closing of the wound.After surgery was concluded, the region was cleaned with 4% chlorhexidine digluconate.
The animals were divided into groups according to the therapeutic prescription.The drugs were administered intraperitoneally, using insulin syringes* 1 hour before intervention, according to the following posological scheme: Group C (control) -15 animals: administration of saline solution IP 1 hour before surgery, in the same volume used for the corticosteroid.
The animals were kept in cages and separated by groups, with food and water ad libitum.They were sacrificed by intoxication with ethyl ether to allow specimens to be obtained referring to periods of time of 2, 3, 4, 7, and 14 days.
Thereafter, the anatomic parts containing the hemi-thorax of each animal were removed.The muscular insertions were removed and each specimen was catalogued and kept individually in 10% buffered formaline.
Bone densitometry was used as the method of analysis.The Digora system (Soredex, Finland) was used, which characteristic is a sensor (phosphorous board) of size and active image area of 30mm x 40mm, similar to a periapical radiographic film.Its matrix (image size) measures 416 x 560 pixels and the size of the pixel is 71 x 71 micrometers having a resolution of 6.1p/mm.The radiographs were taken by placing the specimen on the sensor and using an X-ray machine with 50kVp and 10mA.The focus-film distance was 40cm and the exposure time used was 0.5 seconds.After exposure, the sensor was placed in the Digora scanner for reading and processing.Twenty-four seconds later, the image on the computer screen could be worked on with a linear density reading tool.From the image, the computer mouse was used for determining the line over the fracture stubs (Figure 1).The system presented the maximum and minimum density of this line, as well as the mean density in pixels, the latter being used as the result.From the intensity given by the measurements of the scale in gray color identifiable by the human eye, it was possible to measure the bone density (Figure 1).The maximum degree of density established is 256, which corresponds to white; and the minimum degree of density is zero, which corresponds to Statistical analysis of linear density was done considering the time of treatments: Analysis of Variance (ANOVA) was used (p=0.05).The Tukey test for multiple comparisons of measurements was elected when the analysis of variance detected the significant effect of a factor.It was also defined that the factor time should be analyzed by a polynomial regression in order to check its effect on the response variable (linear density).In all calculations the SAS β System (Statistical Analysis Systems, Institute Inc., Cary, NC, USA) was used though the GLM procedures and the SAS/LAB software 17.

RESULTS
The method made feasible to supply data that were statistically significant in assessing the gain in bone density during the period of time analyzed.Also the doses of employed Dexamethasone did not significantly alter the gain in bone density.
In the Control Group, the linear density increased as time passed, showing evidence of differences, the linear density measurements at the end of the treatment (14 days) being significantly greater than the measurements at the times of 2 and 3 days.
In Group 1, the increase in linear density is evident when one compares the specimens at 14 days to the specimens at 2, 3, and 7 days (Figure 2).
After the polynomial regression, it was possible to cross the information of the previous analyses and observe that in the linear equations for the Control Group and Group 1 gains in linear density are observed (sloping trend lines -Figure 3), showing positive association between time and gain in linear density.

DISCUSSION
There was concern about creating an experimental model that would provide conditions for checking the increase in linear density at the stubs of fractures in rats, with or without the influence of Dexamethasone, by means of Digora digital radiography program tool 8 .
The glycocorticoids act by inhibiting phopholipase A2 and also interfere in the work of cytokines at the level of the regulating genes, influencing not only the repair phase but also the bone metabolism.This influence is related to the dosage and with the time that drug is administered 1 .In the present study, the posology was followed for modulating the inflammatory response made by injuries, in the case of the fracture of a rib.An analysis was made in order to verify if there was an alteration in the density of this location of the fracture, according to the dosage.The doses of Dexamethasone were extrapolated from the dose used for a young, normal reactive adult from 70 to 80Kg, and also from doses used in protocols for reducing the inflammatory effects after surgeries including third molars 4 .Some experimental model used glycocorticoids in large quantities for long periods of time, with the intention of provoking osteoporosis, obtained a greater bone repair inhibitory effect in the group treated with Dexamethasone 9,16 .A reduction in the serum level of calcium showed that the excess of glycocorticoids really provoked not only a decrease in bone trabeculae, but also spacing of these trabeculae 9,16 .The inhibition of the matrix occurs after exposure of the bone to high levels of glycocorticoids 9,16 .
Some investigators have examined the use of several β SAS Institute Inc., The SAS System Release 8.02, 1999.sensor-based systems for direct digital radiography in attempt to improve detection of bone alterations 2,10,11,14,15 .
In our experiment, analyzing linear density in direct digital radiographs, the exposure values were predetermined in a pilot study, using a specimen that was submitted to exposure times of 0.10s, 0.16s, 0.20s, 0.25s and 0.30s with 50kVp and 60kVp.For selecting the ideal regimen, the radiographs were taken together with an aluminum stepwedge.The regimen selected was 50kVp, 10mA and 0.10s for normal radiographic films.In preliminary studies, Borg & Gröndahl 7 (1996) determined that when digital sensors are used, the doses should be smaller than the value of 50kVp, 10mA and 0.05 seconds, thus being established as standard for the Digora system.In the present experiment, analysis of linear density by digital radiography for repairing fractures in rats was done by the sum of the linear density averages of the two stubs of the fracture.It was possible to identify the action of Dexamethasone in sufficient dosages for modulating the inflammatory response as well as the relation of time and their interaction.With regard to figure 3, it was possible to note that the interaction of time with the treatment in Group 1 provided a gain in bone density that behaved as the Control Group, which did not receive Dexamethasone.Therefore, the factor time was the only responsible for the increase in density, and subsequently the dose of Dexamethasone was incapable of diminishing new bone formation.This process minimized concern regarding the need for medicating the patient who has to be submitted to an intervention, whose injury may have import inflammatory consequences and should be modulated for this type of drugs.
There are several methods for bone density analysis.Volumetric CT scan (Maxiscan) is used to analyze bone density around immediately loaded dental implants 3 .This radiological assessment showed that the mean of densitometric profile is a measurement of bone mineralization, and can be used as a new method to analyze bone density reducing the need of histological analysis by human biopsy 3 .This is in agreement with the present results, which demonstrated that Digora tools for density analysis were efficient in order to provide numeric values about bone density alterations.Dental computed tomography is used to analyze bone mineral density in correlation of insertion torques in oral implants 5 .Also, it is observed that noninvasive assessment of bone mineral density employed a low dose protocol and may be used to estimate expected primary stability, helping the surgeon in operation technique 5 .Using digital imaging analyses by Radiograph Workshop Software was able to analyze bone density in patients with generic osteopororosis symptoms, where the severity of changes in the mandibular cortex was significantly related to all measured mandibular bone mineral density values 18 .Following this discussion, we think that digital dental radiograph can in such a way be used for previous diagnosis to surgical procedures for the preservation of post-operative cases as in the present experiment.
Furthermore, the efficiency of the digital radiograph as a method for bone analysis was improved.In our opinion, this imaging methodology can be used as an important diagnostic tool for proservation of post-operative cases not only of fractures, but also of any procedure that involves bone repair and regeneration.

CONCLUSIONS
Direct dental digital radiograph is useful for bone density analysis adding high and quick image attainment for fracture repairing.
Dexamethasone was shown to be incapable of diminishing new bone formation in the protocol used.

FIGURE 1 -
FIGURE 1-Image showing Bone Linear Density analysis by Digora System determining the line over the fracture stubs (arrow), showing the minimum (78.00), maximum (102.00), and the mean (96.80) density profiles for this image set

FIGURE 2 -FIGURE 3 -
FIGURE 2-Means, Standard errors and confidence level of means of Linear Density in the different times and treatments studied.Bars with the same letter indicate means of times that do not differ