Introduction
The aim of this prospective in-vivo study was to investigate the possible effects of temperature changes from various adhesive cleanup procedures on pulpal tissue.
Methods
The materials, consisting of 40 sound maxillary and mandibular premolars to be extracted during orthodontic treatment, were randomly assigned to 4 groups, with 1 group as the control. The teeth in the 3 study groups were etched; brackets were bonded and then debonded. The remaining adhesive was removed with a tungsten carbide bur by using a high-speed hand piece. The teeth in the control group were not etched and bonded. In group 1, the residual adhesive was removed with water cooling, and the teeth were extracted 24 hours later. In group 2, the residual adhesive was removed without water cooling, and the teeth were extracted 24 hours later. In group 3, the residual adhesive was removed without water cooling, and the teeth were extracted 20 days later. The teeth were prepared for histologic examination, and the number of vessels, vessel areas and perimeters, extravasation of red blood cells, vascular congestion, and inflammatory cell infiltration were evaluated to determine pulpal tissue changes.
Results
According to the findings from histologic and immunohistochemical evaluations, the coronal pulps of the teeth in groups 1 and 3 were almost similar to the control teeth, but some distinct pathologic changes were observed in group 2.
Conclusions
Adhesive removal without water cooling caused some vascular and pulpal tissue alterations, but these were tolerated by the pulpal tissues, so the changes were reversible.
In the history of orthodontics, the changeover from banding to bonding with advancing bonding techniques was an important development. Because of the invention of bonding, orthodontic treatment has become widespread, various treatment techniques were developed, the numbers of appointments were reduced, and treatment duration became shorter. With the growing popularity of bonding, debonding procedures have become a subject in the orthodontic literature. Studies about debonding and adhesive cleanup procedures generally focused on instruments, residual enamel surface in terms of enamel loss and fracture lines, and the potential risk of pulpal damage caused by the heat generated during debonding.
Surrounding hard tissues can protect the dental pulp from noxious stimuli, including thermal injury. However, pulpal tissue might be exposed to high temperatures as a consequence of different dental approaches. An increase in intrapulpal temperature can cause some histopathologic changes in the pulpal tissues. This might affect the pulpal vessels, leading to vascular injuries and tissue necrosis. The results of an animal study showed that 15% of dental pulps became nonvital when the temperature in the pulpal chamber increased by 5.6°C, and 60% became necrotic if the temperature rose by 11.1°C. Sato and Schuchard reported that excessive heat adduction could result in structural changes of the hard dental tissues and damage to the dental pulp. These changes and the amount of damage are related to the duration of thermal stimulus and the peak temperature.
Uysal et al evaluated the thermal changes in the pulp chamber during various adhesive cleanup procedures in an in-vitro study. They reported that cleanup with a tungsten carbide bur using a high-speed contra-angle hand piece without water cooling increased the temperature by 7.5°C in the pulp chamber. This increase in temperature exceeds the critical value for pulpal health. However, cleanup with water cooling never produced temperature changes exceeding the critical value. They also concluded that, in clinical settings, cooling procedures such as air-water sprays were essential to ensure prevention of pulpal damage.
Articles in the literature about debonding are generally in-vitro studies. Histologic investigations of pulp after debonding or cleanup procedures are limited to only thermal debonding (electrothermal debracketing) of metal and ceramic brackets. These in-vivo studies were carried out on the pulp of extracted teeth. Histologic sections of the teeth were prepared, and the effects of heat increases on pulpal tissue were evaluated.
To our knowledge, no study has shown the effects of adhesive removal on pulp in the orthodontic literature. The aims of this in-vivo study were to investigate the possible effects of heat during debonding on dental pulp and, if changes were observed, to determine whether they were reversible.
Material and methods
Forty maxillary and mandibular first premolars from 10 patients (5 girls, 5 boys) between the ages 15 and 17 years were used. Approval of this study was obtained from the Research Ethical Committee of Cumhuriyet University in Sivas, Turkey. The patients and their parents were told about the procedures, and informed consent was received. The subjects were selected according to the following inclusion criteria: need for 4 first premolar extractions, and no caries or fillings in the first premolars.
Four groups were formed by using 1 premolar of each patient. The teeth in all groups were randomly selected to eliminate possible differences between maxillary and mandibular, and left and right, teeth. The teeth in groups 1, 2, and 3 (experimental groups) were etched for 30 seconds with 37% orthophosphoric acid, rinsed with water for 30 seconds, and dried with an oil-free source for 20 seconds. Metallic orthodontic brackets (Generous Roth, GAC International, Bohemia, NY) were then bonded with an orthodontic adhesive (Transbond XT, 3M Unitek, Monrovia, Calif). The teeth in the control group were not etched and bonded, but they were light cured the same as the study groups with a conventional halogen light for 30 seconds to standardized the effect of heat from light curing; these teeth were extracted within 30 minutes after curing. The brackets in the study groups were debonded by squeezing the bracket wings gently with pliers. The remaining composite on tooth surfaces was removed with a tungsten carbide bur (H22ALGK314016, Komet/Gebr. Brasseler, Germany) in a continuous manner for 30 seconds by using a high-speed hand piece (T3 Racer, D-64625, Sirona, Bensheim, Germany) at 250,000 rpm. This process was similar to our routine clinical cleanup procedure. A new bur was used for each tooth, and all procedures were performed by the same operator (B.K.A.) using special care to minimize damage to the enamel surface. The differences in the procedures between groups 1 through 3 are given below.
In group 1, the residual adhesive was removed with water cooling by using a high-speed hand piece with a cooling aperture (diameter, 0.7 mm) and an air pressure of 40 psi (about 2.7 bar). After 24 hours, the teeth were extracted and placed in 20 cm 3 of fixative formalin solution.
In group 2, the residual adhesive was removed without water cooling. After 24 hours, the teeth were extracted and placed in 20 cm 3 of fixative formalin solution.
In group 3, the residual adhesive was removed without water cooling. The teeth were extracted 20 days later and placed in 20 cm 3 of fixative formalin solution.
Fixation and decalcification of teeth were previously described. The teeth were immersed in buffered 10% formalin immediately after extraction and were fixed for 24 hours. After fixation, the teeth were decalcified for 3 months in Anna Morse solution until they were soft enough (rubber-like consistency) for cutting. The solution was renewed twice a week. After decalcification, the teeth were rinsed in water, dehydrated, cleaned with xylene, soaked in liquid paraffin, and embedded in solid paraffin. The paraffin-embedded teeth were serially sectioned buccolingually with a rotary microtome. Serial sections, 5-μm thick, were obtained and stained with hematoxylin and eosin. Cell morphology as well as the crown and root pulp were qualitatively assessed by 1 examiner (C.C.O.) blinded to the groups of the histologic sections. The images captured were transferred to SPOT advanced image analysis software (version 4.6, Diagnostic Instruments, Sterling Heights, Mich). To determine vascular proliferation, 4 high-power fields were viewed from the crown of each tooth. The numbers of vessels in each field were counted, and means were obtained. Vascular conditions were also determined for vessel areas and perimeters. Pulpal tissue was examined for extravasation of red blood cells, vascular congestion, and inflammatory cell infiltration by semiquantifying the parameters. The observed changes were investigated by using the following scale: 0, none; 1, mild; 2, moderate; and 3, severe. The groups were also compared for odontoblastic cell zones and cell-free zones of pulpal tissues.
For the evaluation of leukocytes, the CD45 pan leukocyte marker was used. For CD45 immunostaining, fixed sections were deparaffinized in xylene and rehydrated in a graded series of alcohol. For antigen retrieval, the slides were placed in 10 mmol/L of citrate buffer (pH 6.0) and microwaved twice for 5 minutes. Tissue sections were blocked for endogenous peroxidase activity with methanol containing 3% hydrogen peroxide for 10 minutes and for nonspecific binding with Ultra V Block (Labvision, Thermofisher Scientific, Fremont, Calif.) for 7 minutes at room temperature. Rabbit anti-CD45 (Santa Cruz Biotechnology, Santa Cruz, Calif.) primary antibody was applied in a dilution of 2.5 μg of protein per milliliter (1:400 dilution) for 2 hours at room temperature in a humidified chamber. The sections were washed in phosphate-buffered saline solution, incubated with biotinylated anti-rabbit IgG (3 mg/ml, Vector Labs, Burlingame, Calif.) at 1:500 dilution for 45 minutes at room temperature. After several rinses in phosphate-buffered saline solution, the antigen-antibody complex was detected by using an avidin-biotin horseradish peroxidase complex with a Universal LSAB Kit (Dako, Glostrup, Denmark). Diaminobenzidine (3,3-diaminobenzidine tetrahydrochloride dihydrate; Sigma-Aldrich, St Louis, Mo) was used as the chromogen, and sections were mounted with Permount (Fisher Chemicals, Springfield, NJ) on glass slides and then evaluated under a light microscope. To test the specificity of the antibodies, sections were incubated with rabbit serum (Dako) at the same concentration as the primary antibody.
Statistical analysis
Semiquantified parameters, inflammatory cell infiltration, extravasation of red blood cells, and vascular congestion could not be statistically evaluated because of the limited number of teeth in each group. The assessments were done according to the distribution of the scores obtained for each group. Nonparametric statistical methods were used to evaluate the of number of vessels, vessel areas, and vessel perimeters. A Kruskal-Wallis test was performed to identify whether there were any differences in each parameter between the groups, and the Mann-Whitney U test was performed to determine which groups had this significance.
Results
According to the histologic and immunohistochemical evaluations, the following results were observed.
In evaluation of odontoblastic cell zone, a columnar odontoblast cell layer with a normal appearance was observed clearly in the control group. In group 1, the findings were similar to the control group. In group 2, the odontoblast nuclei in the odontoblast zone lost their normal appearance; this layer was composed of fewer cells compared with the control teeth. In group 3, the histology of this layer was similar to the control group ( Fig 1 , Table I ).
| Group 1 | Group 2 | Group 3 | Control | |||||
|---|---|---|---|---|---|---|---|---|
| Histologic feature category | n | % | n | % | n | % | n | % |
| Odontoblastic cell zone | ||||||||
| Normal | 9 | 90% | 5 | 50% | 8 | 80% | 10 | 100% |
| Slightly disrupted | 1 | 10% | 4 | 40% | 2 | 20% | 0 | 0% |
| Severely disrupted | 0 | 0% | 1 | 10% | 0 | 0% | 0 | 0% |
| Cell-free zone | ||||||||
| Absent | 0 | 0% | 2 | 20% | 1 | 10% | 0 | 0% |
| Present | 10 | 100% | 8 | 80% | 9 | 90% | 10 | 100% |
| Inflammatory cell infiltration | ||||||||
| None | 6 | 60% | 0 | 0% | 5 | 50% | 9 | 90% |
| Mild | 4 | 40% | 1 | 10% | 4 | 40% | 1 | 10% |
| Moderate | 0 | 0% | 8 | 80% | 1 | 10% | 0 | 0% |
| Severe | 0 | 0% | 1 | 10% | 0 | 0% | 0 | 0% |
| Extravasation of red blood cells | ||||||||
| Absent | 9 | 90% | 6 | 60% | 8 | 80% | 9 | 90% |
| Present | 1 | 10% | 4 | 40% | 2 | 20% | 1 | 10% |
| Vascular congestion | ||||||||
| None | 6 | 60% | 0 | 0% | 4 | 40% | 7 | 70% |
| Mild | 4 | 40% | 1 | 10% | 6 | 60% | 3 | 30% |
| Moderate | 0 | 0% | 7 | 70% | 0 | 0% | 0 | 0% |
| Severe | 0 | 0% | 2 | 20% | 0 | 0% | 0 | 0% |
The cell-free zone was present in the control group and groups 1 and 3. In group 2, a distinct cell-free zone was lacking; this indicated peripheral pulp damage ( Fig 1 , Table I ).
In evaluation of inflammatory cell infiltration, few CD45-immunopositive leukocytes were seen in the pulps of the teeth in the control group. In group 1, the scores were nearly the same as for the control group. Moderate to severe inflammation was observed in group 2, and there was no or mild inflammatory cell infiltration in group 3, similar to the control group ( Fig 2 , Table I ).
The teeth in the control group showed no extravasated red blood cells except for 1 tooth. The findings of group 1 were similar to those of the control group. The number of teeth with extravasated red blood cells was dramatically greater in group 2. In group 3, the number of pulps with extravasated red blood cells was decreased and nearly the same as the control group ( Table I ).
The control group showed no signs of vascular congestion. In group 1, scores for vascular congestion were nearly same as those of the control group (scores, 0-1). In group 2, moderate to severe (scores, 2-3) vascular congestion was defined histochemically. There was no or mild vascular congestion in group 3 ( Fig 1 , Table I ).
The mean values and standard deviations of the numbers of blood vessels for the groups are shown in Table II . When the groups were compared for this parameter, group 2 had statistically significantly different results from the other groups, whereas there was no significant difference between the other 3 groups. The number of vessels was higher in group 2 ( Table III ).
