الفهرس 
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Abstract
This study was done to evaluate the effect of topical application of carbamide peroxide bleaching agent on gingival epithelium and enamel by immunohistochemical and scanning electron microscopic study in albino rats. Material and methods: Eighty four male adult albino rats weighing 200-250 gm were used in the study. They were divided into two main groups, control group which included 24 animals and experimental group which included 60 animals.
The control groups were subjected to topical application of distilled water on the gingiva and enamel of the upper incisors area from the labial side The experimental animals were divided into 3 groups A, B, and C (20 for each group) and were subjected to topical application of carbamide peroxide bleaching agent on the gingiva and enamel of the same area. Three concentrations of the material were used; 6% for group A, 10% for group B and 20% for group C.
Each product was applied on the area for a period according to the manufacturers’ recommended procedure. To apply the 6% conc paint on (group A), teeth and gingiva were cleaned and the material was applied with a brush to the offending area until dryness (30 sec).
After 20 minutes, varnish is removed. 6% conc paint on was daily applicated for 14 days.
To apply 10% (group B) and 20% conc (group C), insert the tray with the gel into the mouth of the animal.
The application of the gel was repeated daily for a period of 5 weeks.
The animals were sacrificed at the periods: day 0, 1 week, 3 weeks, and 5 weeks after the last application.
The maxillae were dissected and the gingiva of the incisor areas were removed and processed for paraffin sections for histological and immunohistochemical investigation. Other specimens of the gingiva and teeth of the application area were processed for scanning electron microscope to detect surface changes of the tissue.
Histomorphometric analysis and Immunohistochemical evaluations: The histomorphometric analysis was performed for the histological findings by calculating the morphometric indexes (MI) of epithelium in the sections. Immunohistochemical evaluations were performed by measuring the PCNA indexes.
The histological examination revealed the following: The control sections of the gingivae revealed normal thickness of epithelium and keratin, normal rete pegs and normal arrangement of the four epithelial layers and each layer could be distinguished from others.
The basal cells were basophilic and arranged perpendicular to the basement membrane, prickle cells were spherical in shape with normal thickness of the layer and keratin was well adhered to the underlying layer.
The gingival sections of the experimental groups revealed the following: Group A (6% Concentration): The application of 6% conc led to marked increase in epithelium thickness with increase in the depth and number of rete pegs that appeared with different shapes.
Hyperkeratosis, hyperchromatism, dyskeratosis and decohesion of keratin were also evident in this group. Disturbed basal cell layers and an increase in normal mitosis could also be noticed in most durations of this group.
Group B (10% conc): Showed marked increase in epithelium thickness with apparent acanthosis and increase in normal mitosis.
Hyperkeratosis, hyperchromatism, decohesion of keratin, increase in N/C ratio and disturbed basal cells could also be seen.
We should know that, the changes in epithelium were similar to that in group A but were lesser in degree.
Group C (20% concentration): The characteristic changes exhibited in the rete processes that appeared irregular, branching and DROP-shaped with secondary projections.
An increase in the normal mitosis could be detected easily in all durations of this group.
The degree of changes in epithelium appeared to be gradually decreased from the day 0 to the 5 weeks after bleaching, that means; deviation from normal (control) was pronounced in day 0 more than 1 and 3 weeks which were more deviated than 5 weeks.
This observation could be noticed through the values of the morphometric indexes.
The immunohistochemical examination revealed the following: The control sections of the gingivae showed PCNA expression in little number of cells in the basal region. The gingival sections of experimental groups revealed the following: Group A (6% Concentration): Showed marked expression in PCNA in large areas of the sections.
The basal regions gave a positive reaction in all durations of this group and a positive cells could be seen in abnormal sites (upper prickle and sometimes granular layers). Group B (10% concentration): Showed moderate PCNA reaction that exhibited difference in the number of positive cells than that in group A.
Most of the positive reactions presented in the basal area but the periods 3 and 5 weeks resembled that of control section.
Group C (20% concentration): Showed marked expression of PCNA marker in basal and parabasal regions.
The number of positive cells decreased gradually from day 0 to 5 weeks after bleaching and this could be noticed from the PCNA indexes.
Scanning electron microscopic examination of enamel revealed the following: The control sections at enamel of a rat incisors showed smooth and unchanged surface. Enamel surface appears with no morphological irregularities.The enamel sections of experimental groups revealed the following: Group A (6% Concentration): It was considered the most affected group by bleaching agent. The most common feature was marked exposure and destruction of some enamel prisms. Pitting, depressions and irregularities were apparent in enamel of this group. The restoration of normal morphology in enamel at the last period was not complete. Group B (10% Concentration): Showed mild alterations in enamel surface with little eroded areas and depressions. The results of 5 weeks period resembled that of control section. Group C (20% Concentration): The degree of changes was less than that of group A but more than that of group B. It showed an incomplete loss of superficial layer with irregular and rough surface . Scanning electron microscopic examination of gingivae revealed the following: Scanning electron microscope of the gingivae at rats of control groups showed flattened polygonal superficial cells with pitting surface. Intimate contacts between the superficial keratinocytes were also observed. The gingival sections of experimental groups revealed the following: Group A (6% Concentration): The application of 6% carbamide peroxide changed the topography of gingiva greatly. from the first view, it did not resemble a gingiva due to the high degree of destruction and desquamation. The effect did not restricted to the surface cells only but extended to the underlying layers. Complete repair could not be detected at 5 weeks period of this group . Group B (10% Concentration): Showed some changes in the gingiva but with less degree than 6% conc. Erosions and detachment of squamae were present but repair could be noticed at the last period (5 weeks). Group C (20% Concentration): Corrugations, wrinkling and separations between cells were the most prominent features in this group. The effect of the applied material decreased gradually by time where the 5 weeks period had the least changes. The statistical analysis revealed the following: By observation the t-test value (in relation to morphometric index ), we found that: Group A: There was a significant difference between control and experimental group. Group B: There was insignificant difference between control and experimental groups. In other words, difference may occurred by chance. Group C: There was significant difference between control and experimental groups For PCNA index: By observation the t-test value, we found that: Group A: There was significant difference between control and experimental group. Group B: There was insignificant difference between control and experimental group. They were not differ significantly in the rate of proliferation. In other words, difference might occurred by chance. Group C: There was significant difference between control and experimental group. Conclusion: Within the limitations of the present study, the following conclusions were reached: ٭ Short course application of 6 and 20% carbamide peroxide bleaching agents caused significant increase in gingival epithelial proliferation but 10% conc caused insignificant effect . ٭ SEM investigation demonstrated that, 6 and 20% carbamide peroxide caused topographic changes in gingival surface more than 10% did. ٭ SEM investigation demonstrated that, the bleaching agents affected enamel morphology producing porosities, depressions and partial affection of enamel prisms. These defects differed with different concentrations where they were pronounced in 6 and 20% more than 10% concentration. ٭ Re-mineralization by saliva could reduce the destructive effect of carbamide peroxide bleaching agent on enamel specially in case of using lower concentration. Recommendation: ٭ Carbamide peroxide bleaching agent must be used under professional supervision with following the manufacturing advice. ٭ Avoid the use of high concentrations of carbamide peroxide bleaching agents for long time. ٭ Further investigations on the cumulative effects of carbamide peroxide bleaching agent on enamel and gingiva (cell cycle regulation) when used for longer durations. ٭ It is better to use fluoridated carbamide peroxide bleaching agents with shorter periods to aid in remineralization. ٭ Bleaching products with a concentration more than 10% should be used with a care. ٭ The use of carbamide peroxide bleaching agents should be avoided in case of damaged or diseased tissues. ٭ If it is possible, rubber dam can be used to exclude the soft tissues from contact with the material.