| Objective:The condyle is the growth center of the whole mandible, which surface is covered with a thin layer of fibrous cartilage and serves as the growth basis over lifetime. When the mandibular condylar cartilage is stimulated by functional mandibular protrusion orthodontic force, which can be transformed into biological signal stimulation, it triggers a series of signal transduction pathway, to regulate the process of cell mitosis together, showing itself on condylar cartilage cells proliferation. Scholars have conducted many studies around signal pathway existing in the remodeling of condyle after functional treatment, but the cytological mechanism about the signaling pathways into the nucleus to regulate cell mitosis is not very clear. Recent studies have found that Cyclin A2 for cell mitosis is the most powerful stimulating factor, and concentrated in the nucleus, activating protein substrates when combined with the cell cycle dependent kinase CDK1,and also plays an important role in the G1/S phase and G2/M phase of the cell cycle, not only can control DNA replication but also can promote cell division. So the purpose of the experiment was, by using the histological staining and immunohistochemistry staining method, to preliminarily find out the role and significance of Cyclin A2 in the condylar reconstruction through analyzing the expression and the change regulation of Cyclin A2 in condylar caritilage during mandibular protrusion, which could provide experimental evidence for clinical work.Methods: Select seventy 4-week-old healthy male Sprague-Dawley(SD) rats, weighing about 90 g. They were divided randomly and equally into one experimental group and one control group after they were adaptively feed for a week. Considering the period of the experiment process(1, 3, 7, 14, 21, 28 and 35 days), the groups were respectively classified into 7 subgroups(5 in each), so there were a total of 14 subgroups. In the experimental subgroups, self-made mandibular advancement inclined bite plate appliance was cemented on the rats’ incisors by 3M glass ionomer, and then connected with face bow on both sides via nasomaxillary complex through a chain rubber ring, thereby to make the inclined bite plate get a stable retention, wearing 24 hour every day. Given to the damage from rats’ Limbs to the appliance and facial tissue, the rats’ limbs were wrapped tightly with medical adhesive plaster. In the control group, no experimental operation was done. All animals were allowed to soft food and free water. They were reared under the same environment until the end of the experiment. Select materials and treat with conventional fixation, demineralization, dehydration and encapsulation by 1, 3, 7, 14, 21, 28, 35 days after the experiment. Condylar histological changes were observed by routine HE staining after paraffin section. Expression of cyclin A2 was detected by the SP immunohistochemical method. Multifunction color cell image analysis management system was used to grade immunohistochemical staining, with IHS value representing staining intensity. All of the results were processed with SPSS13.0 statistical software, showed with x_±s. T-test was used for the comparisons between the experimental group and the control group when making statistical analysis, while the one-way ANOVA method was employed for comparisons among subgroups. If the p-value was smaller than 0.05, they were statistically significant.The test level was α= 0.05.Results:1 Gross observationRat mandibular moved to protrusive by wearing inclined plate appliance, and mostly upper and lower anterior teeth were of corresponding or anticover. After removing the appliance, the jaw could not recover the formal shape. In the control group, there were no coresponding or anticover and the rats’ anterior teeth covered about 3mm.2 Condylar cartilage morphological changesCondylar cartilage were divided into fiber layer, proliferative layer, transition layer, cartilage layer(hypertrophic layer), cartilage calcification layer under the microscope. With the functions of orthodontic force in experimental rats, the number of cells in the proliferative layer, transition layer increase, the cell volume of cartilage layer(hypertrophic layer) enlarges in middle and posterior, and at the same time, condylar cartilage is more thicker than control group. Thickness in all layers of condylar cartilage stabilized gradually until the 21 th day. On Day 35 th, the thickness of the transition layer and cartilage layer decrease obviously, but still thicker than the control group. And the cell volume of cartilage deep layer enlarges, karyopyknosis, with the cartilage calcification layer, actived entochondrostosis. With age-related in the control group, thickness in all layers of condylar cartilage decreased gradually, especially in the middle and the posterior part, but in the anterior part unconspicuous.3 Cyclin A2 expression level and distributionNo positive colored particles were seen in the blank control group(PBS replaced primary antibodies). In the control group, only a small number of Cyclin A2 were positive in the cartilage layer, and they have been maintaining a low expression level until the end. Cyclin A2 in the experimental group was mainly expressed in the proliferative cell layer and transition layer, positive mainly located in nucleus. On the first day, Cyclin A2 in the experimental group IHS=12.00±3.61, which had no significant difference compared to the control group IHS=10.33±1.53(P>0.05). On Day 3th, the experimental group IHS=70.33±28.50, which had significant difference compared to the control group IHS=7.67±3.06,(P<0.05). On Day 7th, the experimental group IHS=95.67±11.37, positive expression continued to improve, which had significant difference compared to the control group IHS=7.67±3.51(P<0.01). On Day 21 th, positive cells in most samples the experimental group began to decrease obviously, which still had significant difference compared with the control group(P<0.05). On Day 28 st and 35 th, Cyclin A2 in the experimental group and the control group showed no statistical difference(P>0.05).Conclusions:1 Cyclin A2 paticipated in the mitosis process of condylar chondrocytes during functional mandibular protrusion, promoting the rats’ mandibular condylar chondrocytes proliferation in growth development period.2 The expression of Cyclin A2 may have participated in the conversion functional orthodontic force with biological signal. |
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