| Objective: The final effect of the functional appliance has been one of the focus for the orthodontic doctor. Due to the difference of the experimental methods, design ideas, as well as the degree of patients' cooperation, and the results are also different. In the academia, there are two different points.Some scholars believe that the functional appliance could promote the condylar reconstruction and affect the growth and development of mandible by the way of changing the muscles' tone.So that the appearance of ClassⅡmalocclusion also could be improved.While other scholars believe that the functional appliance place a"mortgage"on the maximum growth of mandibular and do not produce significant bone effect. The normal cartilage cells can synthesize small amounts of NO. The mechanical stimulation on cartilage cells can affect the synthesis of NO. iNOS is one of the main factors for the generation of NO. Thus,the purpose of this study was to preliminary find out the role of iNOS and NO in the condylar reconstruction through analyzing the expression of iNOS in condylar caritilage during mandibular protrusion.At the same time, we observed the long-term stability of functional appliance through the dynamic changes of iNOS,which could provide experimental evidence for clinical work.Methods: 60 4-week-old healthy Sprague Dawley male rats were chosed .Their weight was about 90g.They were divided randomly into two equal groups after fed for 7 days by pellets feed: experimental group (S) and control group (D), each group included 30 rats. The experimental group rats wore removable upper jaw inclined bite plate appliance to keep the mandibular protrusion for 10-12 hours during the day,and the rat of the control group was natural growing without appliance. All rats were fed water freely. The experimental group were fed freely, the control group were fed regularly. The purpose was to ensure that animal's weight was the same.The experimental lastde for 6 weeks : 1 week (1w), 2 week (2w), 3 week (3w), 4 week (4w), 5 week (5w), 6 week (6w). By experimental period ,5 rats were randomly chosen to put to death from each groups. Left condyle cartilage was placed in 4% paraformaldehyde which fixed for 24 hours at 4℃. After condyle cartilage was flushed by distilled water,which was placed in 10% of the EDTA to decalcify.The tissue was conventional treated and embeded in paraffin. Made condylar tissue vertical sections, stain with HE.Observed histological features and measured the thickness of condylar cartilage in different experimental periods. After the tissue sections were immunohistochemical stained, utilizing Motic Med 6.0CMIAS to detecte the expression of iNOS of the different experimental period points in the different parts of condylar cartilage .The data was done statistic analysis using the SPSS13.0 statistical software.Then analyzed the change of condylar cartilage thickness and the intensity change of iNOS expressed in the condylar cartilage at the different time points.Results:1 Histological observationMacroscopic observation: The rats in condylar anteroposterior dimension was greater than the exterior and interior dimension. The anterior region of condyly was longer and the posterior region of condyly was shorter.1.1 Histological features of condylar cartilage in control group Microscope showed that a layer of fibrous tissue was disc which surrounded the condylar cartilage surface. In the slight rearward part of disc posterior band was the most thickness of the condylar cartilage. Condylar cartilage gradually thinned from back to front. Under the condylar cartilage was trabecular bone. With the growth and development, the overall thickness of condylar cartilage thinned gradually and had age-related change.Condylar cartilage had its unique boundaries, according to the different of cell morphous which could be divided into:①Fiber layer composed by many fibroblasts which was dense fibrous tissue.②Germinal layer this layer composed by many dense small cells,the cell relatively active and was seen mitotic figure.③Maturity layer in this layer the cell increased, irregular morphology, irregular arrangement, nucleus mostly located on the cell side, cartilage matrix increased.④Transitional layer the cell swelling, nucleus was not obvious, cartilage matrix increased significantly, this layer cell was region where cartilage cells gradually transformed bone.1.2 Histological features of condylar cartilage in experimental groupThe thickness of central and posterior condylar cartilage was increased after functional mandibular protruded for rat. The thickness of condylar cartilage in central and posterior had statistically significant difference compared with the control group from the second week to the sixth week. Thickened cartilage layer showed that germinal layer cells increased and nucleus became large and obvious,maturity layer cells were also thickened and the cell morphology was similar to the control group. The thickness of condylar cartilage anterior had no statistically significant difference compared with the control group from the first week to the fifth week.In the sixth week the thickness was obviously thinner which had statistically significant difference. After the rat weared the appliance for some time, some rats were habit to mandibular protrusion and appeared labial teeth crossbite.2 Immunohistochemical resultThe expression of iNOS was brown which located in cytoplasm. Cell shape was integrity, structure was clarity. The color of positive cells was significantly higher than background color.The black control (PBS instead of first antibody) group had no positive staining granules. iNOS expressed in the whole layer of condylar cartilage, cartilage cells of maturity layer superficial lamella expressed strong. The expression level of iNOS was the most strong in posterior condylar cartilage, followed by the anterior, the center was weakest.2.1 The expression of iNOS in anterior condylar cartilageThe trend of anterior condylar cartilage was the same as posterior condylar cartilage in the control group and experimental group. Pairwise comparison from the second to fourth week had statistically significant difference in the control group. After mandibular protrusion of rat, anterior expression level also was increased. From the first to sixth week, the experimental group and control group had statistically significant difference.2.2 The expression of iNOS in central condylar cartilageThe trend of central condylar cartilage was also the same as posterior condylar cartilage in the control group and experimental group. Pairwise comparison from the second to fourth week had statistically significant difference in control group. Experimental group, iNOS expressed had statistically significant difference in the first and second weeks. From the third to sixth week, the control group and experimental group had no significant difference.2.3 The expression of iNOS in posterior condylar cartilageThe expression of iNOS was gradually decreased in control group, pairwise comparison from the second to fifth week had statistically significant. Experimental group, the expression of iNOS was higher than control group.The trend of expression was the same as the control group. From the first to fifth week, the experimental group and control group had significant difference. The expression of iNOS had no significant difference compared with the control group in the sixth week of experiment.Conclusion:1 The condylar cartilage of growing rat appeared age-related change by the time, the thickness of condylar cartilage was thin gradually. The thickness of condylar cartilage appeared adaptive remodeling after the rat wore functional appliance. In the early of treatment, the thickness increased obviously and thinned gradually with the experimental process .2 Condylar cartilage had iNOS in normal physiological conditions. iNOS decreased gradually with the growth and development which suggested that iNOS involved in the growth and development of condyle.3 Wore the functional appliance for rats, the expression of iNOS increased and reduced gradually with the experimental cycle. The trend was consistent with the control group and stabilized gradually in the late of experiment which suggested that functional appliance should pay attention to maintain for the treatment.
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