| Part one: The remodeling of the condyle following forwardmandibular positioningClass â…¡ malocclusion accounts for 20%-30% among malocclusion. Those patients usually have a strong treatment demand due to their appearance. Thus this disease occurred at youthhood and pre-youthhood can be treated by the adapative remodeling of the mandible and muscles. Since the application of the functional appliance, numerous clinical studies have been made in terms of its mechanism and effectiveness. However, it is still controversial whether the functional appliances could stimulate the growth of mandibular condyle. The purpose of this study was to investigate the effect of mandibular forward positioning in mandibular condyle in animal model by histomorphology, fluorescence staining, and bonehistomorphometry and the relationship between the mandibular biomechanics and tissue remodeling.In part one of this study, 24 8-week-old Japanese white rabbits were randomly divided into the experimental group (n=12) and the control group (n=12). In the experimental group, four titanium screws were planted at the anterior portion of the zygomatic arch, the upper and lower border of the mandible angle, and the chin for the fixation of the functional appliance. The lateral X-ray film of the head was taken before and after the fixation of the functional appliance, respectively. Functional appliances made from polymethylmethacrylate with identical inclined planes were cemented onto the upper central incisors of the experimental group with dental adhesive resin cement, The appliances were worn 24 hours producing a continuous forward and downward positioning of the mandible. The control group was not received any treatment. Two animals in experimental group and control group each time were killed at 3 days, 1, 2, 4, 8, and 12 weeks after the treatment. The specimens from right TMJs were processed for HE staining, while the specimens from left TMJs were processed for scan electron telescope.Our results showed that the mandibles in the animals from the experimental group were moved anteriorly and inferiorly. In the control group, all zones in the condylar cartilage became thinner from 3 days to 2 weeks and stabilized after 2 weeks. Increased osteoblasts and thicker trabecula were found in the marrow inferior to the condylar cartilage. A thin gelatin-like layer was seen covering the smooth surface of the condyle. In the experimental group, the condylar cartilage was relatively thinner compared with the control group at 3 days after treatment. At 1 week after treatment, all layers in the condylar cartilage became thicker, especially in the proliferative zone at the middle and posterior position of the condyle. At 4 weeks, the condylar cartilage was obviously thicker, especially in the transitional and hypertrophic zones. At 8 weeks, the layers in the condylar cartilage became thinner.However, the calcified zone was still significantly thicker, and the bone trabecula and marrow became more matured. In the animals at 3 day and 1 week, the gelatin-like layer at the anterior incline of the condyle curled to the shape of bundle or granule. It began to decrease at 2 weeks and recovered.In part two of this study, Forty rabbits were divided into the experimental group of 24 animals treated with the functional appliance and the control subgroup of 16 animals without the functional appliance, tetracycline, caclein and xylenol orange were injected in all animals at the beginning day of the experiment, 7 days and 1 day before sacrifice, respectively. Six animals in the experimental group and four animals in the control group each time were killed at 2, 4, 8, and 12 weeks after treatment. The specimens from left TMJs were processed for undemineralised sections. These sections were used for fluorescent microscopy and the measurement of bone histomophometry.The parameters measured by histomorphometry showed that from 2 weeks to 12 weeks, BV/TV, Tb.Th, MiAR, I.Ob and OS in subchondral region of the experimental cartilage were significantly increased compared with the age-matched controls (P<0.05). However, Tb.Sp was lower than those in the controls with significant difference (P<0.05). There was no significant difference found in the static and dynamic parameters of the central region between the the age-matched controls (P>0.05).The rabbit animal model of forward mandibular positioning was successfully established in this study. Our results suggested that the biomechanical alteration caused by mandibular forward positioning result in the increase of the condylar cartilage. The remodeling adapt the mandible to the new position. Meanwhile, a significant area difference was found, which might be relative to the new biomechanical environment.Part two: The role of BMP/Smads signal transduction pathway in the condylar cartilage following mandibular forward positioningBone morphogenetic proteins (BMPs) play a key role in the repair of bone and cartilage, differenciation and proliferation of osteoblasts and osteoclasts. Meanwhile, the downstream signaling components of BMPs signaling regulate the signs transfer from cellular plasm to nucleus. Our study found that the mandibular forward positioning could accelerate the new bone formation in the condylar cartilage. However, the role of BMPs and its downstream signaling components in the condylar remodeling are not defined yet. Therefore, To identify the relationship between the expression of BMP/Smads in condylar cartilage and condylar growth modifications in growing rabbits following forward positioning mandibles with functional appliance.Sixty rabbits with age of 8 weeks were randomly divided into the experimental group (n=36) and control group (n=24). Rabbits in the experimental groups were induced to forward their mandibles by a functional appliance. The experimental group and control group were killed after 3 days and 1, 2, 4, 8 and 12 weeks, respectively. The specimens from right TMJs were processed for immunohistochemical staining. The temporal and spacial expression of BMP^ Smadl/^ 4, 6 in condylar cartilage was measured with Image Computer Analysis Software. Date was statistically analyzed by Student's t test and one-way ANOVA.Results showed that BMP-2n Smadl/5> 4 and 6 were mainly expressed at the transitive and hypertrophic zones, while they were also found in the cartilage cells and osteoblasts at the calcified zone. Compared with those of the age-matched controls, the positive signals for BMP-2> Smadl/^ 4N 5^ 6 in the experimental animals were stronger (PO.05) , coincided with the remodeling in condylar cartilage after functional appliance.Through Smad signaling, the biological effect of BMPs transfers from the plasm into the nucleus and regulate transcription through functional cooperativity and physical interactions with other transcription factors. When the mandible were forward positioned, BMP-2, Smadl/5> 4 and 6 were up-regulated obviously, which seemed to be has a close relationship with the condylar remodeling. In conclusion, as the signaling molecules of BMPs, Smadl/5x 4, 6 may participate in the process from the mechanic sign to its biological effect and take an important role in the acceleration of endochodral ossification in the condylar cartilage after functional appliance.Part three: The role of angiopoietin in the condylar cartilage remodeling following mandibular forward positioningBone growth and remodeling is dependent on the angiogenesis of the blood vessels and its distribution. Recently, it is found that the family of angiopoietin(Ang) take an important role in the angiogenesis, maturation, reshape and maintenance of the blood vessels, and has a close relationship with the proliferation and transfer of the tumor cells. However, the role of angiopoietin in the condyar cartilage remodeling is not clear yet. Thus, this study was to investigate the expression of Ang-1 and Ang-2 in the rabbit condyar cartilage following mandibular forward positioning through immunohistochemistry, and T test and ANVA were used for the statistical analysis. The animal model and its grouping were the same as the part two.In the condyles from the control group, strong expression of Ang-1 and Ang-2 were found in the rest four zones with the exception of the superficial zone. With the maturation of the cartilage cells, expression of Ang-1 and Ang-2 increased gradually with the strong expression in the hypertrophic zone. Also, positive signals of expression of Ang-1 and Ang-2 could be seen in the osteoblasts and osteoclasts at thecalcified zone and bony trabecula. In the experimental group, the expression of Ang-1 and Ang-2 began to increase at 3 days after surgery, especially in the hypertrophic zone at the posterior of the condyle. The expression of Ang-1 reached the highest level at 2 weeks after surgery, while the expression of Ang-2 reached the highest level at 4 weeks after surgery. Then both of them gradually decreased. The statistical analysis showed that the values in the experimental group at different time points were lower than those in the control group (P<0.05) .Our results indicated that the temporal and spacial expression of Ang-1 and Ang-2 in the condylar cartilage is relative to the condylar adaptive remodeling following mandibular forward positioning.Part four: The role of apoptosis relative genes-Bcl-2 and Bax in the condylar cartilage following mandibular forward positioningApoptosis, programmed cell death, is concerned with the stabilization of environment in vivo and tissue remodeling. Lots of genes including Bcl-2 and Bax participate in the regulation of apoptosis. The purpose of this study was to investigate the changes of Bcl-2 and Bax gene in the condylar cartilage following the mandibular forward positioning and elucidate the relationship between apoptosis and the condylar adaptive remodeling. The animal model and its grouping were the same as the part 2. The temporal and spacial changes of Bcl-2 and Bax were detected by immuohistochemistric staining approach and the gray values of the expression intensity were measured for the statistical analysis by T test and ANVA.In the control group, positive signals of Bcl-2 gene were seen in the other four zones with the exception of the superficial zone, among which the strongestexpression were found in the transitive and hypertrophic zones. However, the expression of Bax gene was observed only in the transitive, hypertrophic and mineralized zones and the strongest expression were found in the mineralized zone. In the experimental group, the expression of Bcl-2 and Bax gene began to increase at 3 days after surgery. The expression of Bcl-2 reached the highest level at 1 week9after surgery, while the expression of Bax gene reached the highest level at 4 weeks after surgery, especially in the posterior of the condyle. Then both of them gradually decreased. The statistical analysis showed that the values in the experimental group at different time points were lower than those in the control group (P<0.05) . The ratio of Bcl-2/Bax began to decrease at 3 days after surgery and continued until 12 weeks after surgery (P<0.05) .Bcl-2 gene is a kind of protein inhibiting apoptosis, while Bax is a kind of protein promoting apoptosis. Both of them are important regulators in the apoptosis of cartilage cells. The temporal and spacial changes of Bcl-2 and Bax gene suggested that apoptosis might take a role in maintaining the cartilage cells stabilization during the condylar remodeling following the mandibular forward positioning.
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