The Effects Of ClC-7 On Tooth Loss In Animal Study

ClC-7 plays a critical role during the osteoclast-mediated bone resorption. The mutations in CLCN7 gene can cause osteopetrosis, a rare autosomal recessive bone dysplasia. In addition to the typical bone features, such as generalized osteosclerosis, a high rate of fractures and osteomyelitis,CLCN7-related osteopetrosis patients also have some dental problems including disturbance of tooth eruption, hypodontia, malformed teeth, and enamel dysplasia. Clcn7 knockout mice also showed disturbance of tooth eruption. And our previous study found that CLC-7 protein was expressed in mouse tooth germ. However, it was unclear whether the CLCN7-related tooth anomalies were mediated by osteoclasts or not. In order to provide a growth environment without osteoclasts for the tooth germ, we transplanted the tooth germ into the mouse renal capsule. The expression of Clcn7 gene in tooth germ was downregulated by infection of Clcn7 shRNA lentivirus to observe the role of Clcn7 in the growth of tooth germ. Methods:1. Preparation of Clcn7 shRNA lentivirus and establishment of tooth infection system. Tooth germs of the mandibular first molar were taken from the E13.5 days Balb/c mice, and then were infected by the Clcn7 shRNA lentivirus with GFP green fluorescence. We observed expression of the fluorescence after four days of the infection to determine the best multiplicity of infection(MOI). On the basis of the optimal MOI, total RNA was extracted and then quantitative real-time PCR experiments were performed to validate the silencing effect.2. The effect of Clcn7-silencing on tooth morphology. The tooth germs infected with Clcn7 shRNA lentivirus were transplanted under the kidney capsule of adult male mice. The host mice were sacrificed and the transplantation blocks were removing after four weeks. Micro-CT scanning was performed to observe the changes in tooth morphology. Then H&E staining and AZON staining were applied to observe the changes of the roots, dentin, periodontal tissues and other tooth structures.3. The effect of Clcn7-silencing on the expression of teeth related proteins. In order to investigate the possible mechanisms of ClC-7 on tooth development, immunohistochemical staining was taken to further test the expression of CLC-7 and DSP in tooth germs. On the other hand, odontoblast cell line MDPC-23 was chosen for Clcn7 siRNA. And then real-time quantitative PCR was used to detect the expression of Clcn7 and Dspp. Results:1. The MOI were 5000 TU/ tooth germ and infection for 24 hours. Under this condition, the effect of infection was best with strongest expression of green fluorescence.2. The results of RT-qPCR showed that the expression of Clcn7 was reduced by 70% in experimental group compared to the negative control(P<0.05).3. The results of micro-CT scanning showed that the tooth development were intact with integrite structure, normal roots, clear outline of the cusps in the blank control group and negative control group.While it showed malformed tooth structure, crooked or shorten roots and unobvious cusps in the experimental group.4. H&E staining and AZON staining of paraffin sections showed that the tooth structure were intact with normal predentin and periodontal tissue such as clear and regularly arranged periodontal ligament, cementum and alveolar bone in the blank and negative control group. On the contrary, in the experimental group, part of the predentin became thicker, the root became curved, and normal periodontal tissue was replaced by the disordered arrangement of fibroblasts.5. The results of immunohistochemistry staining showed that, CLC-7 and DSP expressed in odontoblasts and predentin.Contrary to the blank and negative control group, the expression of CLC-7 was not reduced, while the expression of DSP was significantly decreased in the experimental group.6.The results of RT-qPCR of odontoblast cell line MDPC-23 showed that the expression of Clcn7 was significantly decreased while Dspp were not changed in Clcn7 siRNA group compared to the blank and negative control group. Conlusions:1. Clcn7 shRNA lentivirus or Clcn7 siRNA can effectively silence the Clcn7 gene, downregulating the expression of ClC-7 whether in the tooth germ tissue or odontoblast cell line MDPC-23.2. In order to study the role of ClC-7 in tooth development, we transplanted the tooth germ into the renal capsule to simulate an environment lack of osteoclasts for the growth of tooth germs. Verify the feasibility of tooth growth and developing in the microenvironment of the renal capsular at the same time, which providing an excellent environment for tooth development.3. CLC-7 was expressed in predentin and odontoblasts.4. Clcn7 gene plays an important role in tooth development, and it can affect the morphology of teeth, the development of dentin and roots. Maybe Clcn7 affects the tooth development through affecting the expression of DSP, then the dentin.