| CLCN7 gene encodes the voltage gated chloride channel 7 (ClC-7) in humans. The mutations in CLCN7 gene have been associated with osteopetrosis in which the abnormal osteoclasts functions are involved. Our previous studies found that some osteopetrosis patients with CLCN7 mutations suffered from impacted teeth and root dysplasia. Dysfunctions of the CLCN7 gene affect osteoclast-mediated extracellular acidification, resulting in disturbed dissolution of bone inorganic matrix and a series of clinical features. In addition, our previous work had shown that there were abundant chloride channels in tooth germ of newborn mice, including ClC-1-7 chloride channels. ClC-7 has also been detected in ameloblast, odontoblast, dental papilla cells and DFCs. Therefore, we hypothesized that ClC-7 plays an important role during tooth development and eruption. As a heterogeneous cell lineage, DFCs may differentiate into several types of cells, such as periodontal-type, cementoblastic-type and osteoblastic-type. To verify our hypothesis, we adoped mouse DFCs as an in vitro model and constructed Clcn7-shRNA lentivirus vector in order to inhibit the ClC-7 expression of DFCs. After that, we tested and screened some significantly changed genes resulted from lowered ClC-7 expression. Then we set up a DFCs/MNCs co-culture system which can mimic the interaction between osteoblasts and pre-osteoclasts, to test the lowered ClC-7 expression’s effect on osteoclasts differentiation and function. Furthermore, we treated DFCs with osteogenic medium or treated dentin matrix medium in order to detect that if there is any changes on their osteo-/cementoblast related genes expression and calcified ability, and if ClC-7 play any roles in these processes.Materials and MethodsExperiment Ⅰ:Using Wise’s method with little modification, tooth germs were dissected from 3-5 postnatal days Balb/c mouse and the DFCs were abtained via the method of twice-enzyme isolation. We observed the characteristics of different generations of DFCs and identified the tissue sources by immunocytochemistry staining of Vimentin and Cytokeratin.Experiment Ⅱ:We constructed Clcn 7-shRNA lentivirus vector and transfected DFCs with it. Then we tested the level of lowered ClC-7 expression and the following changes of other osteoblast and osteoclast related genes through qRT-PCR. Immunocytochemistry of CLC-7 was also performed to observe its expression in DFCs.Experiment Ⅲ:we set up a DFCs/MNCs co-culture system in order to test the effects of lowered ClC-7 expression on osteoclasts formation and function by TRAP stain and dentin resorption assay.Experiment Ⅳ:We treated DFCs with osteogenic medium or treated dentin matrix medium combined or not combined with Clcn 7-shRNA lentivirus treatment, then tested their effects on osteo-/cementoblast related genes expression and calcified ability by qRT-PCR and Alizarin red stain.Results1. With the method of twice-enzyme isolation, we can get a success rate of more than 90% and can see sporadic colonies in the 2nd day and the cells contacted and fused completely after 4-5 days in the culture. The third passage of cells grew fastest, then slowed down and tended to stop in the fourth and fifth passage, respectively. After several times of passage, the cells became larger and polymorphic in shape, some dendrite-like cells were observed. Immunocytochemistry stain showed that the gained DFCs were positive to Vimentin and negative to Cytokeratin.2. Expression of several osteoblast/osteoclast-related fuctional factors were detected in DFCs. After inhibited the expression of ClC-7 in DFCs, the changes of osteoblast related genes were significant while the osteoclast related ones were not. Among three core signal pathway molecules, only Rankl changed significantly while Runx2 and Tgfbl did not.3. After 7 or 14 days’DFCs/MNCs co-culture, TRAP stain was performed and dentin absorbtion pits were observed and pictured under a scanning electron microscope. The lowered expression level of ClC-7 resulted to the decreased number of TRAP positive multinuclear osteoclast-like cells and their impaired dentin absorbtion capability.4. Osteogenic medium showed both osteoblast and cementoblast differentiation inductive abilities while treated dentin matrix medium only showed osteoblast differentiation inductive abilities. But there was no difference of induced calcified capability between Clcn 7-shRNA group and control group. Tgfb1 was significantly increased after 3 days’OM or TDMM induction combined with Clcn7-shRNA treatment.Conclusions1. The method of twice-enzyme isolation helps us get considerable cells in a short time with a high success rate and easy to handle, so it’s an ideal culturing method for mouse DFCs in vitro. The isolated cells from the dental follicle showed the elongate shape and expressed Vimentin instead of Cytokeratin, and the overall characteristics of DFCs could attribute to their mesenchymal origin in our study.2. The lowered ClC-7 level of the DFCs resulted in changes of expression levels of osteoblasts related factors and RANK-RANKL-OPG signaling, suggests that DFCs may work similar to osteoblasts and ClC-7 may affect DFCs via RANK-RANKL-OPG signaling.3. DFCs/MNCs co-culture system can mimic the paracrine and induction effects of DFCs during tooth eruption, and then promote MNCs differentiate into OCLs. The lowered ClC-7 level of DFCs resulted in decreased OCLs number and their impaired dentin absorbtion capability.4. Osteogenic medium showed a more cementoblast differentiation inductive ability than treated dentin matrix medium. ClC-7 may play different roles during these two processes. TGF-β1 may take part in the regulation of ClC-7 in the multi-differentiation processes of DFCs. But the exact mechanism needs to be further elucidated. |
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