Effects Of 1,25-(OH)₂D₃ On The Biological Characteristics Of Porcine Ameloblasts

Background:With the development of molecular biology and tissue engineering, regeneration of tooth has become a research focus all over the world. Tissue engineering of tooth regeneration, including simple construction and whole tooth reconstruction:Mixing part of the tooth cells with corresponding scaffold materials and then implants the mixture into animal tissue is the simple construction of tooth, some progresses have been made in this area currently. However, the whole tooth reconstruction is very complicated, it involves the complex of various tooth-forming cells and the combination of different types of scaffolds, there isn't any big progress in this area. Enamel is the hardest substance in vertebrates. Simulating the enamel mineralization to produce biological false teeth, possessing re-mineralization ability, is a dream of all the dentists. At present, the interaction of ameloblasts and the special proteins is still the basis of bio-mineralization forming tooth enamel. Ameloblast is the enamel-forming cells, which is evolved from the inner enamel epithelium in enamel organ. Studies have shown that ameloblast is the key cell in the enamel formation, it not only synthesizes and secretes the specific enamel matrix:amelogenin, non-amelogenin and protease, but also can reabsorb and degrade the matrix. In addition, it also joins in the active transport of calcium. Amelogenin is the main part of the organic matrix of enamel, it plays an important regulatory role in the start of enamel mineralization, the nucleation, the crystal arrangement and many other factors. Non-amelogenins, including enamelin and ameloblastin, were also considered to play a wider role in promoting the nucleation of crystal and impacting the growth of crystal morphology. Therefore, any factor, which can impact the growth and the protein secretion of ameloblasts, can affect the mineralization of enamel.As the activated form of vitamin D steroid hormone, 1,25-(OH)2D3 plays an important role in regulating the growth, differentiation and protein secretion of cells. The receptors of 1,25-(OH)2D3 are widely distributed in oral tissues. Ameloblast is one of the target cell of vitamin D. Using RT-PCR and Northern Blot, foreign scholars found that the expression of amelogenin and ameloblastin, the special proteins secreted by ameloblasts, is regulated by 1,25-(OH)2D3. However, RT-PCR and Northern Blot only can detect the genes expression at transcription level, rather than the translation level. From the transcription level to the translation level, it still contains a complex process. There isn't a consequential correlation between them. In this study, we propose to culture porcine ameloblasts in vitro, using Western Blot to detect the expression of amelogenin and ameloblastin at translation level in the porcine ameloblasts, which is treated by 1,25-(OH) 2D3 at different concentrations and in different time. This work lays an experimental foundation for bio-mineralization of enamel.Objective:1. Porcine ameloblasts is cultured in vitro for further research.2. To investigate the effects of 1,25-(OH)2D3 on the cell proliferation of porcine ameloblast, which is treated by 1,25-(OH)2D3 at different concentrations and in different time.3. To detect the effects of 1,25-(OH)2D3 on the amelogenin and ameloblastin expression of porcine ameloblast, treating by 1,25-(OH) 2D3 at different concentrations and in different time.Methods:1. Cell culture:Select Tibet mini pig, weight 30kg,6 months old, and remove the molars under sterile conditions; separate dental follicle, dental papilla, and enamel organ. Cut the enamel organ into 1mm3 size, using tissue explant culture technique for primary culture, and enzyme digestion methods for passage culture.2. Cell identification:Using anti-vimentin and anti-keratin antibody, immunocytochemistry was performed to identify the cell origin of cultured cells. Detecting amelogenin, a special protein of ameloblasts, by anti-amelogenin antibody to further identify the cultured cells.3. MTT assay:Porcine ameloblasts was treated by gradient Concentration of 1,25-(OH)2D3 (0,1×10-10,1×10-8,1×10-6mol/L) in vitro, using MTT to detect the cell proliferation of porcine ameloblasts dealing with 1,25-(OH)2D3 in 1 day,3 days,5 days.4. Western blot:Porcine ameloblasts was treated by gradient Concentration of 1,25-(OH)2D3 (0,1×10-10,1×10-8,1×10-6mol/L) in vitro, using Western blot to detect the expression of amelogenin and ameloblastin in porcine ameloblasts dealing with 1,25-(OH)2D3 in 1 day,3 days,5 days.Results:1. Cell morphological characteristics:Porcine ameloblast are epithelioid cells, cell morphology is varied, including round, polygonal etc., multi-core, filopodia, incised borders also can be found. Some cells contain clear granular-like substance, which are stellate reticulum cells probably. Giant cells can be found among the oval epithelial cells, rather than fibroblasts (long-spindle shape, smaller cell body, large nucleus, center-located nucleus, clear nucleolus). With the increase of culture time and successive passages, the giant cells were more prevalent, and the cells could not be passaged further.2. Immunocytochemical results indicated the cells were epithelial origin, cytokeratin-14 is positive, cytoplasm was stained with pale-brown, and on the contrary, vimentin is negative, stained cytoplasm without coloring. Cytoplasm staining is positive, pale-brown, detecting by anti-amelogenin antibody, this indicates that the cells is ameloblasts.3. At same time-checkpoint, MTT test results showed that 1,25-(OH)2D3exerts the inhibiting effects on the proliferation of porcine ameloblasts at 1×10-10-1×10-6mol/L concentration range, the inhibiting effects is correlated to the increase of 1,25-(OH)2D3 concentration. Differentiation, between the experimental group and control group, as well as in each experimental group, is significant (P< 0.05). At same concentration of 1,25-(OH)2D3, with the increase of 1,25-(OH)2D3 treating time, the inhibiting effect of cell proliferation is gradually increased. A significant differentiation can be found between each experimental group (P<0.05). The cell proliferation inhibiting effect is shown as a time-dependent manner.4. At same concentration of 1,25-(OH)2D3, with the increase of 1,25-(OH)2D3 treating time, Western blot detecting results showed that the expression of amelogenin and ameloblastin is gradually increased. Differentiation, between each experimental group, is significant (P< 0.05). At same time-checkpoint, with the increase of 1,25-(OH)2D3 concentration, the expression of amelogenin is gradually increased,...