| PURPOSEIn this experiment, the study was focused on the effect of static magnetic field on the osteoblast growth and function change on basis of rat calvarias osteo-blast culture, which could exclude the intervention of complicated biological environmental factors in vivo. Also the effect of static magnetic field was studied a-bout the structure and function of periodontal membrane by animal model with periodontitis. The purpose laid foundation for choosing the proper magnetic parameter in clinical practice as well as the scientific application of magnetic thera-py.METHODSIn vitro experiments:1. Design of static Magnetic FieldNdFeB was magnetized and fixed in a cuboids shaped tank. North pole of die magnet was upwards and the cell culture plates were laid on the tank. The level of the tank was regulated to attain the desirable magnetic field intensity at the bottom of different cell culture plate: 0Gs, 400 Gs, 620 Gs, 830 Gs, 1080 Gs.2. separation and culture of Rat Calvarias OsteoblastCalvaria bones of neonatal Wister rats were obtained and digested by 0. 25% tripsin and 1mg/ml collagenase â…¡. After fibroblast cells were isolated by differential adhesion method, the osteoblast cells were cultured in cell incubator serial subcultivation was conducted after cell had reached confluence and the passage cell was collected for later use.3. Detection of Osteoblast ProliferationOsteobiast cells were seeded on the 96-- well culture plate and cultured for 24 hours, then exposed to the magnetic field with different magnetic intensity for further culture of 24h, 48h, and 72h respectively. OD value (optical density) was measured at 490 nm in a microplate reader. The experiment was repeated twice.4. Flow Cytometry for Detecting Cell CycleOsteoblast was planted on the culture dish (35mm in diameter) for 72h culture, then exposed to the field with different magnetic intensity of 0 6s, 620 Gs, 830 Gs, and 1080 Gs respectively for another48k culture. Then they were digested by 0.25% tripsin after that PI ( propidium iodide) was addedâ– â– '. Then suspension was subjected to FAGS Flow Cytometry for detecting the DNA content. The experiment was repeated three times.5. Osteoblast ALP staining and activity detectionCells were seeded on coverglass for48h, then changed into 62OGs magnetic field for another 48h,at last stained with ALP enzyme by means of improved Ga- Co method. At the same time the cell was seeded on the 96 - well culture plate, and exposed to the different magnetic intensity field for 24h, 48h, 72h respectively. After the effect of 0.2%TritonX -100,add pNPP, then OD value (optical density) was measured at 490 nm in a microplate reader.6. Immunohistochemical staining and Western blotting analysis of osteoblast Cells were seeded on coverglass for 72h, and changed into 62OGs magneticfield for48h, then fixed by acetone, and immunohistochemical staining with collagen type I by SABC method/Meanwhile cells were seeded on both sides of 24- well culture plate with the density of 5 X 104/ml, added 62OGs magnetic field intensity to one side, cultured for 2 weeks the form of cell and bone tuber could be observed under invert microscope.7. cell calcification in vitroCell was seeded on the 24-well culture plate, and exposed to the 620 Gs magnetic intensity field for 2 weeks. Transparent calcification nod was inspected through phase contrast microscope.In vivo experiments:8. Experiment on animal8.1 animal models in periodontitisThe Wistar rats with periodontitis were obtained by use of the stainless steel wire. After 5 weeks steel wire was removed, cut down skin of cheek on caput, and buried NdFeB with and without magnetism in hypodermis. After 2, 4 and 7 days, the rats were put to death respectively, then took out of the diseased teeth and their Periodontal tissue for specimen.8.2 HE staining for observation of periodontium structure under light microscope and ultrastructure change under electron microscope8.3 Immunohistochemical staining and hybridization in situ detecting of BMP-2Tissue sections were immunohistochemical stained with BMP-2 by SABC method with the first Ab density 1-100. And the other sections were digested by Pepsin, then putted into pre. hybridization fluid for 4h under 42 , added BMP-2 oligonucleotide probe to stay overnight in calorstat.RESULTS1. Observation of primary cultured osteoblast form under phase contrast microscopeAfter fibroblasts were isolated by differential adhesion method, the osteo-blasts were abstained and cultured in cell incubator for 24h, they grew into the shape of triangle, bursiform and multi -angles form which adhered to the wall. And they could cover the floor at about 7days later.2. Osteoblast Cell Proliferation in static Magnetic FieldWhen the magnetic exposure time extended to 48h or 72h, the corresponding OD value of osteoblast with magnetic treatment of 400 Gs or 620 Gs increased obviously with significant difference as compared with OGs group ( p < 0.0S). However, such proliferation effect was not shown in the 1080 Gs group (p>0.05).3. Effect of static Magnetic Field on Cell Cycle of Osteoblast CellAfter magnetic exposure with intensity of 620 Gs, G0/G1 phase percentagedecreased, but S phase and G2/M phase percentage was dbviously higher as compared with control. Proliferation index (p <0.05). However, there was no significant difference in S phase and G2AM phase percentage between the 1080 Gs group and control (p>0.05). ,4. Effect of Magnetic static Field on ALP enzyme of osteoblastThere were black granules deposit in: '5. Effect of Magnetic static Field on collagen type I of osteoblastMale BMP-2 staining is brown in cytoplasn. The BMP-2 staining color of cell in magnetic therapy group was deeper than that of control. Hie result of Weatern protein blot showed that the magnetic static field affected expression of collagen type I of osteoblast. Hie expression in 62OGs and 83OGfi group was increased greater than control, but that of 1080Gs group decreased.6. Effect of Magnetic static Field on cell calcification in vitroCell arrayed compactly in the shape of multi-angles form and fusiform , and they could gather together into colony. Through phase contrast microscope, there could be seen a lot of dispersed high density areas which appeared very strong bright points. Intermediate cefl was embed by extra matrix, and formed transparent calcification nod. However, it could hot be seen in the controls.7. Result of HE staining in periodontal membranePeriondontitis group: periodontal pocket formation, periodontium fibers disordered arrangement, depressed absorption on the surface of alveolar bone, showing up a lot of osteoclast. Periodontitis after magnetic therapy for lweek group: periodontal fiber arrayed in good order, osteoblast increased on alveolar bone, cell on the surface was big and obvious. .8. Periodontal membrane under electron microscopeThe volume of cytoplasn decreased and organell didnt become developed in periodontitis. While in therapy group, the quantity of fibroblast processes and the volume of cytoplasn increased, and they have developed rough endoplasmicreticulum and ribosome. (9. Result of BMP-2 stainingThere was no significant difference in expression of BMP-2 between normal and periodontitis group, but great difference between magnetic therapy and peri-odontitis group, P<0.05. When periodontitis, there were BMP-2 malehybrid-ization signals in periodontium fibroblast, cementoblast and collagen fibers. In magnetic therapy group, the signals increased with different degree, especially in the second day.CONCLUSIONS1. Within a certain range of magnetic intensity, magnetic field enhances cranium osteoblast growth in vitro and this effect was completed by altering cell cycle and raising S phase and G2/M phase percentage.2. Static magnetic field stimulation appears to enhance the differentiation of osteoblast by increasing alkaline phosphatase activity and the production of collagen type I . And static magnetic field also stimulated the calcification of osteoblast. The results indicate that osteoblasts are sensitive to static magnetic field stimulation, which alters cell activity through altering the secretion of BMP-2 growth factor.3. 1200Gs static magnetic field could stimulate the BMP secretion of periodontal membrane cell , increase the number of osteoblasts on the surface of alveolar bone, and activate the function of fibroblast in periodontal membrane. This supposed that a certain range of magnetic intensity could promote the reconstruction of periodontal membrane in rats. Thus static magnetic field may play an important role in treatment of periodontitis.. |
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