| Objective: As is known to us all, the biological basis of orthodontic treatment and distraction osteogenesis were the adaptive reconstruction of canial and maxillofacial bone reposed to mechanical strains. Osteoblast played an important role in the process of bone reconstruction. Osteoblast was one of the main components of the bone tissue, and served as the sensitive cells to stress, and played a center regulation role in various processes of bone metabolism. It was proved that stress stimulus with suitable intensity and action time could stimulate the activity of osteoblast and promote the bone formation. Nevertheless, it was not clear how osteoblasts sensed the mechanical stimulation signal and turned it into biomechanical signals. Previous scholars were confined in researching one or several molecule genes, which was difficult to fully comprehend the molecular mechanism of osteoblasts to mechanical signal. Therefore, this study loaded with cyclic-tension force on mouse osteoblasts cultured in vitro through Flexcell FX5000 Tension system, and investigated the effect of cyclic-tension force on proliferation and differentiation of osteoblasts by MTT method, and screened out optimal tensile value, so as to analyse the gene expression profiles of the optimal force value group and control group using gene chips.(MoGene 2.0 STArray).Through the analysis of differentially expressed gene, the study explored the response mechanism of osteoblast to mechanical signal in molecular level and provided the molecule theory basis for orthodontic treatment and distraction osteogenesis in clinic.Methods:1 The resuscitation and cultivation of osteoblastsAfter having resuscitated the cryopreserved original generation of mouse embryonic osteoblast, microscope was used to observe the growth condition of MC3T3-E1 osteoblast. They were not passaged till monolayer cells spread to more than 80% of the bottom of the culture bottle.2 Osteoblasts seedingThe fifth generation osteoblasts were inoculated into five BioFlexCollagen TypeⅠ-Eachs with 2×105 piece/hole respectively and cultured 24 hours. Then low concentration of serum was used to make osteoblasts synchronization growth 24 hours.3 Torque model of osteoblasts in vitroThe BioFlex-Collagen TypeⅠ-Eachs were randomly divided into A, B, C, D, E group. There were six holes in each group and each hole was added in 2 ml 10% serum culture medium. What is more, five groups were separately subjected to 0%, 6%, 12%, 18% or 22% elongation for 24 h by Flexcell 5000 strain loading system simultaneously. The frequency and waveform were 0.1 Hz and half sine wave.4 The detection of osteoblast proliferation activityAfter withdrawal of traction force, MTT colorimetric method was performed to assess cellular proliferation and Enzyme-linked immune detector was used to test absorbance value of osteoblasts at 490 nm. Then data was recorded and analyzed using the SPSS19.0 statistical software.5 The extraction of total RNAThe total RNA was extracted in the optimum value group and control group cells using TRIZOL method and detected their purity and integrity.6 Gene chip detectionAffymetrix gene expression profile chip technology was adopted to select differentially expressed genes between the optimum value group and control group. Afterwards, transcriptome analysis Console3.0 software was employed to analyze scan findings and screen out differentially expressed genes.Results:1 MTT results:Absorbance values were compared after osteoblasts loaded with cyclic tensile strain for 24 hours:(1)6 % elongation experimental group: 0.4621±0.0120;(2) 12 % elongation experimental group: 0.5423 ±0.0148;(3) 18 % elongation experimental group: 0.4511±0.0369;(4) 22 % elongation experimental group: 0.3623±0.0324;(5) 0 % control group: 0.3931±0.0538. There was statistical differences in the absorbance values between experimental groups and control group(P<0.01). Experimental results indicated that the proliferation of MC3T3-E1 osteoblastic cells were signifycantly increased after 6 %, 12 %, 18 % elongation stimulation, in which, 12 % elongation rate showed the strongest stimulatory effects(P<0.01). When the mechanical strain was 22 % elongation, cell proliferation would be inhibited. Consequently, 12 % elongation was supposed to the optimal value for promoting cell proliferation and differentiation; 2 The identification of total RNA: 1) Ultraviolet spectroscopical detection and analysis indicated that all total RNA OD260/OD280 between 1.9 and 2.1, which showed that the purity of RNA were qualified. 2) By gel electrophoresis, RNA extracted from A and C samples revealed there were three stripes. In addition, 28 s strip was still two times more than that of 18 s strip, and 5 s strip was the darkest. It showed that the integrality of RNA was qualified; 3 The results of gene expression profiles: Microarray was used to analyze the differentially expressed genes. Compared with the control group, there were 722 more than 2 times differential genes in the 12 % elongation experimental group, in which, 560 genes were up-regulated, 162 genes were down-regulated. Gene associated with cell proliferation differentiation mainly included Rho, SOST, Wnt3 a, BMP4, MAPK10, MAPK3, these genes raised 2.35,-2.37, 2.15, 3.19, 2.71, 2.53 times respectively. Furthermore, they were important components of BMP signaling pathway, MAPK signaling pathway and Wnt signaling pathway. These signaling pathways may be involved in the process of cell proliferation differentiation induced by mechanical dynamics.Conclusion:1 The experiment demonstrated that appropriate tensile stress had the effects on stimulating the proliferation and differentiation of cultured osteoblasts in vitro. However, too more force value could inhibit the growth of osteoblasts.2 Genes expression was altered after osteoblasts suffered to appropriate tensile stress. Genes associated with cell proliferation differentiation mainly included SOST, Wnt3 a, BMP4, Rho, MAPK10, MAPK3, in which,Wnt3 a, BMP4, Rho, MAPK10 and MAPK3 were up-regulated, SOST was down-regulated.3 SOST, Wnt3 a, BMP4, Rho, MAPK10, MAPK3 may be regulate the proliferation differentiation of osteoblasts through Wnt, BMPs and MAPK signaling pathways associated with ossification. |
