The Study Of Osteogenesis Induced By MiR-34a Under Mechanical Stress In Vitro And In Vivo

The ideal treatment effect was based on the choice of appliance,the design of orthodontic plan and better periodontal conditions.The tooth accomplished physical movement in the alveolar bone depending on the basic theory of bone resorption in compression lateral and bone deposition in tension lateral.Thus,the conditions and bone remolding of alveolar bone was physic bases of tooth movement.However,in clinical orthodontic the patients with bad alveolar bone were always common,especially adult patients.Because the development of alveolar bone has affected by congenital and aquired factors,the conditions of alveolar bone in adult patients were always absorption,degeneration(or density)and defect.The disadvantages of these alveolar bone could not be remedied by any appliance techniques,and the doctors usually only compromised the orthodontic design which led to non-ideal orthodontic effect.Recently there were still no methods and ideas to improve the bad conditions of alveolar bone in clinical orthodontic and experiment studies.With the rapid development of biotechnology,more and more fields payed attention to gene therapy.In the previous study,the orthodontic study was still limited to the level of factors,proteins and signal pathways,and the study about molecular level was rare.micro RNAs were an endogenous single RNAs and could regulate 60% genes of human,and micro RNAs were hot in field of gene therapy.Under orthodontic force,many micro RNAs were detected by mciro RNA gene chip.The mutual regulation effect between micro RNAs and mechanical force was precise and essential for bone remolding induced by mechanical stimulation.Among them mi R-34 a was a specific and credible micro RNA about bone.The mi R-34 a was widely used in inhibiting cancer.Moreover,mi R-34 a not only inhibited the proliferation,development and migration of cancer cells but also the osseous metastasis,thus in recent five years the veil of mi R-34 a participating in bone metabolism was gradually uncovered.In 2014 year,the Nature reported the essential effect of mi R-34 a on bone remolding,especially about osteoclastogenesis,following by other studies about the effect ofmi R-34 a on kinds of osteocytes.mi R-34 a was a naturel inhibitor of osteoclastogenesis,and also could regulated osteogenesis.Moreover,mi R-34 a participated in development of chondrocytes,bone repairing reaction and tooth development.Due to the disadvantages of instability,negative charge and degradation by nuclease,the micro RNAs always need to be carried by cationic polymers.PEI25 K possessed of advantages of high transfection efficiency,high affinity capacity,better ion buffering which made it be “gold standard” in field of non-viral cationic polymers.But the biggest disadvantage of PEI25 K was its severe cytotoxicity.The derivates of PEI25 K N-Ac-L-Leu-PEI not only inherited the advantages of PEI25 K but also better cell biosecurity.As the N-Ac-L-Leu-PEI had been reported and applied in well-known magazines,in this paper we would apply N-Ac-L-Leu-PEI to accomplish the transfection of mi R-34 a in vitro and in vivo.It was well known that micro RNAs degraded or inhibited target gene by base complementation paring to 3' non-translated regain of target m RNA.Thus,we studied the basic mechanism of bone remolding induced by mi R-34 a and to find the target gene of mi R-34 a.The study of mechanism and target gene may help us understand the pathway of the effect of mi R-34 a on bone remolding under mechanical force,which also allowed us to modify delivery carriers by grafting target gene to improve the specification of mi R-34 a on bone remolding,then increased the regulated efficiency.The content of this research was divided into five parts:Experiment 1: the osteogenesis differentiation and expression of mi R-34 a in alveolar bone were changed by tooth movement in vivo and in vitro.In vivo Wistar rats were used to build experimental tooth movement model,and in vitro the rat bone mineral stem cells loaded by four-point bending mechanical apparatus were used as model.The histologic and immunohistochemical staining,real-time PCR,ALP activity,ALP staining and Western blot were used to detect the osteogenesis differentiation and expression of mi R-34 a in vivo and in vitro.Experiment 2: the detection of carry capacity of N-Ac-L-Leu-PEI with micro RNAThe delivery system N-Ac-L-Leu-PEI was experiment compared with commercial Lipofectamine 2000.The characterization detection,gel retardation assay,MTT assay,flow cytometry and fluorescence microscope in vitro as well as transfection efficiency by q RT-PCR and biosafety by serum biochemical and tissuetest were applied to examine the delivery capacity and biosafty of N-Ac-L-Leu-PEI.Experiment 3: the effect of mi R-34 a on osteogenesis differentiation of BMSCs under mechanical force in vitro.We applied N-Ac-L-Leu-PEI to achieve the transfection of mi R-34 a agomir and antagomir and then examined the effect of mi R-34 a on gene,protein and ALP activity of osteogenesis differentiation under mechanical stress through mi R-34 a overexpression and mi R-34 a inhibition.Experiment 4: the effect of mi R-34 a on rat tooth movement and alveolar bone remolding under orthodontic force in vivo.Similarly,we applied N-Ac-L-Leu-PEI to achieve the transfection of micro RNAs in local alveolar bone in rats.And then we applied real-time PCR assay,histologic and immunohistochemical staining,micro CT assay to detect the displacement of tooth movement,the expression of gene and proteins of osteogenesis differentiation,the indexes of bone metabolism through mi R-34 a overexpression and mi R-34 a inhibition.Experiment 5: the mechanism of the regulation of mi R-34 a on bone remolding under tension stress.We used western blot assay to detect the mechanism.Firstly,we examined the Wnt/?-catenin signal pathway under stress loading.And then we detected the proteins of Wnt/?-catenin signal pathway through mi R-34 a overexpression and mi R-34 a inhibition by N-Ac-L-Leu-PEI under stress loading.Thirdly,we preliminarily presented the target of mi R-34 a by software analysis and luciferase assay.Lastly,applying si RNA to achieve suppression of target gene,and then evaluate the regulation of mi R-34 a on proteins from Wnt/?-catenin pathway.Through the above 5 experiments,the results were as follows:Experiment 1The rats' tooth movement model in vivo and BMSCs force loading model in vitro were successfully built.Under orthodontic force loading,more Runx2-positive cells were found from histologic and immunohistochemistry examination,and increased genes of Runx2,Col I,ALP as well as proteins of Runx2 and Col I were also found in force group in vivo and in vitro.The ALP activity in vitro was also improved by stress.Moreover,the expression of mi R-34 a was augmented under force loading in vivo and in vitro at every test time,which kept a similar increased tendency with osteogenesis differentiation genes.Experiment 2N-Ac-L-Leu-PEI owned lower size and zeta and could completely combine mi R-34 a at mass ratio of 2:1 to form nanocomplex(diameter 200nm).MTT assay showed the relative growth rate of BMSCs were all above 90% after transfection of N-Ac-L-Leu-PEI/mi R-34 a complex which proved N-Ac-L-Leu-PEI to be good biosecurity.Flow cytometry and fluorescence microscope observation showed higher transfection efficiency of N-Ac-L-Leu-PEI/FAM-mi R-34 a.And the better biosecurity and transfection efficiency were higher than commercial Lipofectamine 2000.Moreover,the expressions of mi R-34 a were changed to 29 folds and 0.4 folds after N-Ac-L-Leu-PEI-mediated agomir and antagomir delivery in vivo.The indicators of liver and kidney were normal as well as no abnormal liver and spleen and kidney lung tissue.Experiment 3After force loading,overexpression of mi R-34 a increased m RNA and proteins level of Runx2 and Col I as well as ALP activity.But the effect of inhibition of mi R-34 a on BMSCs under stress was contrary to mi R-34 a overexpression.Experiment 4The overexpression of mi R-34 a could improve the expression of gene and proteins of Runx2 and Col I,and in experimental lateral the indexes of bone anabolism(BV/TV,BMD,Tb.N,Tb.Th)were increased and the parameter of bone catabolism Tb.Sp was decreased,and the displacement of tooth movement was reduced in the lateral of mi R-34 a agomir compared with mi R-NC lateral.While the effect of mi R-34 a inhibition was also contrary to mi R-34 a overexpression.Experiment 5Under force loading,GSK-3? was inhibited and active ?-catenin was improved,which initiated the Wnt/?-catenin signal pathway.After N-Ac-L-Leu-PEI/mi R-34 a agomir transfection,the GSK-3? and active ?-catenin proteins were more downregulated and upregulated under tension condition which acted the Wnt/?-catenin signal pathway.Similarly,the effect of mi R-34 a inhibition was also contrary to mi R-34 a overexpression.Through software analysis and luciferase assay,GSK-3? was the target of mi R-34 a.After applying si GSK-3?,mi R-34 a lost the regulation of proteins from Wnt/?-catenin signal pathway.According to the results above,we can draw the following conclusions:1.The force loading could improve osteogenesis differentiation and bone formation in vivo and in vitro,during this process the expression of mi R-34 a was also increased and changed by time.2.N-Ac-L-Leu-PEI could condense mi R-34 a to stable nanocomplex with higher transfection and better biosafety in vivo and in vitro.2.In vitro study,mi R-34 a could augment the osteogenesis differentiation under stress loading.3.In vivo study,mi R-34 a could reduce the displacement of tooth movement through enhancing the local alveolar bone anabolism around moving tooth.4.In the study of mechanism,we found mechanical stress could active Wnt/?-catenin signal pathway.The osteogenesis differentiation of BMSCs induced by mi R-34 a attributed to its target on GSK-3? which inhibiting the activation of Wnt/?-catenin signal pathway.