Experimental Study Of Simvastatin's Influence To Mandibular Distraction Osteogenesis In Rabbits

Since Ilizarov fully developed the technique of distraction osteogenesis,now it is widely used in curing serious oral and maxillafacial deformity.Traditional distraction osteogenesis has some shortcomings, such as long curing periord,decline of the new bone′s hardness ,density and mechanical function,and infection.So it has not only important theoretical meaning but also practical meaning to improve the quality and speed of distraction osteogenesis.Since Simvastatin can improve osteoblastic proliferation and up-regulate the expression of BMP2 ,and has biological character to decrease bone fracture possibility in postmonoposaul women, We implore Simvastatin in the distraction period of distraction osteogenesis to investigate Simvastatin's influence to distraction osteogenesis and possible mechanism by pathology, X-ray oberservation, BMD and immunohistochemical methord ,to discuss the best way to promote distraction osteogenesisFirst we design a new distractor simple and with good stability and establish a rabbit model for billateral mandibular distraction osteogenesis. The result shows the new distractor has good stability and can extense rabbit mandible successfully. Then we can study distraction osteogenesis in large quantity.Then 18 adult New Zealand rabbits were divided into two groups randomly,one for experiment the other for control. Each group has 9 rabbits.Distraction osteogenesis was applied in every group. From the beginning of distraction period, every rabbit was administrated with Simvastatin 5 mg·kg -1·d-1 at the same time in experimental group for 7days.Every animal was distracted for 5 days by 1mm·d-1.At the end of distraction period,the second week and the fourth week of consolidation period,all the animals were examined by X-ray under anaesthesia,and three animals of each group were sacrificed and fixed and examined by BMD and pathology test.Pathology test shows there are more new formed trabeculars arranged intensively in distraction zone in experimental group compared with that in control group.The quantity of shuttle-like cells,osteoblasts and osteocytes in distrction gap in experimental group is more than that in control group.In the fourth week of consolidation period, narrow fibro district still can be seen in distraction gap in one rabbit of control group,on the contrary the distraction gap was full of bone in experimental group.This implys Simvastatin promote new bone formation in distraction osteogenesis by promoting osteoblastic proliferation and differentiation. In the end of distraction period ,osteoclasts can hardly be seen on the edge of trabeculars in experimental group,while osteoclasts can be seen in control group.This suggest that Simvastatin maybe can inhibits bone resorption by inhibiting osteoclatic differentiation.There are more blood vessels and less Lymphoid cells in distraction gap in experimental group than that in control group.This suggests that Simvastatin promote angiogenesis and it has anti-infection function in distraction osteogenesis.X-ray observation shows that during the end of distraction period very low density image of new bone appears in the distraction gap of experimental group,while nothing can be seen in the same district in control group.At the second week of consolidation period,the width of low density image in distraction gap in experimental group is much narrow compared with that in control group.At the fourth week of consolidation period,the image of distraction gap in experimental group is difficult to identified,while the density of distraction gap in control group is low enough to identified clearly. Bone mineral density test shows during the second week of consolidation period,the BMD in distraction gap in experimental group is much higher compared with that in control group .P<0.05.The difference has statistic meaning.During the end of distraction period and the fourth week of consolidation period, the BMD in distraction gap in experimental group is a little higher compared with that in control group . P>0.05.The difference has not statistic meaning. All these suggest that Simvastatin promotes the formation and mineralization of new bone in distraction osteogenesis.To further study the mechanism of Simvastatin promoting distraction osteogenesis,we detect the expressions of c-fos,OPG,NOS3,OSX,FⅧrAg in distrction gap by immunohistochemical methord.The result shows in the end of distraction period,strong positive staining of OPG is higher in experimental group than that in control group.In statistic analysis, P < 0.05.The difference has statistic meaning.During consolidation period, the positive staining of OPG in both groups wore off gradually,and there are no significant staining difference between the two groups. .In statistic analysis, P>0.05.The difference has no statistic meaning.This suggests Simvastatin maybe inhibit osteoclastic formation and activity in some degree by its influence to OPG or OPG's up-stream factors .But its effect to reduce bone resorption is relativelyweak.In the end of distraction period,the expression of NOS3 in experimental group in blood vessels is very strong,which is much higher than that in control group .In statistic analysis, P<0.05.The difference has statistic meaning. the positive staining of NOS3 in both groups wore off gradually,and there are no significant staining difference between this two groups.This suggest that Simvastatin up-regulates NOS3's expression in the end of distraction period and promotes angiogenesis in distraction gap. In the end of distraction period, the positive staining of NOS3 in shuttle-like cells and osteoblasts in experimental group is a little higher than that in control group. .In statistic analysis, P<0.05.The difference has statistic meaning.This suggests that in the end of distraction period ,Simvastatin can also promote osteoblasts'proliferation by NOS3 to promote the formation of new bone.In every periods of distraction osteogenesis,the positive expression of c-fos in shuttle-like cells,osteoblasts and osteocytes in experimental group is obviously stronger than that in control group .In statistic analysis, P<0.05.The difference has statistic meaning.In consolidation period, the positive staining of c-fos in both group wore off gradually.This suggest Simvastatin up-regulates the expression of c-fos to promote osteoblastic proliferation and differentiation.The positive staining of OSX is weak in the end of distraction period and the second week of consolidation period.In the fourth week of consolidation period , the positive staining of OSX in osteoblasts, osteocytes and shuttle-like cells strengthened in both groups.The expression of OSX in experimental group is obviously higher than that in control group. .In statistic analysis, P<0.05.The difference has statistic meaning.This suggests OSX mainly stimulates osteoblasts in the later course of distraction osteogenesis. It is an important middle-stream transcription factor that enfluence distraction osteogenesis.In the end of consolidation period, Simvastatin promotes osteoblastic differentiation and the mineralization of new bone and inhibits chondrification by up-regulating the expression of OSX .In the end of distraction period, the strong positive staining of FⅧrAg was mainly found in blood vessels.And positive staining of FⅧrAg was found in osteoblasts,osteocytes,shuttle-like cells. The expression of FⅧrAgin experimental group is obviously higher than that in control group. .In statistic analysis, P < 0.05.The difference has statistic meaning.After distraction period, the positive staining of FⅧrAg in both groups wore off gradually. This suggests that Simvastatin up-regulates FⅧrAg's expression in the end of distraction period and promotes angiogenesis in distraction gap of the experimental group. In the end of distraction period ,Simvastatin maybe can enfluence osteoblasts in some degree by FVIIIrAg's over-expression to promote the formation of new bone. Conclusion: Simvastatin enfluences distraction osteogenesis mainly by promoting osteogenesis and angiogenesis and maybe by reducing bone resorption in some degree. Simvastatin promotes osteogenesis by three molecular pathes: BMP2,NOS3 and c-fos。Simvastatin stimulates the expression of BMP2 to upregulate the expression of Runx2 and OSX,then the expression of OCN,OPN,BSP,ON and ALP, colleginⅠwere started by OSX.So the differentiation and maturation of osteoblasts ,and the mineralization of new bone were promoted.At the same time ,the differentiation to condrocytes was prohibited.Simvastatin promotes the over-expression of NOS3 by types of mechanism. The increasing of NO's formation promotes the proliferation of osteoblasts.This mechanism can be explained by following theory:In distraction osteogenesis, Simvastatin maybe increases the expression of NOS3(NO)to upregulate c-fos'expression,so the expression of TGFβand its'downstream factor OSX is also increased.At last the expression of OSX'down-stream factor is promoted. At the same time, c-Fos also stimulates the expression of IGF1and IGF2.So the quantity of mature osteoblasts is increased.And c-Fos its'self can also stimulate the proliferation and differatiation of oateoblasts and promote the expression of OCN.Simvastatin inhibits bone resorption by up-regulating the expression of OPG through these three up-stream factors.And it promotes angiogenesis by up-regulating the expression of NOS3 and its'downstream factor TGFβ1 and also by increasing FⅧ rAgand other angiogenesis factors'expression .In addition , NOS3 maybe co-operate with VEGF and FⅧrAg to promote the proliferation of osteoblasts.Now it is the first time that Simvastatin was applied in mandibular distraction osteogenesis.And it was found that Simvastatin can promote formation and mineralization of the new bone The experiment supplys the basis for clinical applyment of Simvastatin in distraction osteogenesis.It has important practical value.At the same time ,it is the first time that immunohistochemistry methord was implored to investigate the expression of NOS3 and OSX in distraction zone of distraction osteogenesise.And we explain the possible molecular pathes by which Simvastatin influence distraction osteogenesis.This supplys the basis for explaining the molecular mechanism of distraction osteogenesis and for promoting distraction osteogenesis by other methords.It has important theoretical meaning.