The Effect Of Exogenous VEGF And BMP On Revascularization And Bone Activity Of Rabbit Heterotopic Allograft Decalcified Bone

1 Background The repairment of large bone defects caused by trauma,infection and bone tumor has been one of the difficult problems which the surgeon are facing.At present,autologous bone graft has always been considered the gold standard for treatment of bone defects,but autogenous bone repair bone defect after transplantation often face someserious complications,such as pain,infection,nerve injury at the same time,because of the autogenous bone graft materials are less,which limits its application in clinic.With the deepening research of material on bone transplantation,because allograft bone has wide source and has the advantages of bone induction,bone conduction and so on,so it can meet the needs of bone defect repairment of various bone defects and has been widely used in clinic.However,allograft bone in the repairment of bone defect has revascularization,bone reconstruction and other problems,focusing on these problems,many scholars have been exploring the issuses,And in our previous investigation,Luo etal had put the allogeneic bone in the rich blood supply of the rabbit right thigh muscle with the vastus medialis muscle gap near the saphenous artery,after a period of time,the allograft bone can achive revascularization and bacome activated bone,which accompanied the formation of new bone.Acorrdng to the result,in order to accelerate the process of bone allograft vascular transition into activation bone,and shorten the time of bone graft to repair bone defect by creeping substitution,in this experiment,we choose China rabbits as experimental object,adopt allograft bone as bone graft material,by applying exogenous growth factor VEGF composite BMP to accelerate the transformation of allograft bone for ectopic activation speed,shorten the creeping substitution process.2 Objective To investigate the effect of exogenous VEGF and BMP on revascularization and bone activity of rabbit heterotopic allograft decalcified bone.3 Method210 adult healthy China white rabbits were selected,no limitation with sex,30 rabbits as the donor preparation of allogenic decalcified bone,according to the random number table,the rest were randomly divided into experimental group A(allograft decalcified bone +VEGF+BMP)60 and group B(decalcified bone +VEGF)60,control group(allograft decalcified bone)60.For the group A,the prepared 1.5cm long homologous decalcified tibia was placed in rabbit right thigh of rectus femoris and vastus medialis muscle gap near by saphenous artery,and fixed on the femur with two0.8mm Kirschner wire.In the vicinity of the skin,Implanted an osmotic pump which contain the VEGF solution 200 ul with concentration is 0.5?g/ml,and implanted the gelatin sponge contain the BMP solution 1ml with concentration 1?g/ml in the bone,group B received the same treatment with the A group without BMP;control group were equal with allogenic decalcified bone in his right thigh was placed in the corresponding parts of rabbit.Each group respectively at 0,2,4,6,8,10,12 week to death 10 white rabbits,By specimen observation,HE dyeing observation and detection of type I glue protein fluorescence intensity,calcium content and Bone alkaline phosphatase of the examination.and According to the experimental results to analyze the effect of exogenous growth factors on revascularization and bone activity of rabbit heterotopic allograft decalcified bone.4 Results1.general observation of speciman Two weeks after operation,the wound healing of the rabbits in the three group was good,and no obvious inflammatory solution was found.After 4 weeks,observing group A bone surface with a small amount of connective tissue covering,and it is closely linked with the surrounding soft tissue,comparing with 2 weeks,the surface of thebone appeared more superficial bone resorption;the gorup B was observed that the ectopicbone segment with the surrounding soft tissues have certain adhesion,bone surface also appears a little superficial lacuna;For control group,the bone adhesion with surrounding tissue is loose,bone surface is still relatively smooth withuut any obvious lacuna.With the extension of time,the formation of fibrous connective tissue scaffold is loose,the surface of bone lacune deepened tarnish and the bone lacuna of bone surface further deepen.2.HE staining results At 0 week,there was a small amount of connective tissue in the Haversian canal of the bone tissue of the three groups.At 2 week,the group A showed less angiogenesis and osteoclast distribution in Haversian canal,in group B,around Haversian canal without obvious vascular ingrowth and only with a little granulation tissue.At 4 weeks,in group A,it appear more microvascular distribution in the lumen side of Harvard canal;in group B,the lumen of the Haversian canal increased,and the number of new microvessels increased and some of the dense connective tissue;In the control group,there was a little capillary likly structure in the Haversian canal.And with the time it can observe that there are some of the distribution of osteoblasts around the Haversian canal,the new bone formation is less.at the same time,accompanied by a large amount of bone cell distribution around it.The CD34 immunofluorescence staining showed that,the number of CD34 positive blood vessel showing the first increase and then decrease,with the extension of time.and the number of the group A and group B increased higher than control group before 8 weeks,but the increasing btween group A and group B with no significant difference.The number of CD34 positive CD34 positive vascular vessel of the group A and group B reached the maximum number of positive vessels in the sixth weeks,and for control group,it reached the maximum in the eighth week,but the amount of angiogenesis of group in the sixth weeks was significantly higher than that in control group in the eighth week,at the same time the amount of angiogenesis of group A a little more than sixth week in group B,the angiogenesis of vascular production of group B in the sixth week is also more than the control group in the eighth week,but the comparison between all the groups with statistical significance(P<0.05).Within groupA in six week,with the increaseing of observation time,the number of CD34 positive vessels also show an increasing trend,and the number of CD34 positive vessels in fourth week less than taht in sixth week and has statistical significance(P<0.05),the number of CD34 positive vessels at eighth week less than that in 6 week,and has statistical significance(P<0.05).For group B,the comparing results is similar to group A,in control group,comparing the numbers of CD34 positive vascularat at sixth week is also less than that at eighth week,the CD34 positive blood vessel number at tenth weeks is also less than that at eighth week,and it has statistical significance(P<0.05).The trend of the three groups of collagen I fluorescence intensity showing taht: after sixth weeks,the type I collagen absorbance valueof group A has been in a rising state,and it has a slow descending trend from sixth to twelfth weeks.in the first 6 weeks,comapring each observation point between the last observation points,the differemce has statistically significance(P<0.05),There was no significant difference between the sixth week and the tenth week and the 12 week(P<0.05).In group B,the number has been on an upward trend from zerow week to eighth weeks,and the fluorescence intensity of I type collagen decreased from eighth week to twelfth week.In the first 8weeks,there was a significant difference between the absorbance value of each observation point and the last observation point(P<0.05).There was no significant difference between the eighth and the tenth weeks(P>0.05).In control group,the number also has been on an upward trend from 0 week to tenth week,and the fluorescence intensity of I type collagen decreased from tenth to twelfth week.And in the first 10 weeks of each observation point,the absorbance value compared with the observation point on the difference was statistically significant(P<0.05),and comparing the absorbance value between tenth and twelfth week there was no significant difference(P>0.05),comparing between the groups,the fluorescence intensity of type I collagen of group in sixth week is higher than that of group B in the tenth week,In group B,the fluorescence intensity of type I collagen was higher than that of control group at eighth week,but there was no significant difference between the two groups(P>0.05).3.The detection of calcium content With the development of vascularized bone in the muscle space,the calcium content of the three groups showed different degrees of decline.The results of group A showed that the calcium content of the observation nodes was significantly decreased in fourth and sixth weeks,and the differencehas statistically significant(P<0.05),and the calcium contentresult showed a slight increase from sixth to eighth week,but with no statistically significant(P>0.05).From eighth week to 12 week,the content of calcium decreased again.In group B,the calcium content decreased insixth week to eighth week,and there was statistical significance(P<0.05);but in eighth to 10 week,the calcium content has increased trend,but the overall trend is declining with no statistical significance;from tenth to twefth week,the calcium content showing the decreaseing trend.In the control group,The calcium content also showed a decreasing trend with the extension of time,although there was an increasing trend at the individual observation points,but the difference has no statistically significant(P>0.05).4.Bone alkaline phosphatase test With the increase of the time of vascularization and bone activation,the content of BALP in the group A reached the maximum at sixth week,and it showe the decreased trend at the end of sixth week to tenth week.The BALP content in the group B increased gradually and reached the maximumin eighth week,and then decreased slightly in the tenth week to the twelfth week.In control group,the content of BALP increased gradually and reached to the maximum in tenth week.5 Conclusion In the process of ectopic construction of vascularized bone,the application of exogenous growth factors VEGF and BMP can significantly promote the process of heterotopic bone formation.