Experimental Study Of The Osteoinductive Capacity Of Human Acellular Amniotic Membrane In Lumbar Vertebra Of Mature Canine

Objectives:To explore the osteoinductive capability of the human acellular amniotic membrane(HAAM) in lumbar vertebra of mature canine,to provides the basis of HAAM as the bone tissue engineering natural occurring matrix,and provides a new treatment idea for the treatment of bone defects and vertebral fusion.Methods:The HAAM was created by the patented technologies of the amniotic cells from non-acid-base system.L1-L4 vertebra of 15 mature canine were divided into 4 groups of HAAM,CPC,HAAM-CPC and the blank control group,and observed in 2, 4,6,8,12 weeks after implantion.The bone defects were made in lumbar vertebra.In group HAAM,CPC and HAAM-CPC,the defects filled with HAAM,PLGA, HAAM+CPC(volume ratio=1:1)and the blank control group only sutured.The rabbits were sacrificed by the end of 2nd,4th,6th,8th,12th weeks after operation.Obtain the marked site of bone defect and scan by computed tomography.Transsected operative site of the vertebra after decalcification and observed the appearance of the specimen by eyes.Then fixed with formaldehyde,embedded into paraffin,and sliced in routine.The light microscopy was applied as the pathologic morphology observation.The immunochemistry was applied as the specific expression of VEGF and collagenâ…£for explore the potential ability for bone inductivity.Results:CT imaging observation:Density was gradually increase in group HAAM from 2nd to 12th weeks after operation as well as blank control group,and density of group HAAM was higher than blank control group before the 6th week while blank control group higher than HAAM at the 12th week.Density of group CPC and HAAM-CPC were not changed obviously after operation.Observation the appearance of the specimen by eyes:HAAM was well located in the defction,and pykno-tissues can be seen at the junction of HAAM and host in group HAAM,and the area of the defection was gradually diminished after operation but relic HAAM still can be seen.In group contral,we can see that bone defction was filled with blood clot which were gradually substitute by nascent bone trabecula and defaction was healing at the end of 12th weeks.But the defaction area were not obviously change in the other 2 groups,and the bouncary of implant and the host was very clear. Histological morphology observation:After 2 weeks,in group HAAM,lots of vasculars intruded into the space of HAAM.The cells attached to the surface of HAAM,and the cells' mophologic along the amniotic matrix fiber.No inflammatory attacks and liquefaction phenomenon.In the blank control group,hematoma formed in the bone defects,a large number of red blood cells,mesenchymal cells,fat drops and capillaries can be observed.In group CPC and HAAM-CPC,the blood cells intruded into the space of CPC,there was no adhesion between cells and the surface of inplant.No immune and inflammatory response.After 4 weeks,in group HAAM, vascularize was prosperity,new bone trabecula was mult.Lots of new vessels and hemorrhage can be founded in group control.After 6 weeks,close to the edge HAAM layers,mesenchymal cells into cartilage cells,cartilage cells following a thick layers of new formed bone matrix.Cell adhered to the surface of HAAM,and the processes connected forming a network structure.In blank control group,granulation tissue can be observed in bone defects,angiogenesis actively.After 8 weeks,in group HAAM, the amniotic was calcified,new bone trabecula maturitied.In group control,there is no obverious difference from HAAM group.After 12 weeks,remaining HAAM can be founded in group HAAM,there is still lots of new borned bone trabecula and blood capillary at the edge of the HAAM,but the group contral was healed,only individual thin trabecula can be founded.In group CPC,fusion between CPC and host was founded at the end of 8 weeks,lots of macrophage was founded at edge.In group HAAM-CPC,there is some fusion between implant and host at the end of 12 week,macrophages was existence in the boundary,no new vessels seen.Immunochemistry observation:(1) VEGF:In group HAAM,new capillary were productive from 2nd to 12th weeks;in group CPC,the positive expression scattered in hematoma and new capillary were less than group HAAM before 6 weeks, but at the end of 8 weeks,group contral was more over;in group CPC,positive expression can be observed in 8th week after implanted;in the HAAM-CPC group, only fewer positive expression diffused distribution after 12 weeks.(2) Collagenâ…£: only HAAM group of 6 weeks was done and showed positive expression.Conclusions:Compared to CPC and blank control,HAAM have a better osteoinductive vascularize capabilities in lumbar vertebra of mature canine,can be used as material candidate of bone tissue engineering natural occurring matrix,provide a new treatment idea for the treatment of bone defects and for bone fusion,and as a new carrier for genes and medicines.