Experimental Study Of The Osteoinductive Capacity Of Human Acellular Amniotic Membrane In Proximal Femur Of Adult Rabbits

Objectives:To explore the osteoinductive capability of the human acellular amniotic membrane(HAAM)in proximal femur of adult rabbits,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 for bone fusion.Methods:The HAAM was created by the patented technologies of the amniotic cells from non-acid-base system.27 adult rabbits were divided into 3 groups of HAAM,PLGA and the blank control group,and observed in 1,2,4 weeks after implantion.The bone defects were made in dorsal of femur,just below the great trochanter,0.6 cm in diameter.In group HAAM and PLGA,the defects filled with HAAM,PLGA,and the blank control group only sutured.The rabbits were sacrificed by the end of 1,2,4 weeks.Obtain the marked site of bone defect.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 BMPs for explore the potential ability for bone inductivity.Results:Histological morphology observation:After one week,in group HAAM, vasculars and a large number of mesenchymal cells 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 group PLGA,the blood cells intruded into the space of PLGA,there was no adhesion between cells and the surface of PLGA.No immune and inflammatory response.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.After two weeks,in group HAAM,the further absorption and reduce angiogenesis can be observed;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 group PLGA,PLGA began to soften and degradation,granulation tissue formed in the space of PLGA,but no cartilage cells.In blank control group,granulation tissue can be observed in bone defects,angiogenesis actively.After four weeks,in group HAAM,the amniotic organizations disappeared,angiogenesis reduce,the hypertrophy cartilage cells increased,bone defects gradually filled with cartilage.In group PLGA,cells adhered to the residual of PLGA,granulation tissue rich in blood vessels,but no cartilage cells and osteoinductive phenomenon.In blank control group, the bone defect filled with granulation tissue,angiogenesis is still active,but no cartilage and bone tissue.Immunochemistry observation:(1)VEGF:After one week,in group HAAM, the VEGF positive expression at the surface and space of HAAM;in group PLGA, the positive expression scattered in hematoma and;in the control group,the expression rarely.After two weeks,in group HAAM,strong expression can be observed around angiogenesis;in group PLGA,fewer positive expression can be observed;in the control group,positive expression diffused distribution.After four weeks,in group HAAM,the naive cartilage cells' VEGF expression negative, matured and hypertrophy cartilage cells color darked,the hypertrophy cartilage cells in the area of original bone connected to cartilage were more positive.In group PLGA,the expression of VEGF enhance with the intrusion of vascular.In blank control group,the expression highly because of the rich capillaries in granulation tissue.(2)BMPs:After 1 week,in group HAAM and PLGA,positive expression highly can be observed around vessels in every group.After two weeks,in group HAAM,there is a strong positive expression around angiogenesis,in group PLGA, the expression fewer than group HAAM;in the control group,positive expression scattered distribution.After four weeks,in group HAAM,the cartilage cells expressed negative.In group PLGA,the expression highly with the intrusion of vessels.In the control group,the expression increased. Conclusions:Compared to PLGA and blank control,HAAM have a better osteoinductive capabilities in rabbit femur,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.