The Effect Of Platelet-rich Plasma And Vascularized Muscular Fasciae On Tissue Engineered Bone Vascularization

[Objective]1, To explore the feasibility of MSCs as seed cells of bone tissue engineering, which were isolated from the mongrel bone marrow, cultured, proliferated and induced to osteoblast differentiation. 2, To make allogenic demineralized bone matrix of the mongrel and to explorethe feasibility of DBM as scaffold of bone tissue engineering. 3, To explore the effect of PRP and vascularized muscular fasciae on tissueengineered bone vascularization. [Methods]1 , MSCs of the mongrel were isolated,cultured,proliferated in vitro and induced by the medium containing dexamethasone (10nmol/L),β-sodium glycerylphosphate(10mmol/L) and ascorbic acid(50mg/L).MSCs were observed by inverted phase contrast microscope and growth curve was made by the method of MTT.Alkaline phosphatase(ALP) was strained by Calcium-Cobalt methods,and calcifying nodule was strained by Von Kossa and Alizarin-Red methods to verify osteoblastic phenotype. 2, Thighbones of the mongrel were degreased, demineralized, deproteined, freezed, dried and sterilized and then were made into demineralized bone mantrix. MSCs was seeded onto the scaffold and their growth situation were analyzed by inverted phase contrast microscope and scanning electron microscope at different time.3, DBM/MSCs composites were implanted into the back of 12 mongreles.4 groups were divided according to whether the composite was treated with PRP or vascularized muscular fasciae or not.4 mongreles were sacrificed at 4, 8, 12 weeks respectively to undergo examination of gross specimen ,x-ray and histology.[Results]1> MSCs isolated from the mongrel marrow by gradient centrirugation have active proliferative abilities and high purity. The differentiation of MSCs to osteoblastic phenotype was verified by the positive staining of mineralized node and alkaline phosphatase and square or multangular cell configuration. And the growth curve of MSCs and induced cells has the typical shape of "S".2% DBM has a three-dimensional mesh structure. The mean pore diameter is 254.39±88.71 um and the pore rate is 70%.MSCs could adhere to the surface and inner walls of DBM .proliferated well and secreted a large amount of extracellular matrix.3 >The volume of new bone ,bone optical density ,vascularization degree of tissue engineered bone in group A,B,C were significantly superior to group D at different time. [Conclusions]1 n MSCs of the mongrel were isolated by gradient centnfugation proliferated and induced to differentiate into adequate amount of osteoblasts. MSCs are the ideal seeding cells of bone tissue engineering. 2% DBM is a potentially ideal scaffold material for bone tissue engineering. 3 > Both PRP and vascularized muscular faciae can accelerate osteogenesis and vascularization of tissue engineered bone and the two factors have synergistic effect.