Bone Marrow Mesenchymal Stem Cells Combined With Nanohydroxyapatite/Collagen Modified By Fibrin And Fibronectin For The Repair Of Segmental Bone Defects

Recently,great development in tissue engineering has provided a novel cue for the management of critical large bone defects.As common seed cells of bone tissue engineering,marrow mesenchymal stem cells(MSCs) are multipotential cells capabl of differentiating into osteoblasts,chondrocytes,and other connective tissues cells. nanoHydroxyapatite/collagen(nHAC) is a kind of biomimetic materials and has being widely used in clinical application.Its three-dimensional structure is similar with the natural cancellated bone.Based on the study of nHAC,we constructed the composite scaffold by combining nHAC,fibrin and fibronectin in order to make up for each other's deficiencies.In the first part of this sutdy,the aims are to investigate the in vitro biocompatibility between MSCs that have been induced and nHAC modified by fibrin and fibronectin,and to provide a new treatment in the defect of bone.In the second part of this sutdy,we produced the defect models in the femur of SD rats.We repaired the defects with MSCs/(FB+FN)-nHAC.The curative effect was evaluated by radiographic examination,histologic analysis and scanning electron microscopy for providing theory basis for clinical application.PartⅠ:Effect of surface modification on cellular compatibility of nanoHydroxyapatite/collagenMaterials and method:MSCs obtained from SD rat bone marrow were induced and proliferated.After their osteoblastic phenotype was demonstrated,MSCs were seeded onto the nHAC scaffolds modified by fibrin(FB) and fibronectin(FN)(experiment group A),FB(experiment group B) and FN(experiment group C) as well as common nHAC(control group D).The adhesive rates were calculated.At different time points(3, 7,10,14 d),the cells on the scaffolds was counted and alkaline phosphatase(ALP) activity was detected.The cell growth on scaffolds were also observed by scanning electron micrographe(SEM).The differences among groups were compared and analyzed.Results:The differentiation of MSCs to osteoblastic phenotype was demonstrated by the positive stainings of alkaline phosphatase(ALP), collagen type I and mineralized node.The adhesive rates of group A was 74.4%,higher than those of any other groups.The cell numbers in the scaffolds increased with cultivating time for the four groups,and the speed of group A was the fastest.At the same time point compared group A with other groups,the difference in the cell number had statistical significance(P＜0.05).The expression of the ALP in group A was the highest at every time point(P＜0.05).Cells could be observed on every scaffold by SEM,and the cells in group A grew obviously better than those in other groups.Conclusion:MSCs can be induced to express osteoblastic phenotype.(FB+FN)-nHAC has a good biocompatibility with cells through the experiment,and can be used as an ideal scaffold for the management of critical large bone defects.PartⅡ:Repairment of bone defects by composite grafts of bone marrow mesenchymal stem cells with nanoHydroxyapatite/collagen modified by fibrin and fibronectinMaterials and method:MSCs obtained from SD rat bone marrow were induced and proliferated.After their osteoblastic phenotype was demonstrated,MSCs were seeded onto the scaffolds.A bone defect(5 mm in length) was created in each rat and treated with three kinds of implantations:E,composite of MSCs/(FB+FN)-nHAC;F,of MSCs/nHAC;G,blank control.The curative effect was evaluated by radiographic examination,histologic analysis and electron microscopy at 6 and 112 weeks.Results:Roentgenographically,the bone defects in the experimental group E exhibited new bone formation increased with time,which were superior to those of any other groups at 6 and 12 weeks after operation(P＜0.05).Compared group E with other groups,the quality of new bone formation was significantly different by histologic analysis and electron microscopy.At 12 weeks the defect in E got completely repaired and was bridged with the appearance of marrow cavities.The defect in G couldn't get osseous tissue healing.Conclusion:The modification of FB and FN can optimize the repairing effect of nHAC,and(FB+FN)-nHAC can be used as an ideal scaffold for bone tissue engineering.