Establishment Of Mouse Model For Exploring Tissue Engineered Bone’s Osteogenesis Mechanism And Its Preliminary Experimental Application

Backgroud：Large segment defects of bone,which are caused by some factorssuch ashigh-energy trauma,bone and joint tuberculosis and severe infection,resection leaded bybone tumor are very common.Tissue engineering bone constructed by mesenchymal stemcells (MSC) as seed cells is recognized as one of the most promising strategy for repairinglarge segment bone defects.Multiple empirical study of bone tissue engineering focus onthe ultimate effects of osteogenesis and to promote the pre-clinical research.wherea thestudy on the mechanism of seeded cells is rarely involved, some researchers speculate thatseed cells MSCs in the body compensate for the lack of host osteogenesis by directlydifferentiating into bone, so as to promote the repair of bone defects in vivo.In our previousstudies we traced the seeded cells MSCs by marking them with green flourescent,and foundthat transplanted MSCs can participate in osteogenesis, but the large amount of new bonetissue is constitute of the host derived osteogenic cells, suggesting that a large number ofhost cells migrate to the graft site and participate osteogenesis.so we raised a point whichcould be the mechanism how the seeded cells repair the defect: tissue engineering bone istransplanted into the bone defect site, inflammation happened cause of localtrauma.Released inflammatory factor motive seeded cells with the host mononuclear cells,chemokines and cytokine produced by them drive the recruitment of host-derivedosteogenesis related cells and migrate to the injury site.Animal model is the necessarycarrier for bone tissue engineering research,Now although various animal models used forstudy of bone tissue engineering each has its advantage,they are not suitable for study oftissue engineering bone’s osteogenesis mechanism.Objective:(1) Establish mouse femur bone defect model easy to operate, and can beused for bone tissue engineering research;(2) In vitro use MSCs as seed cells compositescaffold for tissue engineering bone, observe cell proliferation, differentiation on the scaffold;(3) engraft well-building tissue engineering bone grafts to mice bone defect model,observe how the seeded cells migrate and homing to injury site. To verify if the mousemodel is scientific and feasible for tissue engineered bone’s study.Methods:(1) MicroCT analyze the anatomy parameters of the mouse femoral. Thirty2~3months old FVB/N mice were divided into3groups, each groups for ten samples.with our own designs of internal fixation, we established defect length for1,2mm and2.5mm of femur bone and middle periosteum defect models, post operation we observe thehealing process through the imaging and histological techniques in different time point.(2)Mouse bone marrow mesenchymal stem cells,were isolated by whole bone marrow cellsadherent method and identified through their specific cell surface antigen markers andmultiple differentiation potential.(3) Use the green fluorescent mouse bone marrowmesenchymal stem cells (mBMSCs) as seed cells with sheep DBM to construct tissueengineering bone, cell proliferation and differentiation on the DBM were observed throughscanning electron microscopy and LSM technology.(4) Wild type of mouse bone marrowmesenchymal stem cells were combined with DBM material to construct tissue engineeredbone. Then TEB were transplanted to the bilateral femoral bone defect model of greenfluorescent mice, one side were implanted with the TEB and the other side with DBMmaterial as blank control, use small animals in vivo imaging system to observe thespecimen, respectively,3days,7days,10days after operation. Digestion of cells on bothsides of the specimen block down the line by flow cytometry for sorting and counting.thedifference between the two groups were compared, we evaluate if the seeded cellsrecruiment more host cells involved in the repairment of bone defect. the differencebetween the two groups were compared, we evaluate if the seeded cells recruiment morehost cells involved in the repairment of bone defect.Results:(1) By measuring the anatomical parameters of the mouse femur: femurlength (15.48±0.73) mm; the middle of the femoral shaft diameter (1.38±0.20) mm;sagittal diameter (2.05±0.05) mm; medullary cavity diameter (0.75±0.80) mm, except forthe part above the femoral condyle and trochanter, femoral shaft length8.5mm.7,28,56,84days after the modeling operation the laboratory mice were observed through X-ray shot,bone defect length of1mm in the experimental group:7days post operation the bone wasfixed well, two ends of bone defects were in correct position;28days post the operation two sides of bone defect were in rigid internal fixation without displacement,one bone defectside can see fibrous callus;56days post operation a large number of bone callus wereformed in the bone defect site.84days after operation bone defects achieved clinical healingwhich has been fulled with continuous callus. Bone defect length of2mm experimentalgroup: bone were fixed well28days after operation,after84d bone defect ends becomeblunt and the bone callus growth is not obvious. Defect length of2.5mm experimentalgroup: continuous observation after the operation,we could see the bone defect were fixedIn good position with the bone ends no angulation and shortening.84d after surgery,significant bone callus replaced by similar to soft tissue image was seen in the bonedefect site.(2) We adopted the methods of whole bone marrow cells sticking wall toseparate mice bone marrow mesenchymal cells.they were seen to stick wall growing frommicroscopy, show the typical fibroblasts form, flow cytometric test shows cellularphenotype of the second generation mice MSCs cultivated by us: high expression of MSCsspecific antigen CD105(92.7%); lower expression of Hematopoietic antigen CD34andCD45; lower expression of Endothelial cell line antigen CD31,; Pathological special dyeingconfirmed that cells have the potential to differentiate to osteoblast, adipose cells.(3) Weput bone marrow mesenchymal stem cells of FVB/N strain of mice on pig derived DBMin vitro to reconstruct TEB, through the confocal laser imaging technology and scanningelectron microscopy (SEM) technology we observe cells being planted on DBM material,with the time going on, the cells’ proliferation and differentiation is good, they can stickclosely on the surface and pore of the material and put out synapses, their fluorescenceintensity is increasing, the cells show fibroblasts form.(4) Establishment of the lateralfemoral bone defect models in mice, no case was failure,3days aftertransplantation,general view of femoral specimens had no obvious difference,by usingsmall animal living imaging techonology, green fluorescent can almost not be seen infemoral defect site of TEB group and DBM group.7days after transplantation,thefluorescent of two groups is enhanced obviously than before,but between the two groupsalso have no obvious difference.10days after transplantation, fluorescent imaging showsthe defect sites of two groups both have strong fluorescence,.fluorescence of TEB group ismore strongly expressed than DBM group. Then we digestive cells down from thespecimen for sorting and counting by flow cytometry,shows that seven days and10days post operation, cell is significantly more in TEB group than DBM group.Conclusions:(1)The mouse femoral bone defect models constructed by us can meet our follow-upexperimental requirements. Without any materials stuffed in the defect site,the bone defectsize for1mm could be restored by the self-repairing mechanism three months after theoperation,but not for the size above2mm even they are fixed in good position.so weconclude that the model can satisfy with the command for critical size defect.(2) The micebone marrow mesenchymal stem cells that we adopted the methods of whole bone marrowcells sticking wall to separate own good potential of the proliferation and differentiation.Weidentified that the cells cultivated by us were bone marrow mesenchymal stem cells neededfor our experiment through flow cytometric test their surface marker and multiple potentialof differentiating to various connective tissues.(3) DBM materials have natural threedimension nets hole bone structure, although there are lots of small pore connected to eachother within internal big pore.they have still relatively large aperture.Scanning electronmicroscopy (SEM) image measurement of DBM pore rate is about76%, DBM material hasgood compatibility and porosity for the cells, cells can proliferate and differentiate well onthe surface and pores of material. Fluorescence of labeled cells can gradually bestrengthened.(4) TEB constructed by bone marrow mesenchymal stem cells of FVB/Nstrain of wild mice and DBM scaffold.they were transplanted into the bilateral femoral bonedefect model of the same strain green fluorescent mice. We can prove that the seeded cellsMSC are able to recruitment more host derived cells to involve the repairing of bone defectthrough small animal imaging technology and flow cytometry sorting methods at differenttime point post operation.so we conclude that the mouse models established by us aresuitable for exploring for tissue engineered bone’s mechanism.