| Part one Preparation and biological detection of the freeze-dried cancellousbone of sheepObjective: In this study, we will prepare the freeze-dried cancellous bone of sheep,observe the gross and microscopic structure and detect and calculate thebiomechanical parameters of the bone, and provide the data of the structure offreeze-dried cancellous bone for application.Materials and Methods: The freeze-dried cancellous bone of sheep was preparedby a series of methods including removing of protein and fat, froze-drying andirradiating. Gross observation of the bone was done including its shape and color.The ultra micro structure of the bone was observed by Scanning ElectronMicroscopy (SEM) and the pore size was measured. The bone was examined byMicro-CT and the main parameters of the bone were calculated, then for statisticalanalysed. Biomechanical testing of the bone was performed. The compressivestrength and Young’s modulus and maximum load were calculated.Results: Deep-frozen and freeze-dried cancellous bone of the10×10×10mm3size was prepared from sheep iliac bone and set aside for later use. Thefreeze-dried cancellous bone is milky and translucent. By the Scanning electronmicroscopy, the structure of the bone was three-dimensional and porous withinterconnected pores. The pore size measured by image analysis was268.60±40.01um. Micro-CT analysis showed that the BMDs of freeze-dried cancellousbone is317.98±46.70mg/cc,TMD is465.41±42.83mg/cc，BVF is0.4861±0.1135，Tb.Th. is0.1601±0.0340mm，Tb.SP. is0.1729±0.0506mm andTb.n.是3.0322±0.3081/mm. MTS858records its maximum load and stress-strain curves which show that the maximum resistance to pressure of thetwo kinds of bones were663.57±20.98Nu and430.41±31.47Nu; their maximumcompressive strengths were8.19±0.80MPa and5.31±0.62MPa and elasticmodulus were289.45±30.10MPa and23.37±3.45MPa.Conclusion: The freeze-dried cancellous bone of sheep is a kind of excellentscaffold according to its pore size and pore distribution and biomechanicalcompression performance.Part two Co-culture of freeze-dried cancellous bone and mesenchymal stemcells of sheep in vitroObjective: To study the biological characteristics of bone marrow derived MSCsand their potential ability to differentiate into osteoblasts, and to investigate thebiocompatibility about the freeze-dried cancellous bone allograft and the BMSCs.Materials and Methods: MSCs isolated from bone marrow can be cultured andpurified with the method of density gradient centrifugation and adherencescreening in vitro. The BMSCs of2,5,8-generation (P2, P5, P8) are cultured incarbon dioxide incubator and the cell growth curves were drawed so theproliferation and the viability were tested. The BMSCs were induced byosteogenic solution and Alizarin Red Staining and Alkaline Phosphatase Stainingwere used to determine the osteogenic properties of the induced stem cells. The P3-generation BMSCs were attached on the cancellous surface of the bone andcultured in vitro. The cells growing on the surface of the bone was detected by theScanning Electron Microscopy.Results: The adherent cells were scattered, mostly round, oval and irregular shapeafter separation for24h. After4d, the cells were typical fibrous cells and began toform small colonies. The colony increased and gradually converted into a filmshape at the6thday. Each cell is gradually connected each other and covered theentire bottom by the10thday. The passaged cells are suspended in culturalmedium. Some cells showed obvious scattered adherent after2h and the next dayall the cells growth adherently and after3-5d reached confluence about90%. Thegrowth curve of MSCs is S-shaped and the growth curve shapes of the P2, P5, P8 cell are of the same basically. There is a period of1-2d for the incubation and thegrowth rate is slow. After3d, the speed of growth of the cells is accelerated namedthe logarithmic phase and stopped in7-8d came into the plateau phase. AlizarinRed Staining was made to detect the sedimental mineralization of the induced cells.There are signs of differentiation showing punctated calcificatio after the BMSCswas osteogenically cultured for12-14d. Mine bone nodules are distributed aroundevenly at16d, and21d, bone nodule formation is obvious. The AlkalinePhosphatase Staining showing that BMSCs has small punctate calcification andcalcium nodules after directly inducing for21d by the inverted phase contrastmicroscope. There were cells growing on the surface of MSCs-FDB complexes invitro at different times. Spherical cells attaching inside the pores and on thesurface of FDBs are visible in2h. The smaller cell bodies, the less tightly thetrabecular bone cells were combined with. As the culture time continues, the cellsbody gradually increased and were combined with material closer with theirsynapse. The cells are irregular shaped and small colonies formed by some cellsand the active cells attached to the trabecular flat surface at48h.Conclusion: Bone marrow mesenchymal stem cells have the ability ofself-replication and proliferation. P2, P5, P8–generation cells showed goodperformance in biology and can be differentiated into osteoblast induced by theosteogenic agents and have excellent biocompatibility capacitive with froze-driedcancellous bone allograft.Part three Experimental study of the osteogenerate behavior ofMSCs-CFDB complexity in sheepObjective: To evaluate the osteogenetic behavior of the MSCs-CFDB complexityin large animals and to provide experimental data for clinical implication.Materials and Methods:9experimental sheep，2.5-3years aged and about40-50kgs were selected. Anesthes was done by intramuscular injection of0.1ml/kginjection of Pentobarbital Ⅱ. The10×10×10mm3bone defect in metaphysealwas performed under the medial tibial articular surface at3-5mm. In group A (theexperimental group) the bone defects in left tibia were filled with the MSCs-FDBsmade by the methods in the part two and cultured for24hours. In Group B (the control group), the bone defects in right tibia implanted with freeze-driedcancellous bone infiltrated in DMEM for24h. General activities and woundhealing were observed after the operation in experimental animals.At4weeks,8weeks and12weeks three sheep randomly selected were induced by pentobarbitaland killed by injection of potassium chloride. The soft tissue covered the graft wasanalysed by histology. Bone block about3cm×3cm×3cm in size was made forthe next study.Micro-CT has performed on specimens and the bone mineral density,tissue mineral density, bone volume to tissue volume, trabecular thickness,trabecular gap width and trabecula number were measured and analysedstatistically. Being demineralized for60days with a neutral10%EDTA, HEstaining and Masson staining was performed to determine the bone formation.Results: All the animals are normal and the incision healed in time. The soft tissueon the graft was observed and no infection was found. At the fourth weeks, thefibrous tissue looked less pink and at8and12weeks, the tissue became dense andthe color gradually turned into normal of periosteum tissue. Micro-CT performedon the specimens and several key parameters concerning of the cancellous bonewere analyzed. The BMD of the experimental group were145.10±19.96mg/cc at4weeks,238.41±33.73mg/cc at8weeks, and215.30±28.50mg/cc at12weeks, inthe control group,there were134.08±23.79mg/cc,240.20±23.02mg/cc and218.70±31.60mg/cc respectively. The BVF were0.3772±0.0474,0.6038±0.1099and0.4016±0.0575in experiment group and in the control group0.3629±0.0510,0.5967±0.0699and0.4453±0.0503.The Tb.Th. in the experimental group and thecontrol group were0.1535±0.0282mm and0.1600±0.0258mm at4weeks,0.2974±0.0448mm and0.2830±0.0445mm at8weeks, and0.1710±0.0239mmand0.1650±0.0224mm at12weeks. The Tb.Sp. of the experimental group and thecontrol group were0.2591±0.0360mm and0.2932±0.0359mm at4weeks,0.1866±0.0254mm and0.2193±0.0382mm mm at8weeks,0.2681±0.0342mmand0.2273±0.0332mm at12weeks. The Tb.n. of the experimental group and thecontrol group were2.5573±0.3052mm and2.2531±0.3610mm at4weeks,2.2149±0.2599mm and2.2469±0.2650mm mm at8weeks,2.4632±0.2844mmand2.8050±0.3384mm at12weeks.Histological examination showed that the trends of the experimental group and thecontrol group are the same basically. Histological analysis showed that the structure of trabecular became sparse and thicker slightly, new bone formed,mainly strips woven bone, in the periphery of the original trabecular bone, vised at4weeks after implantation.At8weeks, structure of trabecular became thicker anddense, the formation of new bone and the degradation of bone graft weresignificantly increased and the new bone appears intertwined state with remainedbone, and at12weeks the structure of trabecular became thinner and the porositysmaller, the new bone increased and transformed into the mature bone tissuegradually. Overall, the trend of bone regeneration of MSCs-FDBs group wassignificantly faster than that of the FDB group. Red fibers were surrounded with asmall amount of blue-green fibers was visible at4weeks in both groups byMasosn trichrome staining. At8weeks blue-green new bone tissue increased,intertwined with red scaffold within the organization. At12weeks, a large numberof blue-green new bone tissue was seen and only a small amount of red fibroustissue in the surface of trabecular bone. By Comparing with the experimentalgroup, the process of change in the control group occurs slowly.Conclusion: The complexity of MSCs-CFDB performed ideal bone regenerationin the defect of the epiphyseal of tibia. But in the process of bone regeneration atthe fourth weeks, the density of bones decreased significantly and its biologicalproperties were affected, so it is noticed in clinical application. |
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