| 1BackgroundWith the rapid advances of modern science, modern life,all kindsof vehicle around us, tissue defects and dysfunction are increasingly common. At thesame time, the requirement of people is keeping pace with the times, notsimply the preservation of life, but the pursuit of a higher quality of life,such asthe pursuit of functional recovery and reconstruction. With the development of modernmedicine, from the mid-1980s, tissue engineering------a new concept， began torise. According to the Committee of National Science Foundation of America, tissueengineering is a science that applies principles of cell biology and biological materialsengineering, of using cell biology, biomaterials and engineering and develop bioactivesubstitutes,which are used to repair or improve the structure and function of tissues ororgans. For trauma, all causes of bone defects seriously affect the quality of patientslife.Traditional autogenous bone graft is not only at the expense of autologous healthytissue,but also can not fully repair the bone defects. Bone tissue engineering sheds light on the solvement of the trouble. In order to repair defects in bone tissue and to achievea safe, effective and no side effects medical realm, it plants MSCs，osteoplast and otherrelevant seed cells in a variety of absorbable stent materials with the help of relativecytokines, such as bone morphogenetic protein (BMP), transforminggrowth factors (TGF).In this issue, we try to improve the traditional CPC and develop a novel bonesubstitute materials.The substitute materials will be osteoinductivity,speed metabolismand high strength,which will provide a new option for a wide range of bone defectsrepairment.2ObjectiveLoad gelatin microspheres which contain rat bone marrow mesenchymalstem cells (MSCs) into calcium phosphate cement (CPC) and make the cellssurvive.Through the experiment,we will develop a new bioactive bonesubstitute materials.3Materials and Methods1)cell isolated and cultureWe selected rat bone marrow mesenchymal stem cells as seedcells. Culture Cells by an adherent method and identify the cells to ensure thatthey have multiple differentiation potential. Preparation contained gelatinmicrospheres for bone marrow mesenchymal stem cells2) Preparation of gelatin microspheres loaded mesenchymal stem cellsTake a dish of the third generation of mesenchymal stem cells and digestthose by trypsin. Dissolve some gelatin in the DMEM to prepare13%gelatinsolution.Add1ml cell suspension in5ml gelatin solution and mix well. Thenpreheate to50℃,the same as olive oil.After adding0.15ml Span-80to oliveoil,add the gelatin solution slowly,stirring for15minutes to900r/min.Afterit,add0.01g genipin,continuely stirring at room temperature. To test different degree of crosslinking on the stability of the gelatin microspheres,the time is12h,24h,48h. After the mixture quenched to4°C and placed in a separatoryfunnel containing5g/L of Tween-80PBS in a1:1ratio, washed3times toremove the olive oil to obtain gelatin microspheres. And stored at37°Cincubator in DMEM medium.3)Preparation calcium phosphate cement contained mesenchymal stem cellsIn order to determine the appropriate proportion, firstly well mix gelatinmicrospheres contained MSCs with bone cement in a mass ratio of2.5%(B),5%(C)10%(D)(w/w). Then mixed it with phosphate buffer at the solid-liquid ratio of2:1,and place the mixture in a pre-prepared cylindermold.Lastly, store in DMEM medium after4h in37°C incubator. Inadditional,preparate a pure CPC (A) as a control group.4)Determination of the physical and chemical properties of gelatinmicrospheres contained stem cellsWatch the shape, dispersion of the gelatin microspheres, and calculate thecell encapsulation efficiency, average particle size and swelling ratio. Thecrosslinking degree of the microspheres was determined with the ninhydrinassay.Observe the degradation time of the microspheres of gelatinmicrospheres.5) Fluorescence stainingAfer immerging the gelatin microspheres in DMEM, stain those withcalcein AM and propidium iodide (PI). After2h,12h,24h, The live (green)and dead (red) cells were observed in fluorescence microscope6) Determination of the physicochemical properties of the new bone cementObserve the new calcium phosphate bone cement in the initial setting time,compressive strength, porosity and metabolic time.Analysis its metabolites, and observe the adhesion and growth of bone marrow mesenchymal stem cellswithin the pore.7) Statistical analysisStatistical analysis was preformed with SPAA13.0software.Using one-way analysis of variance(ANOVA) and SNK-q tests,with significanceassumed at p<0.05.4Results1) The cultured mesenchymal stem cells have been stable spread to the thirdgeneration. By flow cytometry test, the cultured MSCs are in line withinternational standards. CD90expression is positiveand the positive rate is95.24%.But CD45expression was negative.2) Physical and chemical properties of gelatin microspheresThe particles of all groups of gelatin microspheres changed little in24h,respectively (75±15)μm、(81.58±24.68)μm'(108.64±37.44)μm.After24h,the microsphere particles significantly reduced (27.88±7.46) μm, thedifference was significant (P <0.05); The degradation time of cross-linkedgelatin microspheres was significantly prolonged, compared with theuncrosslinked group.the difference was significant (P <0.05).3)Physical and chemical properties of calcium phosphate cementIn each group,MSCs are growing well, and compared with the controlgroup.As the proportion of gelatin microspheres increases, the compressivestrength of CPC is to gradually reduce, the initial setting time extend, theporosity increases and the the macroporous rate increases (P <0.05).5Conclusion1)The cultured MSCs are in line with international standards2)Through the test of physical and chemical properties of gelatin microspheres, the particle size of genipin crosslinked gelatin microspheres ismoderate after24h, the degradation time and the load cell rate meet theexperimental requirements,and MSCs survive well.3)Through the determination of the physical and chemical properties ofcalcium phosphate bone cement,we find when the mass ratio of the gelatinmicrospheres contained MSCs and CPC is5%, the physical and chemicalproperties of CPC is appropriate and the cells grow well.4)The results lay a solid foundation for further vivo experiments. |
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