| Part1: Culture and osteogenic differentiation of children adipose-derivedMesenchymal Stem CellsObjective: To culture the isolated Adipose-derived Mesenchymal Stem Cell(ADSCs)from children adipose tissue. Exploring the differentiation of ADSCs into osteogenic cellsin vitro. Method: Disuesd adipose tissue was collected in surgeons. Adipose-derivedMesenchymal Stem Cells were gained through density gradient centrifugalization andcultivated in vitro.We observed the morphological characteristics of ADSCs underinverted microscope, analyzed the activity by MTT and indentified cells by flow cytometerdetection. Under the induction of osteogenic,the Passage3induced ADSCs wereindentified by Von Kossa calcify nodule staining and alkaline phosphatase staining.Results Children Adipose-derived Mesenchymal Stem Cells were successfully isolated andcultivated for20generation. High proliferation of cells was obseved under MTT dection.CD105and CD44were detected to be positive, while CD34, CD45, were found to benegative. After in vitro induced differentiation, ADSCs exhibited the morphological andbiological characteristics similar to typical osteoblasts, Von Kossa calcify nodule stainingand alkaline phosphatase staining were positive. Conclusion: Highly—purified ADSCscould be successfully isolated from children.and induced to differentiate into osteoblasts.Part2: Manufacture and analyze collagen/hydroxyapatite combinationscaffold.Objective: To manufacture and analyze type I collagen(COl I)/hydroxyapatite(HAp)combination scaffold. Method:Mixture HAp solution and bovine COl I solution accordingto definite ratio to develop3D scaffold which was imitated to the natural bone. Scanningelectron microscope, infrared spectrum and X-ray diffraction were used to analyze thescaffold. Results Excellent bearing capacity and mechanical property was obseved in theCOl I/HAp scaffold.Conclusion The inner structure of COl I/HAp scaffold was homothetic to the natural bone and can be a substitution of natural bone in constructingengeering bone.Part3: Investigation of cell biocompatibility about Culture ofadipose-derived mesenchymal stem cells on collagen/hydroxyapatite scaffoldin vivoObjective: To observe the growth of ADSCs on COl I/HAp compositethree-dimensional scaffold in vivo.Method Adipose-derived mesenchymal stem cellswere osteoinduced and seeded on COl I/HAp scaffold. Cell growth was observed underinverted microscope. Testing cytotoxicity and invesgating the change after intradermallyinjecting COl I/HAp solution.SD rats were taken to establish the leg musle implantationmodel. We set up experimental group (EG) in which implanted cell/scaffold compound,andestabish control group in which implanted simple COl I/HAp scaffold.Generallymorphous, imageology detection and histological detection were used to evaluate theOsteogenic in vivo in periodic time.Results We obseved adherence cells growing activelyin the scaffold without cytotoxicity in vitro.1.No diference was seen in the intradermallyinjecting test.2.In generally investigation, No diference was obseved2weeks afteroperation.Six to ten weeks after operation the scaffold could been seen approximately inEG while the scaffold neerly disapeared in CG.3. We had seen the density of implant in EGwas increased as time went on.The insert in CG became to be vanish.4.In HE staining, nodiference was seen in both group during first4weeks,when all the scaffold were coveredby inflammatory cell and extended by capillary.In8weeks, No inflammatory reaction wasseen in both group. Some kind osteoblasts were seen in the scaffold of EG,while thescaffold of CG was filled with granulation and a few ADSCs.In10weeks new engeeringbone was seen in EG,and the scafford in CG came to be degradation.ConclusionTissue-engineered bone tissue can be formed in vivo by COl I/HAp compositethree-dimensional scaffolds with osteoinduced ADSCs．It is demonstrated that COl I/HApis a kind of favorable scaffold biomaterials for bone tissue engineering. |
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