| Part 1 Isolat ion,Culture and Identification of Beagle Dog Bone Marrow Mesenchymal Stem Cells in VitroObjectiveTo explore the biological characteristics of the isolation,culture and identification of bone marrow mesenchymal stem cells of the Beagle dogs.MethodsAfter the anesthesia successful,in sterile conditions,pentobarbital sodium(30 mg/Kg),use 8%sodium sulfide liquid toremove the hair.then use the 16th bone marrow puncture needle to extraction marrow from the left and right sides of the iliac crestrespectively.The bone marrow mesenchymal stem cells of Beagle were isolated by density-gradient centrifugation methodand increased.they were cultivi ted by 10 mL 10%DMEM(10%of fetal bovine serum).Observed cell morphology and growth under inverted microscope,Immunofluorescence staining was used for identification.The cell growth curve was determined by MTT assay,Cell surface markers were identified using flow cytometry,and the differentiation ability of bone marrow mesenchymal stem cells into chondrocytes was determined by CCK-8.ResultsExchange the cultivate liquid for the first time at 4th day in the primitive cultures,Fibroblast-like cells are adhered to the bottom of the culture bottle,and a few clones are formed,which is derived from the cloning of fibroblasts.During the 7th to 10th day,the clone gradually increased and enlarged,forming a large clone with a diameter of 5mm?8mm and a small clone of<5mm.Flow cytometry showed that CD34 was negative on bone marrow mesenchymal stem cells,while CD44 and CD90 were positive.The cell stabi lity increment by CCK-8;Immunofluorescence staining results:CD44 positive,CD90 positive,and CD34 negative,the nucleus around the yellow-brown.ConclusionThe bone marrow mesenchymal stem cells of the Beagles cultured in vitro are fibroid cells with the ability of cloning and self-renewal.The treatment of bone marrow mesenchymal stem cells provides an experimental basis for the repair of femoral cartilage defects and the application of cartilage reconstruction.Part 2 The complex of Chitosan/Sodium Alginate Membrane Scaffold and Bone Marrow Mesenchymal Stem Cells In vitro studyObjectiveTo investigate the biocompatibility of chitosan/alginate scaffold with Bone Marrow Mesenchymal Stem Cells,and the adhesion,growth,proliferation and chondrogenic differentiation of the cells in scaffold.MethodsThe Bone Marrow Mesenchymal Stem Cells combined with chitosan/sodium alginate scaffold in vitro,to evaluate cell on chitosan/sodium alginate membrane support material of the attachment,growth and proliferation by scanning electron microscopy(SEM),so as to determine the biocompatibility of the scaffold and bone marrow mesenchymal stem cells.ResutsThe scanning electron microscope(SEM)observed that chitosan/alginate scaffold was three-dimensional porous,with porosity of over 75%.Bone Marrow Mesenchymal Stem Cells composite chitosan/sodium alginate membrane scaffold,the cells can attach and proliferate well.After 2 hours of inoculation,Bone Marrow Mesenchymal Stem Cells were observed to be attached and extended on the scaffold.After 1 day of inoculation,the cells were observed to spread evenly,and some cells grew in or across the pores.With the extension of time,the number of cells in scaffold materials gradually increased.ConclusionThe chitosan/sodium alginate scaffolds with suitable three-dimensional porous structure,composite culture of Bone Marrow Mesenchymal Stem Cells showed good cell compatibility,and can be used as membranes of cartilage tissue engineering scaffold.Part 3 Animal experiment research on Repair of Beagle dogs Femoral Head Cartilage Defect with Chitosan/Sodium Alginate Membrane Scaffold and Bone Marrow Mesenchymal Stem Cell ComplexObjectiveTo study the complex of bone marrow mesenchymal stem cells and chitosan/sodium alginate scaffolds implant Beagle dogs femoral head cartilage defect area,observe the Beagle dogs femoral head cartilage repair in the body,to evaluate the treatment of the role and value of Beagle dogs femoral head cartilage defect.Methods36 Beagles with the age of 6 months,surgical operation method is adopted to establish the cartilage defect model of Beagle dogs femoral head.According to the different grafts,they were divided into 3 groups randomly,The experimental group(group A):24 Beagle dogs on the left side of the femoral head to establish the cartilage defect in the not weight bearing area,they were placed the complex which are bone marrow mesenchymal stem cells and chitosan/sodium alginate scaffold.Control group(group B):24 Beagle dogs were placed with a blank chitosan/alginate scaffold.Blank group(group C):12 Beagle dogs with no treatment at the cartilage defect of the bilateral femoral head.the specimens were generally observed and histologically examined in the 8th week and the 12th week respectively.ResulisEight weeks after operation,the results showed that the articular surface of the chitosan/sodium alginate membrane scaffold-bone marrow mesenchymal stem cells group was slightly unsmooth,the cartilage defect area.was locally depressed,and the cartilage defect area was filled with a large number of translucent tissue similar to normal cartilage tissue,which continued with the surrounding normal cartilage tissue.Chitosan/sodium alginate membrane scaffold-bone marrow mesenchymal stem cells group compared with the traditional Chinese medicine extract chitosan/sodium alginate membrane scaffold group,the articular surface is more non-smooth,the cartilage defect area is obviously depressed,cartilage defect area is filled by a small amount of translucent tissue similar to normal cartilage tissue,and with surrounding normal cartilage tissue.In the blank group,there was no obvious filling of articular cartilage tissue in the cartilage defect area,and the subchondral bone tissue was completely exposed.At 12 weeks after operation,the articular surface of the chitosan/sodium alginate membrane scaffold-bone marrow mesenchymal stem cells group was very smooth,and the cartilage defect area was filled with a large number of translucent tissue similar to normal cartilage tissue,which continued with the surrounding normal cartilage tissue;compared with 4 weeks ago,the chitosan/sodium alginate extract of traditional Chinese medicine group was very smooth.In the membrane scaffold group,the articular surface was smoother,but there were still obvious depressions.The defective cartilage area was filled with more translucent tissue similar to normal cartilage tissue,which continued with the surrounding normal cartilage tissue,and the subchondral bone tissue was partially covered.In the blank group,the defective articular cartilage was not filled with articular cartilage tissue.The subchondral bone tissue was still exposed.ConclusionChitosan/alginate is the right scaffold,and the complex combination of bone marrow mesenchymal stem cells in vitro can effectively promote the repair of cartilage defects in Beagle dogs. |
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