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The Experimental Study On The Construction Of Cartilage With Cocultures Of Mesenchymal-derived Seed Cells And Acellular Allogenic Cartilage In Vitro

Posted on:2012-02-27Degree:MasterType:Thesis
Country:ChinaCandidate:M M LiuFull Text:PDF
GTID:2214330338962745Subject:Surgery
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ObjectiveCartilage defects is always a big problem in the clinical work. The limit source of autologous cartilage and the high absorption rates of allograft cartilage are limiting the application of adult cartilage tissue.Recently the development of tissue engineering has brought new solutions to this problem,a large number of studies on cartilage tissue engineering drive the arrival of the era of artificial cartilage. In this experiment,we extracted the rabbit peripheral blood-derived mesenchymal cells first,which received in vitro induction,Acelluar allogenic rib cartilage was obtained by using the detergent-enzyme method at the same time.New cartilage was built by the composite cultivation of cartilage seed cells and acellular allogenic cartilage in vitro,in order to provide experimental foundation for repair and rebuilding of defect organs in clinic.Materials and MethodsWe created a New Zealand rabbit trauma model, extracted rabbit peripheral blood stem cells first, conducted the cryopreservation and resuscitation of the passage stem cells,and then they received in vitro induction. After 14 days of culturing, cartilage seed cells were obtained.The growth characteristics of the cells was observed under inverted phase contrast microscope. Toluidine blue and Type II collagen immunocytochemistry staining were employed on cellular creep plates. Acellular allogenic rib cartilage was obtained by using the detergent-enzyme method, paraffin sections were prepared for HE stain, toluidine blue staining,Ⅱcollagen immunocytochemistry, staining results were observed under microscope; SEM samples were prepared for Scanning electron microscopy morphology and structure of support.Composite cartilage was obtained after 7 days of coculturing of the cartilage seed cells and acellular allogenic cartilage in vitro. The composite cartilage were implanted under the skin of the experimental rabbits,the materials were divided into four groups according to their differences, (A group:implantation of fresh allograft cartilage;B group:implantation of acellular cartilage;C group:implantation of composite cartilage;D group:composite cartilage cultured in vitro.The materials were removed after implanted for six weeks and twelve weeks. Paraffin sections were prepared for HE stain, toluidine blue staining,Ⅱcollagen immunocytochemistry, staining results were observed under microscope; SEM samples were prepared for Scanning electron microscopy morphology and structure of support.Results1. Morphologic changes of the cellsPeripheral blood stem cells were changed while the solution changed after 3-3.5 days,showing a small amount of adherent cells,which were short spindle or spindle-shaped; 80-90% of the cells had covered the dish at the end of 12-14 days. After 1:2 or 1:3 passage,the cells were elongated spindle or polygonal,of which the 80%-90%has covered the dish at the end of 5-6d. After recovery, time for stem cells adherenting of the third generation was longer than normal cells without freezing. Both extend the same shape, showing the length of spindle or polygonal, then can be passaged after 5-7d. After cultured for 7 days, the growth of the peripheral blood mesenchymal stem cells was slower compared with the control group, cells in the central of the cell mass were round or polygonal.The cells remain slow proliferation, round or oval cells gradually increased,while the control cells showed significant contact inhibition. Toluidine blue staining,Ⅱcollagen immunocytochemistry showed more cartilage-specific intracytoplasmic typeⅡcollagen secretion.2. Acellular cartilageThe acellular matrix presented with milk-white, size uniformity with intact stained structure, and there were a lot of acid mucopolysaccharide and collagen typeⅡ. The porosity was (61.31±8.45)%, and the pore diameter was (32.80±5.15)μm. The biocompatibility of the transplanted acellular matrix was good at 7 days after transplantation.3. Result of animal experimentComposite cartilage was obtained after 7 days of coculturing of the cartilage seed cells and acellular allogenic cartilage in vitro. scanning electron microscopy observation showed that seed cells were well adhering to the acellular cartilage, cells extend pseudopodia attached to the stent surface, accompanied by large amounts of matrix secretion. Experimental materials in each group were removed after subcutaneous implantation of rabbit back for six and twelve weeks, the observed results are shown:B, C group materials have good compatibility with organisms. C group material can be gradually degradated and absorbed in vivo, May form a small amount of white tissues similar to cartilage.Conclusion1. Rabbit peripheral blood is extensive, mesenchymal stem cells are good source of seed cells for cartilage tissue engineering, which can be extracted easily and grow well in vitro.The third generation of mesenchymal stem cells grow well for the line induced chondrocyte culture.2. under appropriate conditions,rabbit peripheral blood derived mesenchymal stem cells shows the same growth biological activities in vitro before and after cryopreservation and resuscitation. This feature can be used for long-term storage to support the application for the cells.3. Under the processing of neutral detergent-enzyme,rabbit rib cartilage still has good three-dimensional space frame, good matrix structure, good length and uniform pore size porosity, and good biocompatibility with the seed cells, which can be used as an ideal scaffold for cartilage tissue engineering.4. New cartilage tissue can be initially built by the culturation of autologous mesenchymal cells derived chondrocytes seed cells and allogenic acellular cartilage. The in vivo animal experiments confirmed that the implant has good biocompatibility and biodegradability. As a good Composite tissue engineering scaffold material,it can provide an experimental basis for clinical organ defect repairing and recycling.
Keywords/Search Tags:mesenchymal, stem cells, induction, cartilage, seed cells, Tissue Engineering
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