The Construction Of Tissue Engineering Cartilage In Vivo

Use of Autologous Chondrocytes and Bioinert PerforatedChambers to Tissue-Engineer Cartilage In Vivo1. The study of isolation,culture and identification of rabbit chondrocytesObjective: Auricular chondrocytes were isolated from the earscartilages of newzealand white rabbits with the methods of collagenase Ⅱandtrypsin enzymatic digestion. Methods: chondrocytes were harvested from rabbitears by enzymatic digestion. The morphological changes of cell and growthfeatures were observed through the phase-contrast microscope every day. Thechondrocytes were indentified with toluidine blue staining and chondrocytesgrowth curve was depicted. Results: Chondrocytes could be isolatedsuccessfully with collagenase Ⅱ and trypsindigestion.The primarily culturedchondrocytes were polygonal, and became dedifferentiated after3passages. Thechondrocytes maintained the morphology and histological staining patternwithin the first3passages. Conclusions: The study showed that the method of isolation and culture of chondrocytes was simple and feasible. The secondpassage chondrocytes grow well and are suitable for experiment.2. Design and fabrication of the chondrocytes/Collagen andchondrocytes/PLGA-Collagen In VitroObjective: To compare the effect between the Collagen gel andPLGA/Collagen gel hybrid scaffolds as vehicles for chondrogenesis in vitro.Methods: P2rabbit auricular chondrocytes(RACS) were seeded into thescaffolds;(1) RACSwere seeded into Collagen gel scaffold;(2) RACSwereseeded into PLGA/Collagen gel scaffold.After culturing for14days in vitro，thesamples were evaluated by HE, toluidine blue， safranin-O staining, andelectronical microscope scanning. Results: RACS/Collagen gel andRACS/PLGA gel samples formed white cartilage-like tissue in vitro when theywere harvested14days later. HE，toluidine blue and safranin-O staining showedthe chondrocytes were surrounded by much ECM. It was observed thatchondrocytes attached, growed, and proliferated well on the scaffold by SEM.Conclusions: These results indicated that collagen gel and PLGA gel scaffoldswere suitable for use and the two kinds of scaffolds can be used fortissue-engineering cartilage construction.3. Using bioinert perforated chambers to tissue engineer cartilage in vivoObjective: To explore the potential applications of a chamber for invivo tissue engineering, and to establish a novel model for in vivotissue-engineered cartilage. Methods: Four experimental groups were includedin this study:(A) chambers+chondrocytes/collagen gel;(B) chambers+chondrocytes/PLGA gel;(C) chondrocytes/collagen gel alone; and (D) chondrocytes/PLGA gel alone. Groups C and D served as controls. The sampleswere implanted subcutaneously in the donor rabbit, and the contents wereharvested at8weeks after implantation. Results: Histological andimmunohistochemical staining and RT-PCR results revealed regeneratedcartilage-like tissue in group B and small, irregularly shaped islands ofopalescent tissue in group A. In contrast, the control groups displayed vascularinvasion and inflammatory reaction, which eventually led to fibrosis andabsorption. Conclusions: Reproduced cartilages were obtained in animmunocompetent animal model through the use of a bioinert perforatedchamber.Recreating Engineering-Cartilage Without Scaffolds OnCostal Cartilage Repair and Regeneration1. The study of isolation, culture and identification of rabbits bone marrowmesenchymal stem cellsObjective: To culture and amplify rabbit bone marrow mesenchymalstem cells (BMSCs) by using density gradient centrifugation combiningadherent cultivation method, and to identify them. Methods: The growth andmorphology of BMSCs were observed by phase-contrast microscope. TheBMSCs growth curve was depicted by MTT; chondrogenic inductor, adiposeinductor and osteogenic inductor were used to induce the BMSCs intochondrocytes, adipocytes and osteoblast in vitro. The differentiated cells wereidentified by toluidine blue, oil red O and alizarin red staining respectively.Results: The BMSCs could be obtained by the density gradient centrifugationcombining adherent cultivation method and the BMSCs could be differentiated into chondrocytes, adipocytes and osteoblasts after induction treatment.Conclusions: The density gradient centrifugation combining adherentcultivation method was a relatively simple and feasible way which could isolateand culture BMSCs. The BMSCs could proliferate and multi-differentiate verywell.This suggested that BMSCs were superior seed cells for cartilage tissueengineering.2. Fabrication and chondrogenic differentiation of BMSCs SheetObjective: To obtain BMSCs sheet using continuous culturecombining simple mechanical method and to assess the feasibility ofchondrogenic differentiation. Methods: BMSCs were cultured in the culturemedium supplemented with chondrogenic inductor for14days.The harvestedcell sheets were evaluated by histological and scanning electronical microscope.Results: The intact BMSCs sheet could be gained by continuous culture andsimple mechanical method. HE staining showed that the cell sheet was made of6to8layers of cells. The safranin-O, toluidine blue staining showed positiveresults and scanning electron microscope examination revealed that the cellsarranged closely and the matrix secreted abundantly. Conclusions: The studyindicated that the BMSCs sheet could be generated by using this simpletechnique, and the sheet could differentiate into chondrocytes..3. The Effects of chondrogenic BMSCs Sheet on Costal Cartilage Repairand RegenerationObjective: The purpose of the study was to investigate the effects ofchondrogenic BMSCs Sheet on costal cartilage repair and regeneration. Methods: Sections of the costal cartilage of16male rabbits were removed withthe costal perichondrium and the costal cartilage junction was intactly left. Therabbits were treated with three different methods: Method1: The costalperichondrium was left unsutured; Method2: The costal perichondrium wassutured to form a tubular structure after the implantation of alone BMSCs Sheet;Method3: The costal perichondrium was sutured to form a tubular structure afterthe implantation of chondrogenic BMSCs Sheet. The specimens were harvestedafter16weeks postoperatively. Results: The average width of the costal cartilagein the method1group was significantly wider than that of the control group(P<0.01). The biomechanical strength of the costal cartilage in themethod1,2groups were significantly weaker than that of the control group (P<0.01); No obvious differences in the average widths and the biomechanicalstrength were tested between the method3group and that of the control group(P>0.05).HE-stained results showed the repaired tissues in1、2methods weremainly fibrous cartilages, and new cartilage-like tissue regneneration could befound in the3method group, The chondrocyte-like cells were surrounded bythe abundant cartilaginous matrix in the regenerated cartilage. Conclusions: Ourstudy has indicated that chondrogenic BMSCs Sheet could promote theregneneration of new cartilage, which significantly enhanced the strength of therepaired tissue, strengthened the stability of the thorax, and thus, indirectlyreduced the incidence of thoracic deformities.