Experimental Research On Construction Of Tissue Engineering Trachea With Bone Marrow Mesenchymal Stem Cells

PartⅠ:Experimental Research of Porcine BMMSCs Obtained and Differentiated to Chondrocyte and Vascular Endothelium-Like CellsObjective To harvest,isolate,culture bone marrow mesenchymal stem cells(BMMSCs) from porcine sternum.To investigate the induction and differentiation of BMMSCs into chondrocytes,airway epithelial cells and vascular endothelium-like cells in vitro.To explore the possibility as seed cells for tissue engineering trachea.Methods The porcine bone marrow was aspirated from sternum.BMMSCs were isolated and purified by methods of density gradient centrifugation and adhering to the culture plastic.Chondrocytes derived from BMMSCs which were cultured in DMEM-HG with TGF-β1(10ug/L).Induced the BMMSCs in DMEM-HG with COL-I(25mg/L)and acetic acid(0.1%).Vascular endothelium-like cells derived from BMMSCs which were cultured in DMEM-HG with VEGF(10ng/mL).The cells were observed and identified through morphology,reverse transcription polymerase chain reaction (RT-PCR),immunohistochemistry,toluidine blue staining,and immunofluorescence.Results Methods of density gradient centrifugation and adhering to the culture plastic can harvest mononuclear cells with specific characterics.Both of chondrocyte and vascular endothelium-like cells were cultured successfully.Airway epithelial cell was not culture successfully.Morphology of chondrocyte was changed,heterochromia to toluidine blue. Immunohistochemical staining of collagen typeⅡand RT PCR products for mRNA from collagen typeⅡwere positive.Immunohistochemical staining of cytokeratin was negative. VELCs showed cobble stone-like morphology and sufficient cell organs. Immunofluorescence results showed vWF positive of VELCs. Conclusion 1.Methods of density gradient centrifugation and adhering to the culture plastic can harvest higher purified porcine BMMSCs.BMMSCs can provide stem cells for research of tissue engineering trachea.2.Porcine BMMSCs can be differentiated into chondrocyte and vascular endothelium-like cells in vitro.3.Porcine BMMSCs can not be differentiated into airway epithelial cells with the mothode of Coraux. PartⅡ:Experimental Study on Cytocompatibility of Porcine Chondrocyte and PGA/Collagen ScaffoldObjective To study the compatibility of porcine chondrocyte and polyglycolic acid (PGA) coated with cross-linked collagen(PGA/collagen scaffold) by morphological observation,proliferationof the cells and so on.Methods Rat's tail collagen and PGA coated with cross-linked collagen(PGA/collagen scaffold) was produced and constructed firstly.Chondrocyte derived from BMMSCs were cultured with PGA/collagen scaffold together.There were several experimental groups including PGA/collagen group,simple PGA group,simple cells group and control group. Morphological characteristics were observed by HE staining,toluidine blue staining and scanning electron microscope.Adhesive and proliferating cells were detected by MTT assay.Results The PGA/collagen scaffold has elasticity and tenacity.Histological and ultrastructural observation showed that there were lots of micropores in the scaffold.HE staining,toluidine blue staining,scanning electron microscope and the curve of cell growth from MTT assay showed that chondrocyte seeded on the PGA/collagen scaffold grew as well as simple PGA group and simple cells group.Conclusion 1.Chondrocyte seeded on the PGA/collagen scaffold grew as well as simple PGA group,simple cells group.2.The PGA/collagen scaffold had a good compatibility with VSMLCs. PartⅢ:Construction of Primary Tissue Engineering Trachea in vitro and Strengthening of Trachea Wall in vivoObjective To investigate the possibility of constructing tissue engineering trachea in vitro and strengthening of tracheal wall in vivo.Methods Chondrocytes were labeled by Brdu and vascular endothelium-like cells were labeled by DAPI respectively firstly.The mixture of seed cells and scaffold were produced by separately seeding chondrocytes and vascular endothelium-like cells derived from BMMSCs on PGA/collagen scaffold.The two layers were separated by ECM gel.The cells-scaffold mixture was wrapped around a silicone tube(external diameter=2mm).The mixture was implanted into omentum in cell donor porcines and the porcines were sacrificed in 4,6 and 8 weeks.The tissue engineering tracheas were examined by both gross and histology with HE staining and immunofluorescence.The control group was PGA/collagen scaffold group with no seeded cells.Results Seed cells could be labeled well by Brdu and DAPI.Histological analysis of the tissue engineering tracheal wall revealed two layers.Eight weeks after implantation,the labeled seed cells by Brdu were found in the tracheal wall,suggesting that implanted cells survived and participated the tracheal tissue regeneration.Conclusion 1.The omentum was a good bioreactor to construct tissue engineering trachea.2.The engineering tracheas had two layers.3.Implanted cells participated the tracheal tissue regeneration. PartⅣ:Study on vascularization of the Tissue Engineering TracheaObjective To study the vascularization of the tissue engineering trachea.Methods Chondrocytes and vascular endothelium-like cells derived from BMMSCs, and then the endothelium-like cells were labeled by DAPI.The experimental group:both seed cells were seeded onto PGA/collagen scaffold.The contral group:chondrocytes were seeded onto PGA/collagen scaffold.The cells-scaffold mixture was wrapped around a silicone tube.Both groups were implanted into omentum in cell donor porcines and the porcines were sacrificed in 8 weeks.The tissue engineering tissues were examined by histology with HE staining and immunohistochemistry.Results HE staining showed red cells was red in the capillary and can count the number of the capillary.Immunohistochemical staining of Brdu was positive in the experimental groups,suggesting that implanted vascular endothelium-like cells survived and participated the vascular regeneration.The number of the capillary of the experimental group was significant more than that of the control group(P＜0.001).Conclusion The vascular endothelium-like cells derived from BMMSCs participated the vascular regeneration of tissue engineering trachea.