Optimization Of Cartilage Tissue Engineering And Constuction Of Tissue-engineered Trachea With Pedicled Muscle Flap

Objectives:Surgical treatment of segment tracheal reconstruction longer than 6cm needs trachea substitutes,it remains a great challenge due to the lack of suitable autologous tissue donor,limited source and immuno-rejection of allogenous trachea,as well as inferior biological function of synthetic tracheal grafts.Tissue-engineered trachea?TET?using autologous cells would be an ideal substitute for proper biological structure and function similar to native trachea and autologous availability,providing a promising strategy for reconstruction of long segmental tracheal defect.This project attempts to explore the different programs to build better cartilage tissue,and to construct tissue engineering trachea model using in vivo muscle flap culture method.1.To investigate fit way for rib chondrocytes isolation,expansion and culture on three-dimensional scaffold.2.To investigate optimized method for the construction of tissue engineering hyaline cartilage with chondrocytes,bone marrow stem cells or both kind of cells.3.To describe an approach for engineering of vascularized trachea based on the implantation of cocultured scaffolds surrounded by a muscle flap.Methods and Results1.Costal Chondrocyte Isolationm,Expansion and Culture on Three-Dimensional ScaffoldMethods:Rabbits'rib cartilage were sliced into 1mm³ pieces and treated with sequential enzyme digest or block culture to isolate chondrocytes.After two passages,rib chondrocytes were collected and suspended.The cell suspension was seeded on PLGA or Dacron scaffold at densities of×10⁷/ml,then cultured under a 37??5%CO₂ atmosphere.Chondrocytes'morphology and structure sduring expansion were observed by optical microscope and trnsmission electron microscope,while cells growing and matrices formation were examined by scanning electron microscope.Results:Rib chondrocytes were isolated by sequential enzyme digest or block culture,of which the average passage time was 5 days.However,isolation by tissue block culture was slow and even fibroblasts was found in one flask.Chondrocytes attached well on scaffolds.Extracellular matrices was seen after 2 weeks.After 3 weeks,chondrocytes started to apoptosis.2.Optimization of cartilage tissue engineering with co-culture of bone marrow mesenchymal stem cells and chondrocytesMethods:Isolation and amplification of the rib cartilage cells and bone marrow mesenchymal stem cells of New Zealand rabbits,seeding on PLGA contruct respectively or with different ratio?5:5 and 1:9?and cultured in vitro and in vivo.We used HE staining,Periodic Acid-Schiff staining,Safranin-O staining,Alcian Blue staining and Immunohistochemical staining of collagenase II assess cell yield and cartilage characterss.Results:Cartilage tissue could be fabricated by chondrocytes culturing or co-cluture with BMSCs?5:5?,while tissue most close to normal cartilage structure with appropriate GAG content without Osteogenesis phenomenon was found by co-cluture method?5:5?.3.A Vascularized Tissue-Engineered Trachea Fabricated with Pedicled Muscle FlapMethods:PLGA scaffolds were seeded with chondrocytes and cultured in vitro for 2weeks,and wrapped in a pedicled muscle flap or simply subcutaneously cultured in vivo in rabbits.Harvested after four weeks,prefabricated neotrachea was transferred to the defect as a tracheal replacement.Result:Histological analysis confirmed that cartilage formed in the muscle flap groups contained a more homogeneous and higher extracellular matrix content.The trachea grafts established more stable blood supply for the maintenance of tubular cartilage structure and accelerated epithelialization through intramuscular implantation and transplantation with pedicled muscular flap.Long-term survival of animals was achieved by tissue engineered trachea with pedicled muscle flaps.Conclusion:1.Sequential enzyme digestion is fit for rib chondrocytes isolation.2.Good cartilage formation was observed after chondrocytes seeded on PLGA or Dacron scaffolds.The chondrocytes-scaffold model could be further used to construct tissue engineered trachea.3.Co-culture approach with chondrocytes and BMSCs?5:5?may enable the development of proper cartilage tissue for clinically applicable neo-trachea.4.The graft could obtain enough blood supply after repair operation by pre-vascularization and transplantation with pedicled muscule flap5.Proper vascularization could promote cartilage ECM production,enhance the mechanical strength,accelerate the degradation of scaffolds,benefitting trachea defect repair.6.It is a promising approach for long-term functional reconstruction of tracheal defect with tissue-engineered trachea with pedicled muscle flap.