Fabrication Of Engineered Cartilage Using Nano Yarn Three-dimensional Scaffold Combined With Bone Marrow Stem Cells

Engineered scaffolds,cell sources and growth factors are the three elements of tissue engineering,which is an interdiscipline that based on cellular biology,biomaterials and engineering to develop bioactive substitutes for the repair and regeneration of damaged tissues.Therefore,the core of tissue engineering is the fabrication of engineered scaffolds.Electrospinning is an effective technique to produce nano/micro-fibrous membrane using natural polymers,synthetic polymers and blended polymers.Electrospun fibrous scaffolds are promising in tissue engineering application,as they have a high surface-to-volume ratio and high porosity.In addition,the architecture of electrospun scaffold was similar to native extracellular matrix(ECM).Although there were many reports about the successful uses of electrospun scaffolds in the repair of skin,blood vessel and nerve,researches on cartilage regeneration by electrospun scaffolds were rare.Cartilage tissues have a thickness range from 2.2 to 2.5 mm,however cells experience monolayer growth pattern on the surface of traditional electrospun scaffold which limits their applications in cartilage tissue engineering.In this study,we have fabricated a novel kind of nanoyarn scaffold using electrospinning with dynamic liquid supporting system.Electrospun P(LLA-CL)/collagen nanofibers were deposited and twisted into yarns in a water vortex to form nanoyarns(24 μm)before collecting on a rotating mandrel to obtain nanoyarn scaffold.Filed emission scanning electronic microscope(FE-SEM)images revealed that the scaffold was composed of aligned nanoyarns.Large pores and grooves between the aligned nanoyarns were observed on the surface of the nanoyarn scaffold.FTIR analysis of the scaffold confirmed the absorption peaks of P(LLA-CL)and collagen without any peaks change,indicating that the process of electrospinning with dynamic liquid system had no effect on the materials.In addition,stress-strain testing demonstrated that the nanoyarn scaffold had excellent mechanical properties.Pig iliac endothelial cells(PIECs)and MC3T3-E1 pre-osteoblastic cells were utilized as cell sources to examine the biocompatibility of the nanoyarn scaffolds in vitro.MTT assay showed that the nanoyarn scaffolds supported cell proliferation better than the traditional nanofibrous scaffolds.Histological analysis(H&E staining)illustrated that the cells penetrated into the inner parts of the nanoyarn scaffolds,while on the nanofibrous scaffolds,the cells were restricted to proliferate on the surface.Both the FE-SEM and confocal laser scanning microscope(CLSM)images confinned that the cells exhibited extremely stretched phenotypes and spread out well on the nanoyarn scaffolds.Additionally,the PIEC grew along the nanoyarns and initiated to develop into complex capillary-like structures,indicating that the nanoyarn scaffold may improve angiogenesis in regenerative tissues.Biomimetic three-dimensional hybrid nanoyarn scaffold was obtained using freeze-drying to add collagen/hyaluronic acid into the nanoyarn scaffold.New Zealand rabbit-derived bone marrow stem cells(BMSC)combined with the ultimate hybrid nanoyarn scaffold to fabricate engineered cartilage.Alcian blue staining results demonstrated that the BMSCs were induce into chondrocytes in presence of transforming growth factor-β1(TGF-β1).CLSM images showed that the cells present organized growth pattern along the nanoyarns.Besides,cell infiltration throughout the nanoyarn scaffold was observed by H&E staining analysis.Osteochondral biphasic scaffold was obtained by combining the hybrid nanoyarn scaffold with β-tricalcium phosphate(β-TCP)via freeze-drying.Cell-scaffold constructs were implanted into rabbit osteochondral defects after three weeks’ chondrogenic culture of BMSCs on the scaffolds in vitro.After three months,the toluidine blue staining results showed that the defects were repaired by the cell-scaffold constructs.