Fabrication And Meniscal Tissue Engineering Research Of Biomimetic Structured PCL/Gelatin/Chondroitin Sulfate Composite Aligned Scaffold

Objective:Due to the unique anatomical characteristic,meniscal self-healing capacity is especially challenging in the inner avascular regions.Currently,there is no ideal scaffold in upgrading tissure engineered meniscus to restore the meniscal regeneration both biohemically and functionally.For mimicking the meniscal content and specialized arrangement of collagen fibers,we investigated the use of PCL,gelatin and chondroitin sulfate to produce a novel biomimetic structured cell-seeded composite aligned scaffold.In this study,we evaluated the material property,biocompatibility,fibrocartilage-inductive bioactivity and the ability to load with bone marrow mesenchymal stem cells and to facilitate meniscal repair of this new biomimetic structured scaffold.Methods:(1)Fabrication of composite aligned tissue scaffold and examination of its material properties: In utilization of the technology of coaxial electrospinning and high speed rotating mandrel,we improved the conventional electrospinning equipment and produced the composite aligned scaffold and random scaffold,named as A-PCL/GEL/CS and R-PCL/GEL/CS respectively.Additionally,A-PCL and A-PCL/GEL aligned scaffold were also developed as control groups.A comparative analysis of four scaffold groups was performed involving surface morphology,porosity,hydrophilia,degradability and mechanical properties.Furthermore,the infrared interference spectrum and carbazole method were also applied to evaluate the composition and chondroitin sulfate release behavior of the composite core-shell scaffold.(2)Bioactivity evaluation in vitro: Rabbit MSCs were derived from bone marrow and cultured in fibrochondrogenic differentiation inductive medium involving TGF-β3,CTGF,dexamethasone and ascorbic acid sodium salt.The fibrochondrocyte was assessed by cytophenotype observation,specific staining and immunohistochemistry assay.BMSCs were seeded on different groups of nanofibrous scaffold and cultured in differentiation inductive medium.Scanning electron microscope and CCK-8 were utilized to evaluate the cytophenotype and cytocompatibility.Besides,extracellular matrix ELISA kit and realtime quantitative polymerase chain reaction were performed to assess the fibrocartilage-inductive activity.(3)Biocompatibility and regenerative capacity evaluation in vivo: Scaffold extract liquid was prepared to perform the acute systemic toxicity and intracutaneous sensitization test according to material toxicity evaluation standard.Then,the cell-seeded scaffold was implanted in vivo to the meniscus injured rabbit after culturing in fibrochondrogenic differentiation inductive medium for 14 days;control groups were prepared as explants without scaffolds or cells.After 12 weeks postoperation,the regeneration capacity was evaluated by gross observation,histological scoring system and biomechanics test.Results:(1)Scanning electron microscope analysis revealed that biomimetic structured nanofibrous scaffold appeared to be a high degree of alignment.The core-shell structure of single fibers was uniform and the chondroitin sulfate was distributed in the core of the nanofibers homogeneously by transmission electron microscope.After adding the natural component gelatin and chondroitin sulfate,the biomimetic structured composite scaffold was revealed to show better hydrophilia,porosity.Moreover,the aligned scaffold generated a significantly greater mechanical strength than random scaffold.The release behavior of chondroitin sulfate encapsulated in the core of coaxial nanofibers resulted in avoiding initial burst release effectively.(2)According to cytophenotype,specific staining and immunohistochemistry assay,the BMSCs can succeed in differentiating into fibrochondrocyte by culturing in differentiation inductive medium.The biomimetic structured composite nanofibrous showed better cell adhesion and proliferation.Besides,the results of the extracellular matrix ELISA kit and RT-PCR revealed that the PCL/GEL/CS composite scaffold contributed to greater typical gene expression and extracellular matrix deposition,which exhibited higher fibrocartilage-inductive activity.(3)The novel biomimetic structured PCL/GEL/CS aligned scaffold possessed good biocompatibility in vivo.After implanted in the avascular region of injury meniscus for 12 weeks,the histological and biomechanical analysis revealed that the biomimetic structured composite aligned scaffold can facilitate meniscal repair and regeneration,especially loaded with BMSCs.Conclusion:The novel biomimetic structured PCL/GEL/CS aligned scaffold exhibited good biocompatibility and biomechanics property.The combined application of BMSCs and nanofibrous scaffold is proved to facilitate meniscal repair and regeneration in this study.