Fabrication Of Embeded Electrospun Nanofiber Hydrogels And Its Evaluation On The Repairing Of Cartilage Defect

Object: Articular cartilage injure seriously affects the quality of patients’ life.In recent decades,silk fibroin has been widely used as the scaffold material in cartilage tissue engineering due to its excellent biocompatibility.Electrospinning technology has also been applied to the manufacture of nanostructured scaffolds in various tissue engineering fields.However,electrospun nanofiber mats are not suitable for the scaffolds in articular cartilage defects repairing.In this study,a novel scaffold of embedded electrospun nanofiber hydrogel was fabricated,and its effect on the repairing of articular cartilage defects by compositing growth factor TGF-β1 was evaluated.Materials and methods: The silk fibroin and chitin mixed solution was electrospun into nanofiber mats by electrospinning;thereafter,the mats were grinded into short nanofibers.The short nanofibers were added to the silk fibroin solution for mixing,adding cross-linking agent ethylene glycol diglycidyl ether,and then freezing at low temperature.The embeded electrospun nanofiber hydrogel was obtained.The morphology,composition and silk fibroin crystallinity of the material were tested by field emission scanning electron microscopy(FE-SEM)and Fourier transform infrared spectroscopy(FITR).The pore size and porosity of the hydrogel were obtained by measuring 100 pores’ diameters in FE-SEM photographs.The cytotoxicity of the scaffold on chondrocytes was assessed using the CCK-8 kit.The adhesion of the chondrocytes were measured using FE-SEM observing the cell adhesion number and position on the scaffolds after co-culture for 24 hours.The cell proliferation was measured using CCK-8 to calculate the number of cells on the scaffold at a specific time point(days 1,4,7,14).The cellular growth behavior was measured using the cell live-death kit and observed under a fluorescence confocal microscope.The scaffolds were heterotopic transplanted into the subcutaneous and in suit transplanted into the articular cartilage defects of SD rats,respectively.The subcutaneous samples were harvested 4 weeks after surgery;thereafter they were photographed,fixed,dehydrated,embedded,and sectioned.The samples were stained with HE to observe the cell growth and material degradation levels in the scaffold.To test the immunogenicity of the scaffold,total RNA was extracted from some samples,and the expression levels of inflammatory factors IL-1,IL-6 and TNF-α were detected by RT-PCR.The in-suit transplanted samples were harvested at 6 and 12 months after surgery.After taking a large photograph,they were fixed,decalcified,dehydrated,embedded,sliced,and subjected to HE and oafranin-O/Fast Green staining,and Sox-9 and COLII immunofluorescence staining.The qualities of the new tissues were evaluated according to the ICRS scale.Results: The FE-SEM image illustrated that the pores of the embeded electrospun nanofiber hydrogel were irregular hexagonal,with an average diameter of 79.3 ± 13.7 μm;the puncture-like rough surface was formed by nanofibers;the porosity was 92.56 ± 0.34%.FITR analysis showed that there were characteristic peaks of silk and chitin in the hydrogel;the chains of silk and chitin were covalently bound;and the crystallinity of silk increased after cross-linking.Cytotoxicity test showed that the scaffold has good biocompatibility and nontoxicity.The results of cell proliferation experiments showed that the scaffold promoted the proliferation of chondrocytes,and the number of cells at specific time points was significantly higher than other groups(P<0.05,P<0.01).FE-SEM images showed that chondrocytes were more likely to adhere to the palisade-like rough surface.Fluorescence confocal images showed that chondrocytes grew well on nanofiber scaffolds and had higher cell viability than the other groups.For in vivo experiments,the overall score of ICRS showed that the TGF-β1 group had the highest score at 6 weeks(P<0.001,P<0.01).The results of IC RS histology showed that there was new cartilage tissue in the scaffold,and the quality of TGF-β1 group was the best(P<0.001,P<0.05).12 weeks,the overall score of IC RS showed that the repair effect of the blank scaffold group was significantly improved,and there was no significant difference with the TGF-β1 group(P>0.05).The ICRS tissue score showed that the quality of neonatal cartilage in the blank scaffold group was significantly higher than that in the TGF-β1 group(P<0.05).The semi-quantitative analysis of COLII immunofluorescence showed that the COLII content of the new cartilage tissue in the TGF-β1 group was the highest at 6 weeks.At 12 weeks,there was no significant difference in the COLII content between the blank scaffold group and the TGF-β1 group(P>0.05).Conclusions: In this study,a novel cartilage tissue engineering scaffold was prepared which was componentially mimiced the main component of the extracellular matrix of natural articular cartilage and structurally possessed a unique nanofibrous microstructure.The scaffold can facilitate adhesion and proliferation of chondrocytes and promotes cartilage tissue regeneration in rat knee joint defects.The scaffold compositing TGF-β1 can effectively repair articular cartilage defects in rats,and provides a reference for the repairing of articular cartilage defects.