| Objective: The ideal scaffold not only contains growth factors,but also can functionally simulate the extracellular matrix,thereby regulating cell behavior and tissue assembly.Electrospun nanofiber membranes are widely used scaffold materials.This study aims to prepare slow-release b FGF(basic fibroblast growth factor)orientation and random nanofiber membrane scaffolds by electrospinning,through tendon differentiation in vitro and tendon regeneration in vivo To explore the feasibility of the prepared polycaprolactone nanofiber membrane scaffold for repairing injured tendon,thus providing a promising scaffold for tendon tissue regeneration.Methods: In Chapter 1 of the paper,bovine serum albumin(BSA)nanoparticles(NPs)and b FGF-loaded BSA nanoparticles(BNPs)were prepared by desolvation,and then self-assembled on the prepared nanoparticles by electrostatic self-assembly Chitosan shell to stabilize.The diameter of the prepared nanoparticles was measured by DLS and TEM.BNPs were encapsulated in PCL electrospinning fibers to prepare controlled release orientation and random loading growth factor tissue engineering scaffolds,aligned PCL(a PCL),random PCL(r PCL)and b FGF loaded random PCL(r PCL-b FGF)scaffold as a control group,and the morphology and structure of these nanofiber membrane scaffolds were characterized by SEM and TEM.The drug-loading efficiency,drug-loading capacity and in vitro drug release curve of the fiber were measured by ultraviolet-visible spectrophotometer and human b FGF ELISA kit.After inoculating human amniotic mesenchymal stem cells(h AMSCs)onto different groups of PCL nanofiber scaffolds,the cytotoxicity of the scaffolds and the spreading and migration of cells on the fiber scaffolds were investigated by the CCK-8 method and the staining method of living and dead cells With equal ability,the expression levels of genes of different components tendon were detected by q PCR on days 7 and 14,and the expression levels of tenascin were detected by WB on days 14.In Chapter 2 of the paper,through in vivo animal experiments,the ability of b FGF-aligned nanofibrous membrane scaffolds to repair the 2 mm defect of the Achilles tendon in rats was investigated.h AMSCs were inoculated onto different groups of nanofibrous membrane scaffolds in vitro.After 4 days of cultivation,Implanted into rat Achilles tendon defect model.After 3 days,4 weeks,and 8 weeks after implantation,MRI(nuclear magnetic resonance)was used to observe the repair of Achilles tendon defect,and then specimens were collected at 4 and 8 weeks after surgery,respectively.Gene expression level,WB detection of tenascin expression level in vivo,then immunofluorescence staining,histochemical staining,and then observe the repair effect through light microscope.Results: 1.BSA nanoparticles loaded with b FGF were prepared by desolvation method,and chitosan-stabilized nanoparticles were formed by electrostatic self-assembly.The results of transmission electron microscope and scanning electron microscope showed that the nanoparticles were more uniform.The nanoparticles stabilized by b FGF-loaded chitosan had a diameter of about 146 nm,while the pure bovine serum albumin nanoparticles had a diameter of about 205 nm.2.The nanoparticles were successfully carried into PCL electrospun nanofibers,and b FGF-loaded nanofiber membrane scaffolds with different orientations were prepared.Transmission electron microscopy demonstrated the structure of nanoparticles,and scanning electron microscopy confirmed the arrangement of nanofibers.In vitro drug release confirmed that the stent can achieve sustained release function.The mechanical property test found that the mechanical strength and Young's modulus of all aligned fibers are better than the same non-aligned fiber film.The results of in vitro cell viability and proliferation showed that the scaffolds of each group reached more than 80% relative to the blank control group,indicating that the material was basically non-toxic to the cells and the cell viability was good on each scaffold.The staining results of living and dead cells showed that the cells had good morphology and were basically in line with the direction of fibers.Compared with 1 day,culture for 4 days has obvious proliferation activity.In vitro detection of tendon genes and proteins revealed that the aligned nanofiber membrane scaffold group(a PCL + b FGF)carrying b FGF had the best ability to form tenocytes.3.Then the ability of b FGF-loaded aligned nanofiber membrane scaffold to repair the Achilles tendon defect model in rats was successfully investigated,and different groups of PCL-h AMSC constructs were successfully implanted into the defect model.After 4 weeks and 8 weeks of implantation,macroscopic evaluation of the general shape and anatomical image of regenerated Achilles tendon was performed,and the repair of tendon was observed by MRI.The b FGF-aligned nanofiber membrane scaffold(a PCL + b FGF)was the best.The results of q PCR,western blot and immunofluorescence staining showed that all groups were expressed into tendon-related genes and proteins to varying degrees.Among them,the a PCL + b FGF group had the highest expression and had a strong ability to form tendons.Histomorphological results further showed that the a PCL + b FGF group had the best repair,indicating that a sustained-release aligned nanofiber scaffold to promote tendon healing provided a new biological approach.Conclusions: 1.The aligned and non-aligned nanofiber membrane scaffolds with b FGF nanoparticles were successfully prepared by electrospinning technology,and their physical and chemical properties were characterized.2.Through in vitro experiments,it was confirmed that,compared with other groups,the aligned nanofiber membrane scaffolds carrying b FGF can promote the expression of tendon-related genes and proteins.3.It was confirmed by in vivo animal experiments that the aligned nanofiber membrane scaffold loaded with b FGF can improve the healing of autogenous tendon by promoting tendon differentiation.These results verify the feasibility of electrospinning technology in the preparation of biomimetic aligned nanofiber membrane scaffolds,and provide a promising candidate scaffold for tendon tissue engineering. |
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