Implantable And Degradable Antioxidant Poly(ε-caprolactone)-Lignin Nanofiber Membrane For Effective Osteoarthritis Treatment

Objective:Osteoarthritis（OA）is one of the most common musculoskeletal diseases in the world.Oxidative stress caused by free radicals such as reactive oxygen species（ROS）is the main cause of cartilage degradation and OA.However,traditional injectable or oral antioxidants generally do not provide effective treatment due to their rapid removal and degradation or low bioavailability.A new strategy for the preparation of nanofibers based on poly（caprolactone）and PCL lignin（PCL-G-lignin）copolymer was proposed.The feasibility of preparing nanocomposite fiber membranes from Lignin and polycaprolactone（PCL）by electrostatic spinning was investigated.Lignin provides intrinsic antioxidant activity,while PCL adjusts mechanical properties.Electrospun PCL-lignin nanofiber membranes have good antioxidant activity,low cytotoxicity and anti-inflammatory effects.In this experiment,human chondrocytes stimulated by H₂O₂ and OA rabbits were used as models.Pcl-lignin nanofibers inhibit ROS production and activate antioxidant enzymes through autophagy mechanism.Arthroscopic implantation of PCL-lignin nanofiber membrane has good biocompatibility,degradability and continuous antioxidant activity,which is an effective method for the treatment of OA.Autophagy can be divided into large autophagy,small autophagy and molecular chaperone mediated autophagy according to different species,transport modes and regulatory mechanisms.Macroautophagy refers to the process in which the double-layer membrane from the endoplasmic reticulum wraps the substances to be degraded into autophagosomes,then fuses with lysosomes and degrades their contents.Generally,autophagy is called macroautophagy.Microautophagy refers to the direct degradation of the encapsulated material by the membrane of the lysosome.Molecular chaperone mediated autophagy refers to the process in which soluble protein molecules in the cytoplasm are transported to the lysosomal lumen for degradation after binding with molecular chaperone.For a long time,it has been believed that autophagy has no selective degradation of substrates,but with the deepening of research,it is found that autophagy can selectively degrade certain macromolecules and organelles under certain circumstances,which is called autophagy.Reactive oxygen species（ROS）are the main free radicals in organisms,including hydroxyl radical,superoxide anion,hydrogen peroxide（HO2）and the derived organic peroxide radical alkoxy.And alkperoxy,which,as products of normal REDOX reactions in vivo,participate in sterilization,detoxification and regulation of various metabolic pathways 1.Under normal physiological conditions,the body’s antioxidant system will remove ROS on time,thus maintaining the balance between oxidation and antioxidant in the body.However,when the body is stimulated by different stressors or pathogen infection,the ROS level generated in the body is higher than the antioxidant defense capacity of cells,and the REDOX state is unbalanced.Excessive ROS exists in tissues or cells and induces oxidative stress,leading to oxidative damage,such as DNA hydroxylation,protein denaturation and tissue damage.To prevent progressive oxidative damage,organisms activate a series of defensive responses such as enhancing the activity of antioxidant enzymes and initiating lysosomal degradation pathways.In addition,a large number of studies in recent years have proved that ROS produced by oxidative stress can induce autophagy.Autophagy is a kind of self-eating phenomenon widely existing in eukaryotic cells,which degrades long-lived proteins and damaged cellular organs,so that cells can continue to recycle nutrients under stress conditions.It was found that autophagy can remove mitochondria,endoplasmic reticulum,catalase and protein damaged by oxidative stress,and slow down cell death.However,when the autophagy process is blocked,toxic protein aggregation and mitochondrial function damage will further aggravate oxidative stress.It can be seen that there is a close relationship between oxidation excitation and autophagy.ROS is a direct primer for oxidative stress,and about 90%of ROS originates from the mitochondrial endointimal respiratory chain.Leakage of mitochondrial respiratory chains can produce superoxide radicals,which in turn produce ROS.Numerous studies have shown that ROS from mitochondria is the main inducer of autophagy under oxidative stress.ROS can mediate autophagy.Methods:The first part was the synthesis of nano fiber membrane.Firstly,LP copolymer was synthesized from lignin and PCL,and then mixed with PCL in different proportions,nano-composite fiber membranes were prepared by electrostatic spinning.The material was characterized by scanning electron microscopy（SEM）to observe the fiber morphology and calculate the fiber diameter.The tensile strength and Young’s modulus were measured to verify the mechanical strength of the fiber membrane.The creep recovery test is used to analyze the elasticity of a polymer film.The second part is the cell experiment of nanocomposite fiber membrane in vitro.Six groups of chondrocytes were prepared according to different proportions of nanocomposite fiber membranes as the experimental group and the sliver group as the blank control:PCL,PCL-VE and Pcl-lignin nanofibers with or without 0.4 mmol/L H₂O₂ were pre-inoculated for 24 h.Each group was composed of（1）PCL,（2）PCL-VE,（3）Pcl-lignin 50,（4）PCL+H₂O₂,respectively.（5）H₂O₂（6）PCL-lignin50+H₂O₂.Cytotoxicity and cell proliferation on PCL,PCL-VE and Pcl-lignin nanofibers were detected using the CCK-8 kit（Dojindo,Japan）.Live/death activity was measured using the live/death activity test kit（Invitrogen,USA）.Chondrocytes were counted and imaged using a high content screening（HCS）platform（CX51110,Thermo Fisher Scientific,USA）.Apoptosis was detected using the Annexin V-FITC/PI apoptosis detection kit（Key GEN,China）and analyzed by flow cytometry（BD Biosciences,USA）.The activities of SOD,CAT,GSH and MDA in cell homogenate were detected.ROS detection kit（Shanghai Bitai,China）was used to analyze intracellular ROS levels.Intracellular ROS levels were detected by flow cytometry（EX/EM488 nm and 525 nm）.The secretion of matrix metalloproteinase 13（MMP-13）was detected by immunofluorescence assay.Autophagy identification and autophagy assessment were performed using the HCS platform.Observation by transmission electron microscopy（JEM1230,JEOL,Japan）.Real-time polymerase chain reaction（RT-PCR）was used for RNA extraction and QRT-PCR.Qrt-pcr)detected the expression of IL-1β,IL-6,MMP-13,SOD,CAT,GSH-Px,NRF2,ATG4,ATG7,Keap1 and P62 genes.The third part,OA animal model and PCL-lignin nanofiber treatment,the right knee osteoarthritis（OA）was induced by 500μL 4%papain injection[42].After four weeks of treatment,the New Zealand rabbit was euthanized and sacrificed.The joint was taken for further examination.In vitro and in vivo degradation,MMP-13 and IL-6 were detected by ELISA,visual observation and histological examination,and paraffin-embedded sections were stained after fixation with paraformaldehyde.Statistical analysis:All data were expressed as mean±SD or median（scatter plot）,one-way variance（ANOVA）was used for statistical analysis,and Least Significant Difference（LSD）test was used when the results were Significant.Mann-whitney U test was used for macro score.A P value less than 0.05 was considered statistically significant.All experiments were carried out in triplicate or duplicates.Results:LP copolymer was successfully synthesized and a series of PCL/Lignin nanocomposite fiber membranes were prepared by electrostatic spinning,in which PCL provided mechanical support and Lignin acted as antioxidant.The chemical structure of PCL-G-lignin copolymer was characterized by 1H Nuclear magnetic resonance（NMR）and FTIR spectra.These results confirmed the successful combination of PCL and lignin.In addition,the copolymer is soluble in a variety of organic solvents,which is conducive to subsequent treatment.The free radical scavenging ability of PCL-Lignin 50 was further analyzed using superoxide anion and hydroxyl radical detection kits.It is confirmed that lignin has strong antioxidant capacity.Pcl-lignin nanofibers enhance H₂O₂ stimulation of human chondrocytes.The viability of human chondrocytes was analyzed using Cell Counting Kit-8（CCK-8）.The results showed that the cell viability of lignin-containing nanofiber membrane was higher than that of PCL nanofiber,indicating that it had good biocompatibility.The cell viability of PCL/Lignin50 nanofibers was the highest（82.95%）.Therefore,PCL-Lignin 50 nanofibers have been selected for cell and animal studies because of their excellent antioxidant properties and biocompatibility.These results were further confirmed by live/dead assays,which determine cell viability based on plasma membrane integrity and esterase activity.We also evaluated the apoptosis of chondrocytes on different nanofibers by flow cytometry and showed that PCL-lignin50 was most effective in inhibiting apoptosis in the whole body of H₂O₂.In conclusion,PCL-lignin 50can effectively protect chondrocytes from oxidative stress.Study on inhibition of inflammatory cytokines expression by PCL-lignin50 nanofibers.Immunofluorescence staining was used to analyze m MP-13 level of chondrocytes stimulated by H₂O₂,and to evaluate the degradation of ECM in early OA.The increase of PCL,PCL-VE and PCL-lignin50 nanofibers decreased successively,indicating that PCL-lignin50 nanofibers can alleviate the degradation of ECM induced by oxidative stress.This finding was further confirmed by QRT-PCR analysis,which showed that PCL-Lignin 50 nm fiber membranes inhibit ROS production in chondrocytes in response to oxidative stress（resulting in cell damage）.Oxidative stress,autophagy,epigenetic changes and mi RNAs regulation are potential therapeutic targets for osteoarthritis.Oxidative stress is due to the imbalance between the production of reactive oxygen species（ROS）in mitochondria and the ability of cell scavenging system to scavenge ROS（superoxide dismutase（SOD）,catalase,glutathione peroxidase and glutathione reductase and reduced glutathione level）.Aging is associated with ROS accumulation,which is due to the overproduction of ROS and the decline of the ability of the above system to reduce ROS surplus.ROS 19 and ROS in cartilage are mainly activated by h2o2-peroxygenase（dpon）,respectively.In OA chondrocytes,different stimuli induce the activation of cytokine receptors and TLRs,resulting in the excessive production of these reactive oxygen species,resulting in the slow degradation of cartilage,and the slow metabolic rate of collagen in chondrocytes and cartilage,which makes them highly sensitive to the cumulative effect of oxidative stress.Studies in humans and experimental animals have shown that the antioxidant capacity of glutathione system in OA is related to the decrease of the activities of some oxidative stress related enzymes,such as SOD and catalase.In this environment,the oxidative modification of various intracellular and extracellular components,including transcription factors,membrane phospholipids and nucleic acids,as well as the inhibition of glycosaminoglycan and collagen synthesis and the activation of MMPs and aggrecanases,promote the process of cartilage destruction.The resulting cartilage degradation products can further induce inflammatory response and excessive production of ROS by activating the expression of mitochondrial NADPH oxidase and inducible nitric oxide synthase（i NOS）,aggravate joint degradation,form a vicious circle and lead to the progress of OA.In fact,the excessive production of nitric oxide（no）is considered to be the main cause of OA;ONOO,which is not converted into its derivative,consumes the mercaptan groups available in glutathione,which is conducive to oxidative stress and induces cell damage through oxidation and apoptosis of protein,lipid and DNA.ROS can cooperate with different factors,such as advanced glycation end products（ages）accumulated during aging and OA,modify proteins and hyaluronic acid,and change cartilage homeostasis.Oxidative stress produces post-translational modification of col2 and increases its antigenicity;Participate in various stages of inflammatory response（such as activation of NF KB）;Induce telomere instability,leading to cell senescence of chondrocytes;And interfere with the anabolism of insulin growth factor（IGF）-I,all of which help to promote the catabolic state of OA.A variety of intracellular pathways are activated by ROS in different cells of the joint,including ERK1/2 and p38 MAPK.In chondrocytes,activation of these kinases leads to hypertrophic differentiation.Oxidative stress can also change the activity of AP-1,a heterodimer complex composed of Fos and Jun proteins,which regulates the expression of matrix MMPs.The activity of antioxidant factors（SOD,CAT）and the level of GSH and MDA showed that PCL-Lignin 50 nanofibers could help chondrocytes resist oxidative stress.To further confirm the ability of PCL-lignin50 to trigger autophagy,chondrocytes were instantaneously transfected with autophagy labeled proteins GFP-LC3 and RFP-P62.More autophagosome co-localization was observed in the cytoplasm of PCL-lignin50 nm chondrocytes under fluorescence microscopy,suggesting that p CL-lignin 50 nm fibers were more active in autophagy than PCL and PCL-VE nanofibers.In addition,the expression levels of autophagy markers（ATG4,ATG7,Keap1 and P62）were detected by QRT-PCR analysis.Compared with PCL,PCL-Lignin 50significantly up-regulated the expression of ATG4 and ATG7,inhibited the expression of Keap1 and P62,and negatively regulated Nrf2 signaling pathway in selective autophagy under oxidative stress.These results suggest that PCL-lignin50 can attenuate H₂O₂ activation through autophagy to induce oxidative stress.Pcl-lignin 50 nanofibers can reduce the progression of OA in vivo.The antioxidant enzyme activity of SOD and MDA level in synovial fluid were measured to determine whether PCL-lignin50 could inhibit the oxidative stress of OA joint.The results showed that SOD activity decreased and MDA level increased in untreated group,indicating that papain caused oxidative stress injury.PCL treatment does not help.On the other hand,PCL-lignin50significantly increased SOD activity by 62.28%and decreased MDA level by48.36%.The expression levels of inflammatory cytokines（MMP-13 and IL-6）in synovial fluid were detected by ELISA.The expression levels of MMP-13and IL-6 were significantly increased after papain treatment.The levels of MMP-13 and IL-6 were reduced by 52.28%and 63.20%by PCL-lignin50treatment,respectively.Conclusion:In this study,PCL and PCL-lignin nanofiber films were prepared by electrostatic spinning method,and nanofiber films with good oxidation resistance and mechanical properties were customized by changing the proportion of the two polymers,and were used for OA treatment for the first time.This study demonstrated implantable and in vivo biodegradable scaffolds and sustained antioxidant activity on the basis of providing a new strategy for OA arthroscopic intervention.