| Inflammation is the body’s protective response to pathogens or tissue damage by eliminating harmful irritants in order to maintaining tissue homeostasis.The dangers of chronic inflammation are manifested by the persistence of inflammatory factors and tissue damage,which may even induce a series of inflammatory diseases.Among them,periodontitis and chronic skin wound are both common chronic inflammatory diseases that seriously affect the quality of life of patients.Therefore,regulating the progression of chronic inflammation is essential to alleviate tissue damage and promote tissue remodeling.Macrophages,as essential cellular members of the intrinsic immune system,play an important role in the onset,progression,and regression of inflammation.Studies have shown that macrophages are essential in bone remodeling in periodontitis and in the healing of skin wounds.Macrophages have a certain functional plasticity and can polarize into different phenotypes with different functions in response to different exogenous stimuli.Therefore,it is necessary to apply regulatory strategies to upregulate the pro-healing phenotype of macrophages and improve tissue regeneration.In recent years,biomaterial-based tissue engineering technologies have been rapidly developed.Among them,electrospun aligned fiber scaffolds are able to mimic the natural extracellular matrix structure and exhibit the natural advantages of modulating the biological behavior of cells,which have been widely used in the field of tissue regeneration.It has been reported that aligned electrospun fibers can modulate the morphology of macrophages skeleton and thus affect their phenotype under noninflammatory environment,suggesting that they may have some immunomodulatory functions.However,there are relatively few reports on the influence of aligned nanofibers on macrophage polarization in the inflammatory states.Therefore,it is necessary to investigate this issue and explore the potential biological mechanisms.Therefore,in this study,we investigated the effects of electrospun aligned nanofibers on the biological behavior of BMSCs and macrophage polarization,and explored the possible molecular mechanisms by applying RNA-seq technology.Subsequently,a periodontitis bone defect model and a skin wound healing model were applied for dual validation in vivo,which provided an important theoretical basis for the application of oriented electrospun fiber scaffolds in tissue regeneration.Part Ⅰ.Experimental study on the regulation of biological behavior of BMSCs by the alignment and diameter of electrospun fibers[Objective]To prepare aligned and random electrospun fiber membranes with different diameters(micro-and nanoscale)and investigate their effects on the biological behaviors of rat bone marrow mesenchymal stem cells(BMSCs),and to screen the optimal parameters for subsequent experiments.[Methods](1)Aligned and random electrospun fiber membranes with different diameters(micro-and nanoscale)were successfully prepared by electrospinning technology.They were then named as aligned nanofibers(AN),random nanofibers(RN),aligned microfibers(AM)and random microfibers(RN),respectively.The electrospun membranes were characterized by scanning electron microscopy(SEM)and contact angle measurement.ImageJ software was applied to calculate the diameter of each group of fibers.(2)Phalloidin staining,CCK-8,live/dead cell staining and scratch test were applied to investigate the effects of alignment and diameter of electrospun fiber membrane on the biological behaviors of BMSCs such as adhesion,proliferation and migration.(3)ALP staining and semi-quantitative,alizarin red staining,RT-PCR and immunofluorescence staining of osteopontin(OPN)were applied to investigate the effects of alignment and diameter of electrospun fiber membranes on the osteogenesis of BMSCs.[Results](1)SEM showed that the obtained electrospun fibers were smooth and continuous,and the fiber arrangement direction of the aligned group is highly consistent,while the that of the random group is more disordered.The diameter of the microscale groups(AM and RM group)was about 1.2μm,while the diameter of nanoscale groups(AN and RN group)was about 600nm.The results of water contact angle test showed that the water contact angle of AN group was significantly lower than the other three groups among the four groups(p<0.05).(2)The results of phalloidin staining and SEM showed that the cells culturing on aligned group were arranged along the long axis of the fiber,while the cells on the random group were polygonal and arranged more randomly.The results of live/dead cell staining showed that all the four groups of electrospun fiber membranes were provided with good biocompatibility The results of CCK-8 experiment verified that cells of culturing on all groups exhibited good proliferation tendency,and aligned groups(AN and AM group)significantly promoted cell proliferation at day 7(p<0.05).The results of the scratch test showed that the AN group promoted the migration rate of cells when comparing with the other three groups.(3)The results of ALP staining and semi-quantitative experiments,alizarin red staining,RT-PCR and OPN immunofluorescence staining showed that the aligned group were more favorable to the osteogenesis of BMSCs,with the AN group showing the greatest advantage(p<0.05).[Conclusion](1)In this study,PLLA electrospun fiber membranes with different alignment and diameters were successfully fabricated,and the preparation parameters of aligned electrospun fibers were optimized.(2)This study confirmed that the aligned nanofibers(AN)group could promote the proliferation,migration and osteogenic differentiation of BMSCs.Part Ⅱ Experimental study on the regulation of macrophage polarization by aligned electrospun nanofibers and their mechanism[Objective]To investigate the effect of electrospun aligned nanofiber membrane on macrophage polarization in lipopolysaccharide(LPS)-induced inflammatory environment,and their molecular mechanism.[Methods](1)Aligned and random nanofiber membranes were prepared by electrospinning technology and named as A20 and R20 groups respectively.Their surface morphology was observed by SEM,the hydrophilicity was detected by water contact angle test,and the mechanical properties were detected by mechanical test.(2)Phalloidin staining,flow cytometric analysis,RT-PCR,ELISA,Western blot and immunofluorescence staining were applied to investigate the effects of the effect of A20 and R20 groups on macrophage polarization in LPS-induced inflammatory environment.(3)RNA-seq was applied to explore the differentially expressed genes,then KEGG database was applied to perform signaling pathway enrichment analysis.Subsequently,immunofluorescence staining and Western blot were applied to validate them(p<0.05).[Results](1)The SEM results showed that electrospun nanofibers of the two groups were smooth and continuous,the fibers arrangement in A20 group were more orderly,while that of the R20 group were more random and disorganized.The water contact angle of A20 group was lower than that of the R20 group(p<0.05).And the mechanical properties of A20 group were better than those of R20 group(p<0.05).(2)The results of phalloidin staining showed that RAW264.7 macrophages culturing on the surface of the A20 group were transformed into elongated type,while the cells on the surface of the R20 group were still typical "omelet type".Furthermore,in LPS-induced inflammatory environment,A20 group could upregulate the M2 and downregulate the M1 phenotype of macrophages(p<0.05).(3)RNA-seq results and the enrichment analysis of KEGG pathways showed that MAPK,Toll-like receptor and Nod-like receptor signaling pathways were highly correlated with the effect of aligned nanofibers on macrophage polarization,then the heat map and volcano map showed that A20 group inhibited Ml-type polarization of macrophages by inhibiting JAK-STAT and NF-κB signaling pathways.Western blot and immunofluorescence staining experiments were performed to verify this(p<0.05).[Conclusion](1)Aligned nanofibers can upregulate the M2 and downregulate the M1 polarization of macrophages in LPS-induced inflammatory environment.(2)A20 group inhibited the M1 polarization of macrophages by suppressing the JAK-STAT and NF-κB signaling pathways.Part Ⅲ Experimental study of electrospun aligned nanofibers for guiding bone remodeling in periodontitis and skin regeneration[Objective]A rat periodontitis bone defect model and a mouse skin defect model was constructed to observe the repair effect of aligned nanofiber scaffold on periodontitis bone repair and skin wound healing,to investigate the effect of aligned electrospun nanofiber on macrophage polarization and tissue regeneration in vivo,and to provide a theoretical basis for the application of aligned nanofiber in tissue regeneration.[Methods](1)Arat periodontitis bone defect model was constructed,then aligned and random electrospun nanofibers were implanted into periodontal bone defect model of rats.The defective bone tissue was analyzed by Micro-CT,HE staining,Masson staining and immunohistochemical staining to observe the healing of periodontal bone tissue.(2)A mouse skin defect model was constructed,and electrospun fibrous membranes in A20 and R20 groups were applied in mouse skin defects.HE staining,Masson staining and immunofluorescence staining were performed to investigate the healing of mouse skin wounds,macrophage polarization and vascular regeneration in the skin wounds.(3)Macrophage conditioned medium was prepared and its effects on the migration,proliferation and collagen expression of L929 fibroblasts were assessed by scratch assay,CCK-8,RT-PCR and immunofluorescence staining.[Results](1)Micro-CT reconstruction and imaging results of periodontal bone tissue showed that the bone regeneration in the area of periodontal bone defects was higher in the A20 group,and the BV/TV in A20 group was significantly higher than that of Con and R20 group(p<0.05).The results of HE and Masson staining showed that the new bone tissue in the A20 group was denser,with more obvious collagen deposition and higher quality of bone repair.The results of CD163 immunohistochemical staining showed that the expression level of CD 163 in the area of periodontal bone defects was higher in the A20 group than in the Con and R20 groups.(2)The trend of skin wound healing,HE staining and Masson staining images of the skin tissue sections showed that the remaining wound area was the smallest in A20 group at 7 and 14 d,respectively(p<0.05),with less inflammatory cell infiltration and the most abundant collagen fiber deposition in the wound area.Immunofluorescence staining results showed that the number of M2 type macrophages was increased and the number of M1 type macrophages was decreased in A20 group when comparing with Con and R20 groups,and more neovascularization could be observed in the A20 group.(3)The conditioned medium in A20 group significantly accelerated the migration,proliferation and collagen synthesis of L929 fibroblasts when comparing with the Con and R20 groups(p<0.05).[Conclusion](1)Electrospun aligned nanofibers could modulate the M2 phenotype polarization of macrophages in periodontal bone defects and guide bone remodeling in periodontitis.(2)Electrospun aligned nanofibers could promote the M2 phenotype polarization of macrophages in skin wounds,inhibit their M1-type polarization,and guide the regeneration of blood vessels in wounds to accelerate the healing of skin wounds.(3)Conditioned medium of macrophage culturing on aligned electrospun nanofibers could promote the migration,proliferation and collagen synthesis of L929 fibroblasts. |
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