| Background and Objectives:The excessive local inflammatory response drives the fibrotic capsule formation and determines the ultimate fate of the biomaterial integration.Due to the host’s inherent immune defence mechanisms,implantable biomaterial will inevitably induce tissue damage and repair remodeling.During this process,macrophages,as drivers of the i nflammatory response,play a key regulatory role in subsequent tissue repair and integration.Macrophages are involved in the entire process of FBR,from the initial inflammatory reaction to eventual fibrotic encapsulation formation.The process described above inspired us to design and manufacture biomaterials with immunomodulatory properties.This will avoid severe fibrotic reactions from the source to improve the integration and prolong the implant life.Numerous studies have shown that surface modification of materials is a common method to improve tissue integration by inhibiting severe FBR and fibrosis.Notably,the surface morphology,independent of the mechanical and chemical properties of the material itself,fully mediates the biologic recognition process of the implant interface by inflammatory cells.In recent years,thanks to the continuous development of lithography technology,it has become possible to perform rapid and precise topological modifications on materials surfaces at the micro and nanoscale.Since long-term implantation of materials is usually accompanied by mechanical challenges such as continuous tissue pull and biofluid impact,the modification of micron structures may have more durability than nanostructures.In addition,the perception of the surface morphology by cells is related to the cell size.It is believed that the size of macrophages ranges from 20 to 100 μm in some expert consensus.The length of the fibroblasts measured in the experiments was about 100 –200 μm.Macrophages and fibroblasts have each been shown to play important roles in the capsule formation,the clear role of macrophages in the fibrotic response is currently controversial.Therefore,based on the above background,we have successfully constructed micropatterns on the material surface using soft lithography.We aimed to compare the macrophage phenotype on the surface of PDMS with different microgrooves.Conditioned medium(CM)collected from macrophage was cultured with HFF-1 for evaluating the effect of cytokine on fibroblast proliferation,migration and fibrosis-related protein expression.The material was then implanted into rats for in vivo experiment.The effect of microgroove structure on macrophage polarization and capsule formation was explored,which provided a new idea and reference for the design of material surface modification methods to meet the different functional requirements of implants.Methods:1.Preparation of microgroove structures on PDMS surfaces and study on physical and chemical properties of material(1)Fabrication of microgroove structure on PDMS surface: First,the PDMS prepolymer was cured and consolidated on the mold to obtain a PDMS film with a smooth surface on both sides.The sample was set as the control group and named gro up 0–0.Next,the lithography machine(Karl Suss MA6 UV)was applied to the photoresist(PR)-coated silicon micropattern mold to fabricate four additional samples with different sizes of microgrooves on one side of the film.The groove depth was 50 or 10μm,and the bottom width was 50 or 200μm.According to the depth and width,they were marked as group50-50,group 50-200,group 10-50,and group 10-200,respectively.The width of the protruding ridge in each group was 50μm and the thickness of the single film was 0.5 mm.Finally,the smooth sides of the two PDMS films were completely sealed and bonded together to obtain a sample with the same microgroove structure and uniform thickness on both sides.Single-sided PDMS films were used for cell experiments and double-sided PDMS films were used for animal experiments.(2)Detection of the surface morphology of PDMS: A confocal laser scanning microscopy(Olympus,OLS4100,Japan)was used to observe surface morphology and compare the roughness parameters(Ra,Rq and Rz)of material surface.(3)Detection of material surface chemical properties: In order to analyze the molecular structural characteristics of PDMS after surface modification,the samples were scanned using ATR-FTIR spectroscopy.2.The effect of substrate topography on the regulation of the biological behavior of human fibroblasts via modulating macrophage polarization(1)The adhesion of macrophages: THP-1 cells were cultured on PDMS surfaces.At time points 6 and 12 h after seeding,cells were stained with DAPI for 5min.Cell images were taken on a fluorescence microscope and counted using Image J software.(2)The morphology of macrophages: To determine whether the surface topography on PDMS induced macrophages differentiation,the typical cell morphology was observed by ordinary optical microscopy at 1,3 and 5 days.(3)The polarization gene expression: THP-1 cells were cultured on PDMS surface for3 days.The gene expressions of M1 phenotype: i NOS,TNF-α,IL-6 and M2 phenotype:CD206,TGF-β,IL-10 was detected using real-time PCR.(4)Release of inflammatory factors: THP-1 cells were cultured on PDMS surface for 3days.The concentrations of TNF-α and IL-10 in the macrophages supernatants were determined by ELISA kits following the manufacturer ’s instructions.(5)Immunofluorescence staining of i NOS in macrophages: THP-1 cells were cultured on PDMS surface for 3 days.We assessed the difference in macrophage polarization by the immunofluorescence staining of i NOS.The samples were viewed under the fluorescence microscopy and the fluorescence intensity was analyzed using Image J software.(6)HFF-1 cell proliferation assay: HFF-1 cells were cultured in CM for 1 and 3 days.The CCK-8 assays were used to compare the proliferation activity of cells.(7)HFF-1 cell migration assay: When HFF-1 cells merged into a monolayer,a straight scratch was created.The cells were cultured with CM.The scratch areas were photographed under an inverted microscope at 0h,12 h and 24 h and analyzed using image J softwa re.(8)Western blotting: HFF-1 cells were cultured with CM for 5 days for protein blotting experiments.The expressions of fibrosis-related proteins FN,PCNA,α-SMA and COL-I in HFF-1 cells were detected.3.Effects of different PDMS surfaces on inflammatory response and capsule formation(1)Immunofluorescent staining: CD206 immunofluorescence staining was performed on macrophages in the surrounding tissues to compare the inflammatory response at 1 week and 4 weeks after implantation in rats.(2)HE staining: The tissue around the implants was stained with hematoxylin and eosin(H&E)to compare the thickness of the fibrotic capsule at 1 week and 4 weeks after implantation in rats.Results:1.The laser scanning confocal microscopy and fourier transform infrared spectroscopy showed that the surface roughness of PDMS could be changed by morphology modification without changing the chemical properties.The deeper and narrower groove structure of PDMS films meant a rougher surface,and the maximum was 50-50 groups.2.Macrophages tended to adhere and aggregate on group 50–50 surface.The microgrooves on PDMS surfaces partially enhanced the fusion of macrophages and promoted the formation of FBGCs.The deeper and narrower the microgrooves,the more pronounced this effect was.3.Each group of microgroove topographies induced macrophage polarization toward both pro-inflammatory M1 and anti-inflammatory M2 phenotypes,while the rough 50-50 surface was superior to M1 macrophages polarization and secretion of early pro-inflammatory cytokines.4.We found that the CM promoted fibroblast migration and proliferation to varying degrees as time increased.Compared to the control group,the 50–50,10–50 and 10–200groups had a stronger ability to accelerate wound healing at 12 and 24 h and proliferation at1 and 3 days.The 50–50 group had the most pronounced promotion effect with a statistically significant difference at both time points.5.Western blotting results manifested that CM from macrophages culture on PDMS differentially up-regulated the expression levels of fibrosis-related proteins in fibroblasts.Among them,group 50–50 and 10–50 had a stronger capacity for synthesizing FN,PCNA,α-SMA and COL-I.6.The obvious inflammatory reaction in the surrounding tissue was induced at 1 week after implantation in rats,but at this time histological analysis showed no significant difference in the capsule thickness of all groups except for the group 50 –50.At 4 weeks,the inflammatory response decreased,while the capsule thickness continued to increase.The rough material surface was more inclined to develop a severe fibrotic encapsulation.Conclusion:1.Each group of microgroove topographies induced macrophage polarization toward both pro-inflammatory M1 and anti-inflammatory M2 phenotypes,while the rough 50-50 surface was superior to cell adhesion and secretion of early pro-inflammatory cytokines.In addition,CM from macrophages differentially upregulated the fibroblast functions including cell adhesion,proliferation,migration and fibrotic responses.2.In vivo,we observed the thicker capsule formation around rough implants with more severe inflammatory response.Despite the stronger anti-fibrotic properties of smooth surfaces,the optimum morphological modification to meet the different clinical needs at the same time requires continuous optimization and evaluation.3.Micron-scale groove of PDMS surfaces using photolithography and polymer molding techniques is an effective way of surface modification without changing the chemical characteristics of the material itself.Our study suggested that highly modifiable microgrooves on PDMS provided a suitable platform to mediate macrophage polarization and regulate fibroblast behavior and function. |
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