Research On Murine Macrophage M2 Polarization Regulated By Micro/Nanotopography To Promote Osteogenic Differentiation And Its Application

The surface topography of the implant can effectively improve osseointegration,and macrophages play an indispensable role in the osteogenesis process.Macrophages are polarized to different phenotypes?proinflammatory M1-type or anti-inflammatory M2-type?in response to the signals from their microenvironment.The polarization direction affects osteogenesis,that is,M2 polarization is favorable for inflammation regression and bone tissue formation,and M1 polarization induces inflammatory reaction and promotes bone tissue absorption.Previous research by our research group found that the micro/nanotopography formed on the surface of pure titanium can effectively promote the phenotypic and functional changes of macrophages.However,it is not clear how the micro/nanotopography regulates macrophage polarization.In this study,we found that micro/nanotopography can induce M2 macrophage polarization and systematically explore molecular mechanism from autophagy.At the same time,we also chemically modified the micro/nanotopography,that is,dexamethasone loaded on the surface of the micro/nanotopography surface could enhance the function of M2-type polarization of macrophages,improve the osteogenic microenvironment,and promote osteogenic differentiation of mesenchymal stem cells,which provided new ideas for improving implant osseointegration.In this study,firstly,different micro/nanotopographies were constructed on the surface of pure titanium by means of acid etching and anodic oxidation.Secondly,by detecting the expression levels of polarization-related genes and proteins in macrophages,NT-30 that could induce the M2 macrophage polarization were screened out.Thirdly,molecular biological methods were used to study the molecular mechanism of NT-30 regulating the M2 macrophage polarization.Lastly,mesoporous silica nanoparticles?MSN?loaded with dexamethasone?DEX?were synthesized and deposited on the surface of NT-30 by electrophoretic deposition?EPD?.The M2 polarization promoting ability of the samples was tested,and the osteogenic differentiation of BMSC was evaluated by indirect co-culture with primary bone marrow mesenchymal stem cells?BMSC?of mice.In this study,the molecular mechanism of macrophage polarization mediated by topography was preliminarily explored,providing a new target for the directional regulation of macrophage polarization,and a new idea for the development of new targeted drugs and the design of targeted biomaterials.Some attempts were made to optimize the surface topography of the materials,which provided new ideas for the design of bone biomaterials that can inhibit inflammation and promote bone formation.Objective1.To observe the effect of different micro/nanotopography on the biological behavior of murine macrophage line RAW264.72.To explore the molecular mechanism of M2 polarization induced by micro/nanotopography of murine macrophage line RAW264.7,so as to provide theoretical basis for guiding the optimization design of material surface3.To jointly regulate macrophage M2 polarization to enhance osteogenesis of different surface topographies through the combination of topography and chemical factorsMethods1.The micro/nanotopography of TiO₂ nanotubes with different diameters was constructed by acid etching and anodization,respectively labeled as NT-30 and NT-100,and the control group P was set at the same time.The samples were characterized by field emission scanning electron microscope,atomic force microscope and contact angle measuring instruments.2.The murine macrophage cell line RAW264.7 was inoculated on the surface of different micro/nanotopography.At different time,the morphology of RAW264.7 was analyzed by field emission scanning electron microscopy and confocal microscopy.Expression of M2 polarized markers?CD206 and ARG1?and M1 macrophage markers?CCR7 and iNOS?was detected by cytometry,qRT-PCR and western blot.3.Compared with the P group,the NT-30 group was able to induce autophagy in murine RAW264.7 cells.Autophagosomes were detected by transmission electron microscopy and MDC fluorescence staining,and expression levels of autophagy related proteins?LC3I,LC3II,Beclin1?and ERK,p-ERK protein detection by western blot.Autophagy inhibitor 3-MA,autophagy activator RAPA and ERK inhibitor U0126 were used to detect autophagosomes by confocal microscopy.Western blot was used to detect the expression of autophagy-related protein and M2 polarization-specific protein Arg1.4.Mesoporsous silica nanoparticles?MSN?were synthesized by the conventional“sol-gel”method of mixing tetraethyl silicate orthosilicate?TEOS?with the template surfactant cetyltrimethylammonium bromide?CTAB?in an aqueous alkaline solution.The dexamethasone?DEX?was loaded into MSN and the release of DEX from DEX@MSN was also determined.The particles were characterized by transmission electron microscope?TEM?,scanning electron microscope?SEM?,atomic force microscope?AFM?observations and dynamic light scattering?DLS?analysis.The CCK-8 array was used to test the cytocompatibility in RAW264.7 while the qPCR was performed to measure the arginase-1?Arg1?expression,a specific marker of M2-type macrophage.Finally,the Ti implant was pre-treated with anodization under 5V to generate the titania nanotubes with³0 nm diameter?NT-30?and the DEX@MSN was introduced onto NT-30 surface via electrophoretic deposition?EPD?,with the aid of chitosan.After optimizing the deposition parameters through balancing cytocompatibility and Arg1 expression,the supernatants of RAW264.7 were collected to stimulate the murine primary bone marrow mesenchymal stem cells?BMSCs?.The osteogenic differentiation of BMSCs was evaluated by Alizarin Red staining and alkaline phosphatase?ALP?production analysis.Results1.The micro/nano composite topography of 30 nm and 100 nm nanotube arrays was successfully constructed on the surface of pure titanium.AFM roughness obtained:P group<NT-30 group<NT-100 group.The contact angle measurement results showed that the wettability and hydrophilicity of P group<NT-30 group<NT-100 group,and NT-30 and NT-100 were superhydrophilic surfaces.The overall trend of protein adsorption level was:P group<NT-30 group<NT-100 group,and the deadsorption took about 12 hours to reach the equilibrium.2.The Murine macrophage cell line RAW264.7 was cultured on the surface of micro/nanotopography and it was found that the cells adhered on P group were the most in the initial stage,followed by the NT-100 group.SEM and CLSM showed that topography could affect macrophage morphology.The surface cells of P group had the fastest proliferation,followed by NT-100 group.Both groups showed M1 phenotype?‘star'type?,while the NT-30 group has the fewest surface cells,presenting M2 phenotype?‘spindle'type?.The results of qRT-PCR,WB and FC indicated that the topography of NT-30 group had a stronger ability to promote the M2 polarization of RAW264.7,while the P group and NT-100 group promote more M1 polarization.3.Compared with the control P group,the NT-30 group was able to induce autophagy in RAW264.7 cells.Through MDC and CYTO-ID staining and TEM observation,it was confirmed that the number of autophagy vesicle?AVs?on the surface of NT-30 significantly increased.In addition,WB confirmed that LC3-II and Arg1 expression were weakened or enhanced by autophagy intervention with 3-methyladenine?3-MA?or rapamycin?RAPA?,MDC and CYTO-ID staining,and TEM detection,respectively.In addition,AVs formation and LC3-II expression were enhanced in the presence or absence of rapamycin by blocking the fusion between autophagosomes and lysosomes with BAF,but Arg1 expression was significantly decreased.Lastly,MAPK signaling pathway was detected on the surface of NT-30,and it was found that NT-30 surface selectively up-regulates ERK phosphorylation and beclin-1 expression.With the inhibition of ERK activity by U0126,the number of AVs on the surface of P group and NT-30 group was significantly decreased,and the expressions of LC3-II,beclin-1 and Arg1 were also significantly decreased.4.MSN showed a characteristic¹00 nm spherical shape with a nano-tunnel of about4 nm diameter inside.After DEX loading,the nano-tunnels were invisible while the particle size was not changed.The average drug loading of DEX@MSN was about 5.32%,and the average encapsulation efficiency was about 54.5%.DEX@MSN could consistantly release DEX in vitro and showed favorable cytocompatibility at the appropriate concentration.DEX@MSN group showed a 10-fold higher Arg1 expression compared to the untreated group.DEX@MSN and chitosan were co-deposited on the surface of NT-30,and different groups of?DEX@MSN?Chi-Ti samples were obtained with different loading times.?DEX@MSN?Chi-Ti in each group showed good cytocompatibility on day 1 and day 3.Compared with other groups,the expression of Arg1 was significantly enhanced on the surface of the?DEX@MSN?Chi-Ti 5min group by more than 5 times.In addition,macrophages of the NT-30 group or the EPD group were co-cultured with BMSCs and then alkaline phosphatase and alialiin red staining were performed.Dark blue crystals and dark red mineralized nodules were observed in each group,in which the EPD group was significantly more than the NT-30 group,and the osteogenic differentiation was significantly enhanced.Conclusion1.The topography of nanotube arrays with diameters of 30nm and 100nm was successfully constructed on the surface of pure titanium by acid etching and anodic oxidation.AFM roughness obtained:P group<NT-30 group<NT-100 group.The contact angle measurement results showed that the wettability and hydrophilicity of group P<NT-30 group<NT-100 group,and group NT-30 and group NT-100 were superhydrophilic surfaces.The overall trend of protein adsorption level was:P group<NT-30 group<NT-100 group,and the deadsorption took about 12 hours to reach the equilibrium.2.The surface of NT-30 promotes the M2 macrophages polarization,while P group and NT-100 group induced more M1-type polarization.3.NT-30 group promotes the M2-type polarization,and forms a microenvironment conducive to tissue repair.The mechanism is to promote M2 polarization through ERK pathway-mediated autophagy,to provide new insights into the mechanism of M2polarization induced by topography,and to propose new strategies to interfere with the osteogenic microenvironment by the signaling pathway.4.The MSN was used as the carrier to construct the drug delivery system,and the micro/nanotopography with DEX was combined to coordinate the macrophage polarization towards to the direction of anti-inflammatory phenotype?M2?,so as to optimize the osteogenic microenvironment.This study not only enriched the research on the delivery vector of sustained-release drugs,but also inspired us to use the relationship between immune cells and bone tissue regeneration,thus providing new ideas for the design of new biological materials.