Arg-CD-based CHG Composite Hydrogel Selectively Kills Bacteria And Regulates Immune Microenvironment For Infected Bone Repair

【Objective】Implant-associated infection is a major threat affecting the success of orthopedic surgeries.Although various materials scavenge bacteria by generating reactive oxygen species（ROS）,the intrinsic inability of ROS to distinguish bacteria from cells which will cause oxidative stress damage to surrounding healthy tissues significantly limits the therapeutic effects.Herein,we found that the arginine carbon dots（Arg-CDs）which were transformed from arginine exhibited supreme antibacterial and osteoinductive activity.We further added Arg-CDs into aldehyde hyaluronic acid/gelatin methacryloyl hydrogel（HG）to obtain Arg-CDs-containing composite hydrogel（CHG）.This thesis aims to explore the physicochemical properties and biocompatibility of Arg-CD and CHG composite hydrogels.Moreover,the antibacterial and osteoinductive activities of CHG composite hydrogels will be explored.Then,the regulatory effect of CHG composite hydrogels on ROS in cells and bacteria will also be studied.Finally,we will explore the mechanism of bone promotion of CHG composite hydrogels.【Methods】Preparation and characterizations of materials:Arg-CDs were prepared by pyrolysis.The morphology of Arg-CDs was observed by transmission electron microscope（TEM）.The particle size distribution of Arg-CDs was detected by Zeta particle size analyzer.To further explore the synthesis of Arg-CD,the light absorption of Arg-CDs was detected by UV-vis spectrophotometer.The cytoskeleton staining,live-dead staining and CCK-8 were performed to measure the biocompatibility and the promotion of cell proliferation of Arg-CD.The antibacterial and osteoinductive activities of Arg-CDs were detected by bacterial smear count test and alizarin red staining.Oxidized hyaluronic acid（HA-CHO）was obtained by treating hyaluronic acid（HA）with sodium periodate.The formation of HA-CHO was observed by Fourier transform infrared spectroscopy.Arg-CDs were added to HG and irradiated by a blue light source with a wavelength of 405 nm to obtain CHG composite hydrogels.Scanning electron microscope（SEM）was used to observe the cross-sectional morphology of CHG composite hydrogels.The mechanical strength and Arg-CD release rate of CHG composite hydrogels at p H 5.5 and p H 7.2 were measured by mechanical testing machine and UV-visible spectrophotometer,respectively.Rat bone marrow mesenchymal stem cells（BMSCs）were seeded on the surface of CHG composite hydrogels.And the adhesion and growth of cells on the surface of hydrogel were detected by cytoskeleton staining and SEM scanning.CCK-8 was performed to measure the effect of CHG composite hydrogels on the proliferation of BMSC.Characterizations of antibacterial and osteoinductive activity:Staphylococcus aureus（S.aureus）was seeded on the surface of CHG composite hydrogels,and the growth of bacteria on the surface of CHG composite hydrogels were observed by AO/EB staining and SEM.CHG composite hydrogels were placed in the center of an LB agar plate which was coated by S.aureus suspension to observe the inhibition zone around the composite hydrogels.The antibacterial activity and antibacterial kinetics of CHG composite hydrogels were detected by bacterial smear count test.BMSCs and S.aureus were simultaneously seeded on the surface of CHG composite hydrogels at the ratio of cell:bacteria=20:1.The growth of BMSCs and S.aureus on the surface of CHG composite hydrogels were observed by SEM.BMSCs and S.aureus were labeled with Di O（green fluorescence）and Di D（red fluorescence）fluorescent particles,respectively,and then seeded on the surface of CHG composite hydrogels.Using gentamicin(60μg m L^-1)to remove the bacteria outside the cells,and then digesting the cells with trypsin and reseeded in a new cell culture plate to observe the internalization and growth of bacteria inside the cells through fluorescence microscope.BMSCs and S.aureus were cultured with medium containing 100μM hydrogen peroxide（H₂O₂）.After 30 min,DCFH-DA staining was performed.The ROS level in BMSCs and S.aureus was detected by fluorescence microplate（excitation wavelength 488 nm）.After 24 h,the expression of superoxide dismutase（SOD）and catalase（CAT）in BMSCs and S.aureus were detected according to the kit.Alkaline phosphatase（ALP）staining and alizarin red staining were performed to detect the production of alkaline phosphatase and calcium nodules during the osteogenesis of BMSCs induced by CHG composite hydrogel in vitro.The expression of antioxidant and osteogenic related genes and proteins was detected by PCR and Western blot.CHG composite hydrogels were implanted into the infected femoral condylar defects of rats.At 4 and 8 weeks after operation,the regenerated bone tissue in the bone defect was observed by Micro-CT,H&E staining and Masson staining,and the bacteria in the bone defects were observed and analyzed by Giemsa staining.Exploration of osteogenesis mechanism:BMSCs were treated with CHG composite hydrogels in vitro,and then the osteogenic mechanism of CHG composite hydrogels was explored by RNA sequencing.Bone marrow mononuclear cells（BMMs）were cultured using BMSC conditional medium.The expression of Interleukin-10（IL-10）in BMSC was measured by Elisa kit,and the expression of CD206 in BMMs was upregulated by conditioned medium which have treated BMSC with CHG complex hydrogel.Further,mechanism mining was conducted to explore how CHG composite hydrogels up-regulated the expression of IL-10 in BMSC to promote osteogenesis.【Results】Preparation and characterizations of materials:Arginine can be converted into Arg-CDs by pyrolysis,the obtained Arg-CDs showed grid structure.The particle size of Arg-CDs was uniform and about 7 nm.Arg-CD showed a significant UV-visible absorption peak at the wavelength of 220 nm and 305 nm.BMSCs which were cultured with the medium containing Arg-CD（500μg/m L）showed good activity and could adhere to the bottom of the culture dish.In addition,Arg-CD significantly promoted the proliferation of BMSC.Moreover,the bacterial smear count test shows that Arg-CD effectively inhibited the activity of S.aureus and exhibited excellent antibacterial properties.The results of alizarin red staining show that Arg-CD promoted the production of calcium deposition on the surface of BMSC,promoting BMSC osteogenic differentiation and exhibiting good osteoinductive activity.Arg-CDs were added to HG and irradiated by a blue light source with a wavelength of 405 nm to obtain CHG composite hydrogels.The CHG composite hydrogels showed an obvious interconnected microporous structure,but the microporous structure was collapsed at p H 5.5 medium,and the mechanical strength also decreased,indicating that the Schiff-base bond in the CHG composite hydrogels was ruptured in response to the acidic microenvironment.The results of Arg-CD release curve also show that the release rate of Arg-CDs in CHG composite hydrogels was significantly faster at p H 5.5 medium than that at p H 7.2 medium,which once again proved that the Schiff-base bond in CHG composite hydrogels was ruptured in response to the acidic microenvironment to intelligently release Arg-CDs.BMSCs were seeded on the surface of CHG composite hydrogels.BMSCs adhered to and grew well on the surface of composite hydrogels,indicating that CHG composite hydrogels showed good biocompatibility.Moreover,CHG composite hydrogels significantly promoted the proliferation of BMSCs,which could be used as a good biomaterial for tissue engineering.Characterizations of antibacterial and osteoinductive activity:S.aureus was seeded on the surface of CHG composite hydrogels,and the AO/EB staining results show that S.aureus seeded on the surface of CHG composite hydrogels was significantly killed.SEM results show that there were a handful of S.aureus growing on the surface of CHG composite hydrogels,and the cell membranes of S.aureus were destroyed,showing a shrunken shape.CHG composite hydrogels were placed in the center of an LB agar plate which was coated S.aureus suspension to observe the inhibition zone.The results suggest that S.aureus could not grow close to the CHG composite hydrogels,thus forming an obvious inhibition zone around the CHG composite hydrogels.The results of the bacterial smear count test show that the CHG composite hydrogels significantly inhibited the activity of S.aureus,and the bacterial killing activity was time-dependent.After 24 h,CHG composite hydrogels could kill more than 80%of the bacteria.The above results not only show that the CHG composite hydrogels exhibited a good contact antibacterial effect,its non-contact antibacterial effect was also powerful.After tissue infection,bacteria and cells often grow together in the body.In order to simulate the infected microenvironment after bone injury,BMSCs and S.aureus were simultaneously seeded on the surface of CHG composite hydrogels.SEM images show that BMSCs adhered to and grew well on the surface of CHG composite hydrogels,and a handful of S.aureus was found on the surface of BMSCs,showing good cellular activity.To further test the antibacterial activity of CHG composite hydrogels,the results of fluorescence microscope scanning show that S.aureus could not internalize into BMSCs after the treatment of CHG composite hydrogels,which indicated that CHG composite hydrogels could prevent the occurrence of chronic infection.Further detection of the ROS level in BMSCs and S.aureus in the co-culture system shows that the CHG composite hydrogels significantly up-regulated the ROS level in S.aureus and while inhibiting the expression of antioxidant enzymes.Thus,S.aureus was unable to eliminate excessive intracellular ROS and was more vulnerable to oxidant-induced damage caused by CHG composite hydrogels.Therefore,CHG composite hydrogels showed good antibacterial activity.Unlike S.aureus,the CHG composite hydrogels up-regulated the expression of antioxidant enzymes in BMSC.Moreover,CHG composite hydrogels promoted the expression of antioxidant genes and proteins,thus protecting cells from oxidative stress damage induced by infectious bone injury.The in vitro osteoinductive activity of CHG composite hydrogels indicates that CHG composite hydrogels significantly promoted the expression of alkaline phosphatase in BMSCs,and a large number of calcium nodules were generated on the surface of BMSCs.Furthermore,the CHG composite hydrogels significantly up-regulated the expression of osteogenic related genes and proteins,showing a good osteoinductive activity in vitro.The CHG composite hydrogels were implanted into the site of the infectious femoral condyle defects of rats,and samples were collected at 4 and 8 weeks after the operation.The results of Micro-CT scanning show that the CHG composite hydrogels significantly promoted bone regeneration at the defect site.The results of H&E and Masson staining also show that the CHG composite hydrogels effectively promoted bone formation of infected bone injury.Giemsa staining indicated that CHG composite hydrogels significantly eliminated S.aureus and could be used as an ideal scaffold for the repair of infectious bone injury.Exploration of osteogenesis mechanism:To explore the osteogenic mechanism of CHG composite hydrogels,RNA sequencing results show that CHG composite hydrogels significantly up-regulated the expression of Il10.The BMSC conditional medium could up-regulate the expression of CD206 in BMMs,effectively promoting the M2 macrophage polarization.Furthermore,CHG composite hydrogels could up-regulate the expression of Il10 through TLR/JAK-STAT-c-Maf axis,thus promoting the M2 macrophage polarization and improving the immune microenvironment of infected bone injury for the promotion of bone regeneration.【Conclusions】（1）Arg-CDs are successfully prepared by pyrolysis,and the obtained Arg-CD shows grid tructure.CHG composite hydrogels are obtained by adding Arg-CDs into hydrogels. CHG composite hydrogels are response to acidic microenvironment and intelligently release Arg-CDs,showing excellent biocompatibility,antibacterial and osteoinductive activity.（2）When cells and bacteria are simultaneously seeded on the surface of CHG composite ydrogels,CHG composite hydrogels significantly inhibit the expression of antioxidant nzymes in bacteria,and produce excessive ROS to effectively resist bacteria.However,CHG composite hydrogels up-regulate the expression of antioxidant enzymes in cells, cavenging ROS and promoting cell adhesion and growth.Moreover,CHG composite ydrogels up-regulate the expression of antioxidant genes and proteins in cells and rotect cells from oxidative stress damage,showing excellent osteogenic effect for bone egeneration.（3）CHG composite hydrogels significantly promote the repair of infected bone injury,up- egulating the expression of Il10 through TLR/JAK-STAT-c-Maf axis.Therefore,CHG omposite hydrogels promote the M2 macrophage polarization,improving the immune njury microenvironment and promoting bone regeneration.