| Background and objectiveBone related infection causes excessive inflammatory response,progressive osteolysis,and continuous bacterial load,which is detrimental to the bone healing.Due to the lack of timely and effective treatment and difficulties in transporting the wounded,the officers and soldiers in overseas bases and oceangoing ships suffer from high disability and mortality,which seriously affects the smooth conduct of combat and training tasks.In addition,in the harsh environment of the front line,open fracture is prone to infection,which brings great pain to the wounded.Traditional antibiotics can reduce bacterial load,however,fail to inhibit bone resorption and inflammatory response.In order to improve bone healing during bacterial infection,the ideal therapeutic approach should not only clear the bacteria but also inhibit inflammation and osteolysis.In this study,we fabricated a multi-biofunctional coating,named as MSNs-BMP4-EN,by covalently immobilizing mesoporous silica nanoparticles(MSNs)with human bone morphogenetic protein 4(BMP4)and loading them with enoxacin(EN)for antibacterial,inflammation and osteolysis inhibition.Antimicrobial assays demonstrated that MSNs-BMP4-EN significantly inhibited bacterial adhesion and colonization,while further in vitro assays showed that MSNs-BMP4-EN could facilitate bone mesenchymal stem cells osteogenic differentiation and prevent early osteoclastogenesis.We also found that MSNs-BMP4-EN could effectively inhibit the secretion of inflammatory factors,thereby reversing the inflammatory microenvironment triggered by infection and facilitating bone healing.Together,these findings suggest that MSNs-BMP4-EN exhibit excellent antibacterial and anti-inflammatory properties and inhibits bone resorption in the early stages of bonerelated infection,thereby further improves bone healing.Consequently,this bioactive nanocomposite has excellent application prospects for treating bone infections.MethodsMesoporous silica nanoparticles(MSNs)were prepared by sol-gel method.Bone morphogenetic protein 4(BMP4)was covalently grafted on the surface of ammoniated MSNS and enoxacin(EN)was loaded in the pores of MSNS.Scanning electron microscope(SEM)and transmission electron microscope(TEM)were used to observe the surface structure of the coating.After dispersing in anhydrous ethanol under sonication,the particle size of nanoparticles was measured using Particle Size Analyzer instrument.To study the drug release ability of MSNs-BMP4-EN by drug release experiment.The minimum inhibitory concentration(MIC)of MSNs-BMP4-EN was determined by in vitro antibacterial experiment and the growth curve of MSNs-BMP4-EN against S.aureus was recorded.The antibacterial property of MSNS-BMP4-EN in vitro was evaluated by bacterial activity kit,spread plate method(SPM)and live/dead staining assay.The effect of nanoparticles on the proliferation of rat bone marrow mesenchymal stem cells(r BMSCs)was investigated by cell counting kit-8(CCK-8)assay in vitro.The effects of MSNsBMP4-EN on the osteogenic differentiation of r BMSCs were studied by alkaline phosphatase(ALP)staining and quantitative experiments,collagen staining and quantitative experiments,Alizarin Red S(ARS)staining and quantitative experiments,and quantitative analysis of osteogenic differentiation-related gene RNA and western blot analysis.Tartrate-resistant phosphatase(TRAP)staining,quantitative analysis of RNA related to osteoclast differentiation and bone resorption assay were used to evaluate the effect of MSNs-BMP4-EN on osteoclast differentiation.Sanger sequencing and enzymelinked immunosorbent assay(ELISA)were used to evaluate the effect of MSNs-BMP4-EN on local inflammatory immunity in the infection state.The anti-infection and osteogenesis ability of MSNs-BMP4-EN in vivo were evaluated by micro-CT analysis,microbiological analysis,tissue sections and Van Gieson staining.ResultsThe nanoparticles were analyzed using SEM and TEM,which confirmed the different nanomaterials have a uniform spherical shape with a size of about 50 nm and many uniformly distributed holes on the surface.Particle Size Analyzer instrument demonstrated that the nanoparticles formed particle clusters of 210.7 nm,220.7 nm and 187.4 nm in absolute ethanol,respectively.Enoxacin has a characteristic peak at the absorbance of286 nm and a good linear relationship between concentration and absorbance.The encapsulation efficiency and drug loading were 43.25% and 30.10%,respectively.The drug release assay showed that MSNs-BMP4-EN had a high drug release rate and sustained release for 5 days after the burst release of enoxacin within 12 hours.The cumulative release rate of enoxacin was 67.4% at day 5.MSNs-BMP4-EN significantly inhibited the adhesion and proliferation of S.aureus in vitro.In vitro cytotoxicity experiments and osteogenic differentiation related experiments showed that MSNs-BMP4-EN could promote the proliferation of r BMSCs at early and late stages of osteogenic differentiation.In vitro osteoclast differentiation related experiments showed that MSNsBMP4-EN could prevent the formation of early osteoclasts and inhibit osteolysis.In vitro inflammation-related experiments showed that MSNs-BMP4-EN could inhibit the secretion of inflammatory factors in mouse monocyte-macrophage leukemia cells(RAW264.7),thereby changing the immune microenvironment and promoting osteogenic differentiation and extracellular matrix mineralization.In vivo experiments showed that MSNs-BMP4-EN could effectively inhibit infection around the femur in rats,promote bone formation,reduce bone resorption and inhibit inflammation caused by infection.ConclusionMSNs-BMP4-EN has the advantages of simple preparation,low cost,high drug loading,long sustained release performance and good biocompatibility.These experiments demonstrated that MSNs-BMP4-EN possesses excellent antibacterial and antiinflammatory properties and can inhibit bone resorption at the early stage of implant infection,thereby further promoting bone healing.Therefore,this nanocomposite with multiple biological functions has good clinical application prospects in the treatment of bone infections. |
