| Background:Open reduction and internal fixation are important strategies for unstable long bone fracture treatment,which can effectively stabilize the fracture and create stable condition for fracture healing.However,for patients with osteoporosis and bone metabolism abnormality,even under stable condition,there are still risks of fracture nonunion or delayed union.In addition,implant-related infection is also an important and intractable clinical problem.The emergence of antibiotic-resistant bacteria leads to difficult control and prolonged infection.One of the important reasons is that bacteria are prone to form biofilm on the surface of internal implant,and with the extension of time,the sensitivity of drug-resistant bacteria in biofilm to antibiotics decreases gradually,which brings serious difficulties to treatment.Therefore,how to promote fracture healing and control implant-related infection are two major medical problems in clinical practice,and the application of internal fixation coating brings hope to solve these problems.However,the common internal fixation coatings only have single function of promoting fracture healing or antibacterial activity,and few coatings have multiple functions mentioned above simultaneously.Therefore,is it possible to use biological materials to prepare new coatings so that the implant can simultaneously have dual functions of fighting bacterial biofilm and promoting new bone formation and fracture healing? The emergence of a new two-dimensional nano material named black phosphorus nanosheet(BPs)provides us with a new idea to solve the above problems.Similar to other mature two-dimensional materials,BPs has good photothermal conversion effect and photodynamic effect,and has the photothermal antibacterial potential of two-dimensional materials such as graphene and graphene oxide(GO).In addition,previous studies had proved that BPs was gradually oxidized to form phosphate in the physiological environment,which can start the biological mineralization of "phosphorus-driven,calcium extraction",and phosphorus ion can promote the proliferation and osteogenic differentiation of BMSCs,and promote fracture healing.Therefore,this study assumed that BPs can be loaded on the surface of internal fixation to form coating by special technique,and BPs can be brought to the fracture site with the help of the implant,so as to destroy bacteria biofilm and promote fracture healing by above mechanisms.Firstly,BPs hydroxyapatite composite coating was prepared by vacuum plasma spraying technique and layer-by-layer self-assembly technique,and the photothermal conversion performance and degradability of the coating were verified.The antibacterial effect of the coating was tested in vivo and in vitro.The cell compatibility,biological activity and biosafety of BPs hydroxyapatite composite coating were observed and analyzed by cell experiments and animal experiments,to provide a theoretical basis for potential clinical application value.Objective:1.To explore the preparation methods of BPs hydroxyapatite composite coating,and verify whether the coating was successful prepared or not.To characterize and analyze the coating,and study the in vitro photothermal conversion performance and degradation performance of BPs hydroxyapatite composite coating.2.To investigate the anti-bacterial biofilm effect of BPs hydroxyapatite composite coating through in vitro and in vivo experiments on the basis of the successful preparation of BPs hydroxyapatite composite coating.3.To study the biocompatibility of BPs hydroxyapatite composite coating and its effect on the proliferation and osteogenesis differentiation of BMSCs.4.To evaluate the effect of BPs hydroxyapatite composite coating on the healing effect of long bone fracture and to test its biosafety based on in vivo experiment.Methods:1.The vacuum plasma spraying technique was used to spray hydroxyapatite on the surface of titanium alloy and Kirschner wire to form hydroxyapatite coating,then the hydroxyapatite coating reacted with APTES,grafted amino groups on the coating surface to make the coating surface positively charged.The layer-by-layer self-assembly technique was used to load BPs on the hydroxyapatite coating surface through electrostatic adsorption to form BPs hydroxyapatite composite coating.The morphology and particle size of BPs were observed and detected by transmission electron microscope(TEM),the morphology and thickness of BPs were observed by atomic force microscope(AFM).Fourier transform infrared spectrometer(FTIR)was used to test whether amide reaction was successful or not.The phase composition of the coating was analyzed by X-ray diffraction(XRD),and the composition of the coating was analyzed by Raman spectroscopy,scanning electron microscope(SEM)was used to observe the surface characteristics,element composition and content of the coating.The bonding strength between the coating and the substrate was tested by ultrasonic oscillation method.The photothermal conversion property of different coatings was tested by near-infrared light irradiation,and the degradability of BPs hydroxyapatite composite coating in simulated body fluid was tested.2.According to the purposes of the experiments,Staphylococcus aureus and Escherichia coli were used as test strains for in vivo and in vitro antibacterial experiments.In vitro photothermal antibacterial effect of BPs hydroxyapatite composite coating was explored by plate colony counting method,live/dead bacterial fluorescence staining,and PI positive rate was explored by flow cytometry.The morphology of bacteria in each group was observed and analyzed by scanning electron microscopy and transmission electron microscopy to evaluate the damage of bacteria caused by photothermal antibacterial effect and explore the damage mechanism.Three kinds of titanium rods(uncoated titanium rods,hydroxyapatite coated titanium rods,and BPs hydroxyapatite coated titanium rods)were implanted into the tibial condyle defect infection model of SD rats,and the antibacterial effect of BPs coated titanium rods was evaluated by plate counting and bone histopathological examination.Pathological examination was performed on heart,liver,spleen,lung,kidney,and other tissues of the rats after operation to evaluate the biosafety of the material.3.In vitro bioactivity evaluation: BMSCs from SD rat was selected for in vitro cell test.According to the experimental purposes,the experimental groups were divided into blank control group(Control),pure osteogenic induction group(Induce),titanium tablet group(Induce+Ti),hydroxyapatite titanium tablet group(Induce+HA@Ti),and black phosphorus nanosheet hydroxyapatite coated titanium tablet group(Induce+BPs@HA@Ti).BMSCs were co-cultured with the materials,and their compatibility in vitro was detected by CCK-8 test,live/dead staining,cell immunofluorescence staining,and cell proliferation activity was detected by Ed U staining at 1,3,and 5 days after inoculation.ALP activity was detected by ALP staining and activity kit 7 days after osteogenic induction.Alizarin red-S staining and quantitative analysis of mineralized calcium nodules were performed on cells 21 days after osteogenic induction to evaluate the osteogenic differentiation of cells in each group.RT-PCR and Western Blot were used to explore the expression of osteogenic genes in each group.4.In vivo evaluation: 60 male SD rats were selected to prepare the model of femoral closed fracture,and three kinds of Kirschner wires were used for internal fixation of fracture,respectively: blank Kirschner wires,hydroxyapatite coated Kirschner wires and BPs hydroxyapatite coated Kirschner wires.Four weeks and eight weeks after the operation were selected as the time points to evaluate the fracture union effect.Micro-CT and three-dimensional reconstruction were used to evaluate the fracture union.Tetracycline,alizarin red and calcein fluorescence staining of bone tissue was used to explore the formation of new bone,and Van-Gieson staining was used to evaluate the microscopic effect of fracture union.The biomechanical strength of each group was explored by three points bending test to evaluate the degree of bone healing indirectly.Blood routine,liver and kidney function and electrolyte test were performed at 3 and 6weeks after surgery,respectively,to evaluate the biosafety of BPs hydroxyapatite composite coating.Results:1.Fourier Transform infrared spectroscopy(FTIR)verified that the amide reaction was successful.XRD,Raman spectroscopy and SEM confirmed that the BPs hydroxyapatite composite coating was successfully prepared,and the bonding strength between the coating and the substrate was good.With the increase of BPs concentration,the amount of BPs loaded on the hydroxyapatite coating increased gradually,and there was significant statistical difference in the content of BPs among all groups(P<0.05).BPs had good photothermal conversion performance.With the increase of BPs concentration,the photothermal conversion effect of coating in each group gradually increased,and there were significant statistical differences among groups(P<0.05).BPs could be gradually degraded to phosphate ions in simulated body fluids,and the concentration of phosphate ions was gradually increased with the increase of BPs concentration(P<0.05).2.In vitro antibacterial experiments confirmed that BPs hydroxyapatite composite coating had good photothermal antibacterial effect,compared with the material without BPs hydroxyapatite composite coating,the antibacterial rate had significant statistical difference(P<0.05).With the increase of BPs content,the antibacterial rate increased gradually(P<0.05).In vivo antibacterial experiment also confirmed that BPs hydroxyapatite composite coating had good antibacterial effect.Pathological examination showed that bone tissue without BPs hydroxyapatite composite coating had a large number of inflammatory cell infiltration,while bone tissue around BPs hydroxyapatite composite coating had no obvious inflammatory cell infiltration.There were no obvious pathological changes in heart,liver,spleen,lung,and kidney.3.CCK-8 test,cell death and live staining and Ed U staining were used to explore the biocompatibility of coatings and the cell proliferation ability of cells in different groups.The results showed that the number of BMSCs in BPs hydroxyapatite composite coating group was more than that in other groups(P<0.05).Cell immunofluorescence staining showed that the cell morphology of each group was good.ALP staining showed that the cells in BPs hydroxyapatite composite coating group stained deeper,and the OD value was higher investigated by ALP activity quantitative(P<0.05).Alizarin Red-S staining and quantitative results showed that the formation of calcium nodules in BPs hydroxyapatite coated group were significantly more than that in other groups,and the OD value was also higher than that in other groups(P<0.05).RT-PCR results showed that there was no significant difference in the expression of osteogenic transcription factors Osterix,ALP,OPN and Runx2 in BMSCs of BPS hydroxyapatite coated group after 4 days of osteogenic induction(P>0.05),but there was significant difference in the expression of osteogenic transcription factors Osterix,ALP,OPN and Runx2 after 7 days of osteogenic induction,the expression of the above four genes in BPs@HA@Ti group was significantly higher than that in the other four groups(P<0.05).The expression of osteocalcin(OCN)and type I collagen(Col I)genes related to osteogenic differentiation after 7 and 14 days of osteogenic induction in BPs@HA@Ti group was significantly higher than that of the other four groups(P<0.05).The results of Western blot were similar to those of RT-PCR,the osteogenesis related genes and protein expressed in the BPs@HA@Ti group was more obviously.4.Micro-CT and three-dimensional reconstruction showed that the fracture healing effect of BPs hydroxyapatite coated Kirschner wire group was better than that of other groups at 4 and 8 weeks after operation;The results of bone tissue fluorescence staining showed that the bone mineral apposition rate of BPs hydroxyapatite coated Kirschner wire group was higher than that of the other two groups(P<0.05);The biomechanical results showed that the maximum stress of femoral specimens in BPs hydroxyapatite coated Kirschner wire group was better than that in the other two groups at 4 and 8weeks after operation(P<0.05).Staining results were similar to Micro-CT results,the fracture healing degree of BPs hydroxyapatite coated Kirschner wire group was better than that of the other two groups.There was no significant difference in blood routine,liver and kidney function and electrolyte between rats underwent operation and the non-operated rats three weeks and six weeks after the operation(P>0.05).Conclusions:1.BPs hydroxyapatite composite coating could be successfully prepared by plasma spraying technology and self-assembly technique.BPs hydroxyapatite composite coating had good bonding strength with substrate,good photothermal conversion performance and degradation performance.2.BPs hydroxyapatite composite coating had good photothermal antibacterial property,which could effectively remove the bacterial biofilm attached on the internal fixation surface.3.In vitro cytological test showed that BPs hydroxyapatite composite coating not only had good biocompatibility,but also enhanced BMSCs proliferation and osteogenic differentiation.4.BPs hydroxyapatite composite coating had good effect on fracture healing and biological safety,and had certain clinical application potential. |
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