| Part ? Preparation and detection of Silk Fibroin-Silver Nanoparticles scaffoldObjective:Chronic osteomyelitis is a common clinical disease with long treatment cycle,easy recurrence,poor curative effect and high disability rate.In recent years,the increasing number of multidrug-resistant strains(especially Methicillin-resistant S.aureus,MRSA)makes the treatment of chronic osteomyelitis more difficult.In order to overcome the drawbacks of existing materials(PMMA),it is an urgent task to develop new materials with biodegradable absorption,better biocompatibility and antibacterial properties for the treatment of chronic osteomyelitis.Therefore,we have tried a new process to prepare Silk Fibroin-Silver Nanoparticles(SF-AgNPs)scaffold for the study of chronic osteomyelitis.Methods:Silk fibroin scaffolds with different concentrations(0,0.1%,0.5%and 1%)of silver nanoparticles(AgNPs)were prepared by in situ direct reduction of silver ions to form AgNPs according to the double reduction of silk protein and formic acid.The morphology,structure and properties of the prepared materials were characterized by SEM,EDS,IR and XRD.Results:Scanning electron microscopy showed that silk fibroin scaffolds with different concentrations of AgNPs had homogeneous porous interconnection structure,and the pore size was about 130?460?m.AgNPs could be dispersed uniformly on the surface of silk fibroin scaffolds.EDX spectra clearly confirm the existence of silver on the surface of materials.Infrared spectrum analysis showed that AgNPs had no effect on the(acyl)amino bonds of silk protein scaffolds,and formic acid and silver nitrate used in the preparation process did not destroy the structure of silk fibroin.X-ray diffraction further confirmed that the scaffolds were porous SF-AgNPs compounds with stable crystal structure.Conclusion:Silk fibroin porous scaffolds loaded with AgNPs can be prepared by double reduction of silk protein and formic acid.The preparation process is simple and efficient.Characterization tests showed that the loaded AgNPs did not destroy the tissue structure of silk fibroin scaffolds,and AgNPs could be more uniformly dispersed on the surface of silk fibroin materials.Part II Cytotoxicity of SF-AgNPs scaffoldObjective:To verify the biocompatibility of SF-AgNPs scaffold,osteoblasts(MC-3T3)were selected as the research object for cytotoxicity test in vitro.The activity and biological response of osteoblasts on silk fibroin scaffolds with different concentrations of AgNPs were systematically studied.Methods:The growth of MC-3T3 cells on silk fibroin scaffolds containing different concentrations of AgNPs was observed by laser confocal microscopy.The cytotoxicity of scaffold materials was detected by indirect test based on ISO 10993-5 standard.CCK-8 proliferation assay and ALP/Runx2 activity protein assay were performed in MC-3T3 cells treated with supernatant after immersion in scaffolds at different time intervals.Results:Confocal laser scanning showed that silk fibroin scaffolds containing different concentrations of AgNPs could support the adhesion and growth of osteoblasts.Osteoblasts grow orderly along the outline of porous scaffolds,and most of them are elongated cells.CCK-8 assay showed that silk fibroin scaffolds with 1%AgNPs significantly inhibited the proliferation of MC-3T3 cells after 7(relative proliferation rate 90.5±2.7%)and 10 days(88.9±1.2%)of immersion.In addition,silk fibroin scaffolds with 1%AgNPs showed inhibition of ALP and Runx2 activity after immersion for 10 days.Conclusion:SF-AgNPs scaffold could support osteoblast adhesion and growth.The silk fibroin scaffolds with low concentrations(0.1%and 0.5%)of AgNPs showed good biocompatibility.Part III Study on antibacterial activity of SF-AgNPs scaffold for MRSAObjective:This part of the study was designed to verify the antibacterial properties of SF-AgNPs scaffold against MRSA in vitro.Methods:Kirby-Bauer method and growth curve method were used to detect the antibacterial effect of silk fibroin scaffolds with different concentrations of AgNPs on MRSA.Results:The inhibitory zone showed that the silk fibroin scaffold with 1%AgNPs had a 13.2 mm regional inhibitory range,showing more obvious antibacterial properties.The growth curve test showed that the inhibitory rates of silk fibroin scaffolds with 1%and 0.5%AgNPs were 77.5%and 72.3%,respectively.Compared with silk fibroin scaffolds with 0.1%AgNPs(51.8%),the antibacterial effect was obvious.The antibacterial diameters of SF+gentamicin group were 16.8 mm,and those of different concentrations AgNPs(0.1%,0.5%and 1%)-SF+gentamicin were 17,4 mm,18.4 mm and 24 mm respectively.Conclusion:The antibacterial effect of SF-AgNPs on MRSA has obvious dose-dependent of AgNPs concentration.Silk fibroin scaffolds of 0.5%and 1%AgNPs had obvious antibacterial effects on MRSA.Gentamicin has a strong killing effect on ATCC43300 strain.AgNPs and gentamycin had a superposition effect on the antibacterial effect of MRSA.Part IVStudy on the treatment of chronic osteomyelitis model induced by MRSA in rats with SF-AgNPs scaffold loaded with gentamycinObjective:To verify the efficacy of SF-AgNPs scaffold as filling materials for chronic osteomyelitis,we designed SF-AgNPs scaffold loaded with gentamicin in a rat model of chronic osteomyelitis induced by MRSA in order to explore new filling materials and drug carriers for efficient treatment of chronic osteomyelitis.Methods:According to the results of the previous three parts,we finally selected 0.5%SF-AgNPs scaffold for animal model experiment in vivo.Rats in the proximal tibia were chosen to undergo chronic osteomyelitis,then debridement and implantation of scaffold materials were performed.The experiment was divided into five groups:1.blank control group(after debridement);2.silk fibroin scaffold group;3.silk fibroin scaffold+gentamicin group;4.SF-AgNPs scaffold group;5.SF-AgNPs scaffold+gentamicin group.The therapeutic effect of the scaffold was evaluated comprehensively from general condition,Radiology,histology and microbiology.Results:After successful modeling of chronic osteomyelitis in all rats,body weight and appetite decreased significantly,and even white blood cell count increased significantly.After debridement and scaffold implantation,the nutritional status of rats improved significantly(body weight gradually increased),and the white blood cell count gradually returned to normal range.The osteolytic cavity enlargement accompanied with bone mineral density decrease,cortical bone destruction and bone formation were observed in the blank control group and the silk fibroin scaffold group.HE staining also showed a large number of infiltration of neutrophils and macrophages,even abscess formation and osteocyte necrosis and apoptosis in the blank control group and silk fibroin scaffold group;SF-AgNPs scaffold group showed a significant reduction in inflammation and abscess,but still a small amount of inflammatory cell infiltration,accompanied by periosteal reaction.Inflammation and abscess in gentamicin treatment group were evidently subsided,with subperiosteal fibrosis and angiogenesis.Microbiological examination showed that gentamicin loaded scaffolds could significantly kill MRSA bacteria in osteomyelitis.The bacterial load of silk fibroin scaffold+gentamicin group(4×103CFU/g)and SF-AgNPs scaffold+gentamicin group(3.5×103CFU/g)was significantly lower than that of other groups(P<0.01).In addition,the SF-AgNPs scaffold group(6.16×105CFU/g)was significantly different from the blank control group(5.14×106CFU/g)and the silk fibroin scaffold group(5.85×106CFU/g)(P<0.01).The results showed that SF-AgNPs scaffold material could inhibit the growth of MRSA in the tibial bone marrow of rats.Conclusion:SF-AgNPs scaffold loaded with gentamycin are effective in the treatment of chronic osteomyelitis.SF-AgNPs scaffold could be used as a potential filling material and drug delivery platform for the treatment of chronic osteomyelitis. |
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