Study Of Slow-release And Antibacterial Properties Of Nano-silver In Bone Cements

【Background】In implantation,a variety of reasons (alveolar bone atrophy after exodontia,trauma, infection, cancer, birth defects, etc.) can cause lack of bone mass in dentalimplants implanted area,which will affect the implant and long-term effect.Implanted bone substitute materials become the essential means to solve this problem,but how to control for postoperative infection is a growing difficulty andhotspot in the treatment of implantation. Currently,widely research of antibioticssuch as vancomycin, tetracycline, levofloxacin due to load onto the bone technologiessuch as bacterial drug resistance and antibiotic release duration is short, but failed toachieve the goal of the ideal control bone infection. Silver, zinc, copper and otherinorganic antibacterial agent doping into bone powder materials can effectively inhibitbacterial infections, but its toxicity to mammalian cells become concerns in theapplication, and how to make its sustained and effective work is not very good solve.In addition, how to effectively use nanotechnology to delay the release ofantimicrobial agent still need further discussion.【Aim】This study try to use beta-cyclodextrin (β-CD) and chitosan (CS) as ligandsynthesis of silver nanoparticles, then different ligands of silver nanoparticles (NP)load in the artificial bone, combined the technology of emulsification, Polylacticacid hydroxy acetic acid(PLGA), PLGA outside the package made antibacterial bonebiomaterial to delay the release of inorganic nano silver. This experiment use thecharacteristics of cyclodextrin, chitosan to cover silver nanoparticles and enhance itsantimicrobial properties in different levels, and nano silver hydroxyapatite wrappedby Polylactic acid hydroxy acetic acid(PLGA),made into bone powder materialcontrol the infection, and slow release of silver nanoparticles. Through theexperiment, hopely can get sustained release of nano silver, until autologous boneformation, achieve the objective of the ideal control bone infection.【Methods and results】1.Preparation and properties of CD-NP Reference literature[1]in the synthesis of silver nanoparticles synthesis nano silverof β-CD ligand, the method is: different concentrations of beta-CD aqueoussolution as a ligand, sodium borohydride as reducts, reduct silver nitrate aqueoussolution to CD-NP. By scanning electron microscopy (sem), transmission electronmicroscopy (sem) observate CD-NPs’ surface morphology, particle diameter andparticle size of aggregate, the stability of the observation period.Spectrophotometerobservate CD-NPs’ absorption peak.Results：As reported in literature[1], with cyclodextrin concentration increased, theobtained nano-silver antibacterial properties enhanced.so that20mM β-CD as ligands,and2mM silver nitrate solution, stirring after20minutes, add sodium borohydridesolution of20mM, get dark solution，30s later, brown solution was formed，after24hs’ stiring, can obtain good stable nano-silver. Electron microscopy (sem)results showed that nano-silver particles into a round shape, particle size, between24to33nm are uniformly distributed and no obvious aggregation and test antimicrobialproperties of different concentrations of silver nanoparticles.. Ultraviolet absorptionspectra showed that CD-NP absorption peak at410nm. Bacteria experiment showedthat chitosan ligands nano-silver have obvious antimicrobial properties, and alongwith the increased concentration of silver nanoparticles, antimicrobial propertiesenhanced.2.Preparation and properties of CS-NPReference literature[2],chitosan as ligand in the synthesis of silver nanoparticles.The method is using chitosan as a ligand and reductant, Sodium hydroxide andacetic acid as adjusts to adjust PH value, obtained CS-NP. CS-NP soluble in0.1%acetic acid solution by scanning electron microscopy (sem) and transmission electronmicroscopy (sem) observate of CS-NP surface morphology, particle diameter andparticle size of aggregate, the stability of CS-NP and test antimicrobial propertiesof different concentrations of silver nanoparticles. Spectrophotometer observateCS-NPs’ absorption peak.Results showed that chitosan(degree>75%) is soluble in1%acetic acidsolution, the chitosan solution was1mg/ml, heating up to45℃and stir until completely dissolved to form chitosan homogeneous solution,24ml chitosanhomogeneous fluid mixed with1ml100mM silver nitrate solution,and add75ml of0.1M sodium hydroxide solution slowly to the mixture, obtaining brown solution.Electron microscopy (sem) results show that CS-NP size ovoid, particle size between5to25nm, have gathered phenomenon, particle size distribution is not uniform.Ultraviolet absorption spectra showed that CS-NP absorption peak at400nm.Bacteria experiment showed that chitosan ligands nano-silver have obviousantimicrobial properties, and along with the increased concentration of silvernanoparticles, antimicrobial properties enhanced.3.Preparation and properties of CD-NP-HA/CS-NP-HAMethods:Different concentration of CD-NP/CS-NP load into the hydroxyapatite bylyophilization.By scanning electron microscopy (sem) and transmission electronmicroscopy (sem) observate CD-NP-HA/CS-NP-HA ‘surface morphology,particle diameter and particle size of aggregate, stability of the observation period,and test antimicrobial properties of hydroxyapatite loading silver nanoparticles indifferent ligand and different concentration.The nano-silver hydroxyapatiteimmersing in simulated body fluid, test slow-release performancein vitro.Results showed that:0.1umol,0.2umol,0.5umol,1.0umol,2.0umol,4.0umol,10.0umol CD-NP to join100mg hydroxyapatite (10mg/ml) in aqueous solution,lyophilization after stirring4hours.0.1umol,0.2umol,0.5umol,1.0umol,2.0umol,4.0umol,10.0umol CS-NP to100mg of hydroxy apatite (10mg/ml) inaqueous solution, lyophilization after stirring4hours. After lyophilizationhydroxyapatite loading nano-silver is into white powder. Electron microscopy(sem) results showed that CD-NP-HA/CS-NP-HA into granular size, particle sizein9nm-60nm, particle aggregation, stability is good. Bacteria experiment resultsshow that the load0.1umol CD-NP-HA/CS-NP-HA have antimicrobial properties.Prolonged-release experimental results show that: on the14th day,releasing is intothe peak.4.Preparation and properties of CD-NP-HA-PLGA/CS-NP-HA-PLGAMethods: Polylactic acid hydroxy acetic acid (PLGA) dissolved in trichloromethane until completely dissolved, CD-NP-HA/CS-NP-HA dissolved in the solution above,violent stir until completely dissolved, above solution quickly into the distilledwater containing0.4%methyl cellulose, stir until chloroform volatilize completely todo. After centrifugal, washing, the solid freeze-dried.By scanning electronmicroscopy (sem), transmission electron microscopy (sem) observation of CD-NPHAPLGA/-CS-NP-HA PLGA surface morphology, particle diameter and particlesize of aggregate, observe its stability, and test load different ligand concentration ofsilver nanoparticles hydroxyapatite antimicrobial properties. PLGA nano-silverhydroxyapatite immersing in simulated body fluid, test slow-release performanceinvitro.Results:200mg of PLGA will dissolve in20ml chloroform,50mg hydroxyapatiteloading nano-silver in different concentration，dissolved in above solution，then allpoured into200ml of distilled water containing0.4%methyl cellulose, getting thebone powder that slowly release silver nanoparticles. Electron microscopy (sem)results showed that the product is into the ball size, diameter is disparity. Maximumcan reach more than100nm in diameter, minimum can be below10nm. Bacteriaexperiment results show that:0.1umolCD-NP and0.1umolCS-NPslow-releasemicrospheres have obvious antimicrobial properties. Slow release experiment resultsshow that:To42days, the release of nano-silver is still on the rise, without significantdecline.Conclusion:1.Through different methods for synthesis of CD-NP、 CS-NP bylyophilization,which load into the nano-hydroxyapatite and wrapedd bypoly(lactic-co-glycolic) acid (PLGA) after freeze-dried,getting target bone meal.Hydroxyapatite containing0.1umol NP and NP-HA-PLGA containing0.1umol NPhave obvious antimicrobial properties.Slow-releasing experimental in vitro resultsshow that the nano-silver hydroxyapatite in14days to release up to peak, afterwraped by PLGA，until42days release the nano-silver is still on the rise,withoutsignificant decline.2.It still need to cell-related experiment testing different concentrations of silver nanoparticles hydroxyapatite and NP-HA-PLGA effects on cells, and model of theinflammation around the implant in animal experiments test whether it can benormal osteogenesis and anti-infection,it is expected to become a good slow-releasecarrier material.