| BackgroundPercutaneous species in the clinic has been used in a very wide range,such as bone repair and bone anchored hearing aid,dental implants,ventricular assist device and so on.But different from the oral implant,due to the skin-integration of the percutaneous implants and the nonsterile environment on the skin surface,percutaneous implants have far higher infection rate and failure rate than oral implants.The main reason is that the percutaneous implant break the skin barrier,which makes the percutaneous implant hard to integrate with the skin,and,once the percutaneous implant cannot integrate with the skin,it leads to downward,infection,all kinds of pathogens colonization on the percutaneous implant,and eventually lead to failure.In addition,the skin appendages such as sebaceous glands,sweat glands,sweat pores etc.are conducive to bacterial adhesion,resulting the nonsterile environment around the implant,which is also an important reason for the failure of percutaneous implants.Both human tissues and cells have nano modules.Experiments have shown that the nano-scale surface morphology of implant surface is conducive to the formation of skin integration,so that creating nano-scale morphology on the surface of implant is a feasible way to promote skin integration and percutaneous closure.The structure of titanium dioxide nanotube can be used to load the drug,and the nanotube structure itself also has some antibacterial properties.But even after disinfection and infection control strictly,bacterial invasion after percutaneous implant surgeries still occur frequently.The infections around percutaneous implants are mainly caused by Staphylococcus aureus andcoagulase negative staphylococci,also because the healing process after percutaneous implant surgeries are nonsterile,therefore only change the surface morphology of the implants is insufficient to meet the demand of antibacterial.Antimicrobial peptides are kinds of self defensive small peptides produced by biological organisms.The antimicrobial peptides,one kinds of the antimicrobial peptides,has the advantages of less cytotoxicity,almost no drug resistance,high efficiency and broad spectrum.It is an ideal choice for the antibacterial layer of implant.Although the implant itself has a certain drug loading capacity,it generally has no controlled-release ability,and it is difficult to maintain minimal inhibitory concentration at a high risk of infection after the percutaneous implant surgeries.For the inhibition of the burst release,polymer,polymer microspheres,such as PLGA,PGLA,poly malic acid and graphene microcapsules etc.are often elected to be controlled release agent.Among these agents,the biocompatibility of PDLLA and PLGA polymers is well,and can be degraded to non toxic and non irritating substances to the body.These are the most commonly used kind of contrlled release agent.Aims:In order to solve the problem of high infection rate of percutaneous implant,two aspects,surface treatment and antibacterial coating,are going to be used to make the modification of the implant surface in this study.Choose the most common pure titanium material as the drug-coated carrier.Manufacture two different coatings with HHC-36 peptide loaded by PDLLA/PLGA.Test the in vitro release performance,biocompatibility and antibacterial performance of the drug loaded specimens.Methods:Surface treatment of specimen and preparation of the antibacterial coating1.The nanotube structure was prepared on the surface of pure titanium by anodic oxidation.The surface morphology of the samples was observed by field emission scanning electron microscopy,and the diameters of the tubes formation under differentvoltages were tested,so as to prepare suitable parameters for drug loading.2.The solvent-casting technique was used to prepare the HHC-36 antimicrobial peptide coating with PDLLA as the sustained-release agent on the surface of the treated titanium dioxide nanotubes.3.Using a dual-solvent emulsification and volatilization method,the HHC-36 antimicrobial peptide coating with PLGA microspheres as a sustained release agent was prepared on the surfaces of the treated titanium dioxide nanotubes specimens.4.The surface morphologies of the two antibacterial coatings were observed under field emission scanning electron microscopy.Performance test of the antibacterial coating on the surface of the specimen in vitro1.The antibacterial peptide HHC-36 was dissolved in PBS solution by concentration gradient preparation,and the absorption peak wavelength of HHC-36 antimicrobial peptide was confirmed by UV spectrophotometry.The regression relationship between the absorbance and the concentration of the antimicrobial peptide was calculated according to the absorbance obtained from the gradient concentration,and the regression curve was drew.The prepared Ti O2 nanotubes drug loaded specimens were immersed in PBS solution,and after the ultrasonic shock,the absorption peak wavelength absorbance of the antimicrobial peptide was measured by ultraviolet spectrophotometer,and the concentration of HHC-36 antimicrobial peptide was calculated according to the regression equation,then the drug loading of each specimen was calculated.2.The prepared Ti O2 nanotube drug loading specimens were placed in PBS solutions and heated at 37 centigrade water bath.After placing 1h,2h,3h,4h,and 5h on the first day,2ml of each solutions was removed and 2ml PBS was rejoined in the solutions.The samples were labeled and the absorbance at the wavelength of the absorption peak of the antibacterial peptide was obtained using the UV spectrophotometer.According to the regression equation obtained by experiment 1,the concentration of HHC-36 antibacterial peptide was calculated and the sustained release curve was calculated.Test of biocompatibility and antibacterial properties of drug loaded specimens in vitro1.Specimen eluent cytotoxicity testThe 3-8 generations of human skin dermal fibroblast primary cell cultures were used as experimental cells.The leach liquor of drug loaded specimens fabricated in the first part was used for CCK-8 cytotoxicity test.2.Determination of minimal inhibitory concentration of HHC-36 antibacterial peptideAccording to the results of the pre experiment,the HHC-36 antimicrobial peptides are prepared in a concentration gradient into a 500ug/ml-4mg/ml solutions.These solutions were addd to the center of the MH agar plate with Staphylococcus aureus prepared before respectively.37 degrees in the incubator for 24 hours,the bacteriostasis effect was observed and the minimum inhibitory concentration of the HHC-36 antimicrobial peptide was determined.3.Specimen inhibition zone testStaphylococcus aureus,a common pathogen of percutaneous implants,was used as indicator bacteria.Inoculate the resuscitation strains to the blood agar plate before the test,and incubated for 24 hours at 37 degrees.Place two independent colonies of Staphylococcus aureus into the turbidimetric tube and the 10^8CFU concentration of Staphylococcus aureus was diluted by turbidimetry.The diluted Staphylococcus aureus liquid was evenly coated on a MH agar plate using a cotton swab.Each group randomly selected 4 drug-loaded test specimens and the unloaded Ti O2 specimens were used as blank control.After disinfecting with epoxyethane,placed the drug loaded surface of the specimens in the center of MH agar plates containing Auricum aureus.37 degrees in the incubator for 24 hours,the bacteriostasis effect was observed and a caliper was used to measure the size of the inhibition zone.4.Antibacterial Test of Sample EluentStaphylococcus aureus was incubated in 80 ml BHI in a 37 degree water bath for 24 hours.The gradient concentrations of 1x10^5,10^4,10^3,10^2CFU bacteria solutions were diluted by turbidimetry.The drug loaded samples were oscillating in 5ml TBS by ultrasonic oscillation for 30 min,and 1ml of these solutions were taken to mix with thebacterial solutions prepared before.After 3 hours of 37 degrees water bath,each sample was coated with 100 ul to the blood agar plate,and after 37 degrees in the incubator for 24 hours,the number of colonies were recorded.Results:1.Anodizing experiments were carried out on pure titanium specimens at different voltages.Under 20 V,the diameter of the tubes was 100 + 10 nm,and the array was well arranged and well aligned.2.On the specimens manufactured before,the HHC-36 antibacterial coating with PDLLA as the release agent is constructed using the solvent casting technique.3.On the specimens manufactured before,the HHC-36 antibacterial coating with PLGA as the release agent is constructed using the double solvent emulsification and volatilization.4.The stable absorption peak of the HHC-36 antibacterial peptide at 280nmwavelengthwas was tested by a UV spectrophotometer.5.The 15 days in vitro release of the specimens with PDLLA/PLGA coatings weretested and the sustained release curve were drew.The two drugs had a stable drugrelease within 15 days.The drug release rate of the PDLLA carrier was 47% at 15 days,and the drug release rate of the PLGA as the carrier was 34%.6.Adult dermal fibroblasts were used as experimental cells to test the CCK-8cytotoxicity test of the 3-8 generation of primary cultured cells.The cytotoxicity testresults were classified as grade 0 or 1,which accorded with the requirements ofbiological materials.7.The gradient concentration solution of HHC-36 antibacterial peptide was prepared,and the inhibition zone test was carried out.According to the experimental results,theconcentration range was gradually narrowed,and the minimum inhibitory concentrationwas determined around 1.5mg/ml-2mg/ml.8.The inhibition zone of the specimens prepared before according to the groups.The PDLLA group average inhibition zone size was 15.2mm,and the PLGA group averageinhibition zone size was 12.4mm,and there were no colonies of bacteria was observed in both groups.5.9.In the antibacterial Test of sample eluent,the test group showed obvious bacteriostasis effect.The colony number of both groups under CFU of 10^2 were 2,which was obviously lower than that of the control groups.Conclusion:Ti O2 nanotubes were successfully prepared.The 100 nm diameter specimen was selected as the drug carrier,and the drug delivery controlled-release coating was successfully prepared by solvent casting technique and double solvent emulsification volatilization method.It has good contolled release performance in vitro.The specimense eluent was tested by CCK-8 cell toxicity test to prove that it had no cytotoxicity,and had good biocompatibility.The antibacterial properties of the drug loaded specimens showed good and sustained antibacterial properties. |
PDF Full Text Request