In Vitro Testing Of Antibacterial Properties Of The Nano-Silver Coating On The Titanium Surface To The Staphylococcus Aureus

BackgroundWith the prolongation of the average life of Chinese and the intensification of the aging society, patients with common clinical age-related diseases, such as osteoarthritis of the knee, hip fracture, and degenerative diseases of the spine increased year by year. As the conventional and effective treatment approaches of orthopedic surgeons, joint replacement surgery and spinal fixation were gradually increasing in the number of times used, which relieved pain for many patients. Nevertheless, the infection, as a serious and frightening surgical complication of orthopedic surgery, was also increasing each day along with the increase of the surgical volume. Currently, the surgical infection of implants was described as disastrous consequences in orthopedic clinical because it can lead to limb dysfunction, thus seriously affecting the quality of patients lives, and its treatment is quite difficult and greatly expensive. Especially for the internal fixation of the elderly spine and the replacement surgery of the elderly hip knee, the risk of reoperation was significant enlarged since the patients had poor tolerance ability for the orthopedic revision surgery, and some patients had even almost lost their opportunities for surgical treatment due to their poor physical condition. Therefore, the peripheral infection of the implants was one of the most serious complications of the joint replacement of joints and spine orthopedics and the internal fixation of the spine. According to the first statistics, the infection rate of the joint replacement surgery was from1%to5%, and that of the joint repair surgery was even higher. Hence, the prevention for the peripheral infection of the implants became critical. The peripheral infection of the orthopedic implants had its own characteristics. According to the authoritative statistics made by the American Centers for Disease Control and Prevention, the medical implant infection was ranked in the fifth place among death causes of the U. S. hospitalized patients, of which65%bacterial infections were related to the formation of biofilms. Therefore, how to prevent the adhesion and colonization of bacteria around the prosthesis and the formation of biofilm was the key to preventing infection in orthopedic implant surgery.There were mainly two kinds of the recognized infection mechanism and pathogenesis of orthopedic surgery. The first one was that the postoperative bacteria plant in the joint space (blood type) through the bloody spread, which explained why the advanced infection was more typical. The second one was that the intraoperative bacteria plant around the prosthesis, which often showed the signs of infection after a few days to a few months after. If the bacteria could not settle down during this time, the risk of joint sepsis was reduced quickly. If successful, the bacteria would multiply and gather rapidly, and adhere to the surface of the prosthesis via the extracellular matrix of the high-intensity hydration to form the bacterial biofilm. The produced metabolites were accumulated in the biofilm. The drug tolerance of microorganisms of the biofilm was1000times of those free microorganisms. From the production process of orthopedic surgical procedure, it could be clearly known that the inhibition for the intraoperative and postoperative bacterial plantation and the bacterial biofilm formation is the key to preventing orthopedic postoperative infection.In orthopedics, there was no effective method to prevent the bacterial plantation and the bacterial biofilm formation at home and abroad. As shown in the studies, the antimicrobial coating formed on the orthopedic implant surface had good inhibitory effect for the bacterial adhesion and biofilm formation. Silver, as an antimicrobial agent, had long been used in the medical field. The nanotechnology could change the silver into nano-sized, which thus enhances the surface area and increases the better contact with the microorganisms, thereby having good antibacterial properties. This paper forms nano silver/hydroxyapatite composite sterile coating on a commonly orthopedic material-titanium surface. And as the bacterial adhesion carrier, the paper makes in vitro studies for the acquisition of staphylococcus aureus strains by the hospital, and studies the effect of nano-silver/hydroxyapatite composite sterile coating on the formation of staphylococcus aureus biofilm and its antibacterial effect, which provides the experimental evidence for the future application of the nano-silver /hydroxyapatite composite sterile coatings to the clinical orthopedic implants.PART IProduction and Testing of Nano-Silver/Hydroxyapatite Composite CoatingObjective:To produce the nano-silver/hydroxyapatite composite coating on the polished titanium surface and test the physical characteristics of the coating.Methods:Raw hydroxyapatite powder with the particle size of lOμm and raw silver powder with the particle size of5μm were mixed averagely according to the mass ratio of20:1. And the planetary ball milling was applied to grind the powder in ball type so as to mix the particle size of the powder to reach the nanometer level. The acquired nano-level powder was placed into the blender with the powder, which was stirred and blended to disperse it uniformly. Taking the titanium in a constant-temperature bath oven as the cathode and the stainless steel sheet as an anode, a mixed powder of the nano-silver and the hydroxyapatite was ultrasonically dispersed in the ethanol solution. Taking the pH value of the dilute hydrochloric acid as4,20V DC was given for deposition. Then, the samples were placed in a tubular resistance furnace for heat treatment. The titanium samples of the nano-silver/hydroxyapatite composite coating were finished the production. The infrared spectroscopy and the scanning electron microscope were used for the detection of coating components and characteristics.Results:The particle size of the nano powder produced by the original silver powder and hydroxyapatite powder via the ball-milling technology was averaged into about100nm. Through the infrared spectroscopy and the scanning electron microscopy, it could be seen that the hip-joint powder of the nano-silver and the nano-level hydroxyapatite coating was evenly dispersed in the coating, and the coating was fixed and stable on the titanium-based surface.Conclusion:In this section, the production process of the nano-silver hydroxyapatite composite coating was given a detailed discussion. First, the ball-milling technology was used to prepare silver powder and hydroxyapatite powder into the nano-level composite powder and stir and mix well. Then, the chemical precipitation method was applied to prepare nano-silver hydroxyapatite composite coating on the treated titanium plate. By means of the instrument, it was analyzed that the nano-silver particles were distributed in the hydroxyapatite carrier uniformly. Nano-silver was in free state and nanoparticles showed no obvious aggregation, which indicated that the ideal nano-silver hydroxyapatite composite antibacterial coating could be prepared successfully by the above production technology and production process. PART ⅡIn Vitro Testing of Antibacterial Properties of the Nano-Silver Coating on the Titanium Surface to the Staphylococcus AureusObjective:To test the antibacterial properties of the nano-silver/hydroxyapatite composite coating on the staphylococcus aureus and compare the antibacterial properties of the cefuroxime by means of bacterial culture on different carriers under the same condition, so as to mainly detect the adhesion number of the staphylococcus aureus in the biofilm and the morphological differences of the biofilms under the laser scanning confocal microscope.Methods:The staphylococcus aureus isolated, cultured and purified clinically was taken as the experimental bacterial strains. All samples had underwent strict disinfection and set four comparison groups, namely, the hydroxyapatite coating group of the titanium surface, the nano-silver hydroxyapatite composite coating group of the titanium surface, the hydroxyapatite coating+cefuroxime group of the titanium surface, and the nano-silver hydroxyapatite composite coating+cefuroxime group of the titanium surface. And under the same experimental condition, the samples were placed into the bacterial liquid for the culturing, each group with12samples. And the biofilm on the coating surface was detected at6hours,24hours,48hours and72hours respectively. The laser scanning confocal microscope was applied to the biofilm for the morphological observation. The number of the staphylococcus aureus on the sample surface at24hours was compared with that at48hours.Results:The nano-silver hydroxyapatite composite coating+cefuroxime group of the titanium surface did not form the biofilm, the nano-silver/hydroxyapatite composite coating group and the hydroxyapatite coating+cefuroxime group of the titanium surface appeared the biofilm at the48hours, and the hydroxyapatite coating group just appeared a mature biofilm after12hours. The nano-silver/hydroxyapatite composite coating group and the hydroxyapatite coating of the titanium surface showed significant differences in the adhesion amount of the coating surface at24h and48h (p<0.01)Conclusion:Nano silver/hydroxyapatite composite coating could inhibit the biofilm formation significantly; the antibacterial effect of the nano-silver and the cefuroxime had a good synergy, and the nano silver coating had excellent antibacterial properties. According to literatures associated, various factors should consider before development of new nano-silver material and products. To ensure the safety of nano-silver products, several times in vitro and animal experiments.must be repeated before the products entering clinical. In terms of process engineering, some papers have reported that nano-silver in the absence of protective agents conventional conditions, the loss of the aggregation prone nano particle characteristics as a brown oxide or silver oxide, which can affect the antimicrobial effect of the material, and therefore the need to strengthen further studies to overcome the technical deficiencies. With the study of the mechanism of nano-silver antibacterial broad spectrum of in-depth, will develop a safe and effective broad-spectrum antimicrobial nano-antibacterial drugs and materials to provide a theoretical basis and experimental basis, to provide a new approach for a broad spectrum of human bacterial infections prevention and treatment and strategies.The experimental result of a single kind of bacteria Staphylococcus aureus is not sufficient to detect a bacterial nano-silver/hydroxyapatite composite coating antibacterial properties.Secondly, our experiments are done under in vitro conditions, rather than idealized environment, because under in vivo conditions in accordance with the diffusion gradient, and fluid flow can significantly reduce the local concentration of nano silver. Third, the lack of experimental bacterial sample counts lead to less credible to the results. Fourth, the study of nano-silver/hydroxyapatite composite coating for prosthetic bone ingrowth affect pending. Although these findings could not enter the stage of in vivo studies yet, we believe that with the maturity of the conditions and the in-depth studies, such as researches on animal bodies and processing of large samples, the nano-silver/hydroxyapatite composite coating will be greatly applied to the orthopedic implants.