The Mechanical, Bio-safety And Antibacterial Adhesion Properties Of The Carbon Ion Implanted Silicone Rubber

Background and Objectives:In plastic surgery, The malformation and coloboma of tissue or organ in consequence of the congenital and trauma are repaired and reestablishment by the surgical operation. The main therapy method is the soft tissue filler implantation, and it conform to development tendency of modern medicine that noninvasive repair replace repair the trauma with trauma. The main properties of ideal material for clinical medicine are below: first, good biocompatibility; second, good biomechanical characteristic; third, stable chemical property; fourth, non-magnetic, be liable to manufacture and disinfect. Now many biomaterial such as silicone rubber, e PTFE, HA, β-TCP and PLA are widely used in the plastic surgery, but these materials are made by single component or single structure, and have their own advantages and disadvantages, it also cannot satisfied the need of body for the diversity of material property, cannot become the ideal soft tissue filler and exist disadvantages in the clinical applications.SR is a kind of soft tissue filler, and the main factor of affect SR in the clinical applications is the hydrophobicity of the surface. The effective way to enhance the biocompatibility and lower the infection risk is surface modification of SR to hydrophilic. In the preliminary study, we use the carbon ion implanted in the surface of SR, and obtained the carbon ion implanted silicone rubber(C-SR) and found that it enhance the biocompatibility. In this study, we use the shore durometer(type A) and electronic universal testing machine to evaluate the mechanical properties of C-SR, The bio-safety were performed by the erythrocyte haemolysis test, systemic toxicity test, subcutaneous implant test, allergic and dermal irritation test, pyrogenic test according to the national standard. At same time, we conducted a series experiments to investigate propagate and adhesion of different bacterial which including Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis. This study are main for the research and development of soft tissue filler which with the good biocompatibility and lower antibacterial adhesion, and promote the clinical applications of C-SR.MethodsThere are three parts in this study.Part 1: SR preparation, C ion implanted and the testing of mechanical properties of samples.1. A two-component silicone system was used compound the pure SR sheet with 100mm×100mm×1mm mould under the room temperature, and then implanted carbon ion with dosge that 1×1015 ion/cm2 、3×1015 ion/cm2 、1×1016ion/cm2, and marked for C1, C2 and C3.2. The mechanical properties were performed by shore durometer(type A) and electronic universal testing machine, to gain parameters of the shore A hardness, tensile strength, elongation at break and tear strength.Part 2: According to the national standard that biological assessment of medical material, we conduct a series experiments to evaluate the bio-safety.1. According to the national standard of the erythrocyte haemolysis test, we obtain the hemolysis ratio of SR, C1, C2 and C3.2. According to the national standard of the systemic toxicity test, we injected the leach liquor of SR, C1, C2 and C3 to the enterocoelia of SD rats, and evaluate the toxicity of this kind of material through test the liver and renal function change before and after injection.3. According to the national standard of the subcutaneous implantation. We implanted the SR, C1, C2 and C3 in SD rats subcutaneously, take out the material and its surrounding tissues after 7 days, 30 days, 90 days and 180 days, then the HE, Masson and immunohistochemical were used to discuss the inflammatory response and capsular thickness.4. According to the national standard of dermal irritation test. We use the leach liquor of SR, C1, C2 and C3 to test the dermal irritation of guinea pig.5. According to the national standard of dermal allergic test. We use the leach liquor of SR, C1, C2 and C3 to test the dermal allergic property of guinea pig.6. According to the national standard of dermal pyrogenic test. We injected the leach liquor of SR, C1, C2 and C3 to the rabbit to test the changes of body temperature.Part 3: The antibacterial adhesion property of C-SR and SR1. Plate colony-counting methods: After the samples incubation in bacterial culture for 1 or 24 h, the bacteria on the each sample were gently rinsed with PBS respectively and ultrasonically detached in 1 ml of PBS solution. The bacteria in the PBS were re-cultivated on agar plates for colony counting.2. Live and dead fluorescence staining: After incubation for 1 or 24 h, the various samples were gently rinsed three times with PBS before staining the bacteria on the samples. The staining was performed by applying SYTO 9 and propidium iodide dye(LIVE/DEAD® BacLight? Bacterial Viability Kit, Molecular Probes®, OR, USA) for 15 min in darkness and was examined by laser scanning confocal microscopy(LEICA TCS SP5, Germany). The areas of green and red color in the pictures were then analyzed by using Image-Pro Plus version 6.0(Media Cybernetics, Inc., USA) and then calculating the proportion of red coloration based on the following relationship: red proportion(%) = red area /(green area + red area).3. Scanning electron microscopy(SEM) observation: After bacterial incubation for 1 or 24 h, the samples were rinsed thrice with PBS to remove free bacterial cells and then fixed in 2.5% glutaraldehyde for 3 h at room temperature. The samples were then progressively dehydrated in a series of ethanol solutions(15%, 30%, 50%, 70%, 80%, 90%, 95% and 100%) for 15 min each. After that, the specimens underwent critical point drying and coating with a thin conductive layer of Au. Finally, the morphology and adhesion of the bacteria on the various samples were determined by SEM(VEGA 2 SEM, TESCAN Inc., Brno, Czech Republic).Results1. We have obtained the pure SR sheets, and successfully implanted the carbon ion into the surface of SR and made three kinds of new material(C1, C2 and C3).2. The results of the shore A hardness, tensile strength, elongation at break and tear strength, show that the carbon ion implanted SR has the same mechanical properties with the pure SR.3. The erythrocyte hemolysis test show that the rate of hemolysis was 0.1%, 0.3% and 0.4%, which lower than the national standard, it was indicated that the in vivo transplantation of the materials could not cause the hemolytic reaction of the body.4. The liver and kidney function of SD rats have no significant change before and after injected the leach liquor of SR, C1, C2 and C3, no death happen. It shown that the materials have nontoxicity.5. After the samples implanted subcutaneously of SD rats, the HE, Masson and immunohistochemical(CD4, CD68, TNF-α, α-SMA and elastin) were performed. The all results shown that C-SR have a better effect that SR, especially in the inflammatory response and capsular thickness.6. The HE stains after dermal irritation test to the guinea pig shown that the leach liquor of all samples have no dermal irritation effect.7. The HE stains after dermal allergic test to the guinea pig shown that the leach liquor of all samples have no dermal allergic reflection.8. After injected the leach liquor of SR, C1, C2 and C3 in the body of rabbits, there were no changes of body temperature. It shown that the C-SR has no dermal pyrogenic effects.9. It shown from the plate colony-counting that the C-SR has the lower bacterial adhesion and the number of bacterial adhesion decrease with dose of carbon increase.10. The live and dead fluorescence staining of bacteria shown that C-SR also has the lower bacterial adhesion and the number of bacterial adhesion decrease with dose of carbon increase.11. From the scanning electron microscopy(SEM) observation results, the bacteria shown C-SR also has the lower bacterial adhesion and the number of bacterial adhesion decrease with dose of carbon increase.Conclusion1. The carbon ion implanted methods is good and convenience, and the C-SR have keep the good mechanical properties of SR.2. The erythrocyte hemolysis test show that the all rate of hemolysis was lower than the national standard, it was indicated that the in vivo transplantation of the materials could not cause the hemolytic reaction of the body. The subcutaneous implant test indicated that it has good safety and compatibility in the body. The rate of allergic and dermal irritation test was 0, which had no significant difference from negative control, suggesting that the materials have no allergic and dermal irritation. In the pyrogenic reaction, the body temperature of the rabbits averagely increased by 0.1℃, indicating that the materials have no pyrogenic effect, in accordance with the demands of medical materials. All results shown that the C-SR has good bio-safety and biocompatibility compare with SR.3. The C-SR lowers the bacterial adhesion and the number of bacterial adhesion decrease with dose of carbon increase. It reflected that C-SR have the lower risk to biomaterial centered infection, and have a good prospect in clinical application.