Electrophoretic Deposition Of Antibacterial Macromolecular Coatings On Titanium Surface:Fabrication, Characterization And Application

Background and Objective:In recent year, due to its excellent mechanical property and biocompatibility with human tissues, titanium and its alloys have been widely applied in dental implant, orthopedic joint replacement, fracture retention and regeneration of bone defect. However, owing to the inertness of titanium surface, titanium implants are more prone to failure when the patients are suffering from infection, diabetes, osteoporosis, cancer and other systemic diseases. Especially for the infection condition, complex clinical treatments need to be applied to control the infection, and the patient may suffer from complete removal, prolonged hospitalization, financial burden, and even death. In order to overcome the problem caused by the inert titanium surface, a growing number of studies have been focused on the functionalization of titanium surface by surface modification techniques.In our previous study, chitosan/gelatin (CSG) coatings have been successfully prepared on titanium substrates via electrophoretic deposition (EPD). These coatings are biocompatible, and could promote osteointegration of titanium implant, which make them to be suitable carriers for functional agents. Therefore, in the current study, tetracycline (Tc), a model functional agent, was incorporated into the CSG coatings to fabricate antibacterial coatings on titanium surface. Then characterization of surface morphology, physical and chemical properties, mechanical property, swelling and degradation property, tetracycline entrapment and release profile, preparation mechanism, in vitro and in vivo biological performance of the Tc loaded CSG coatings were investigated, which will provide theoretical and experimental basis for titanium surface functionalization using CSG based coatings.Materials and Methods:The CSG electrophoretic solution containing different amount of Tc (0, 1mg/mL and 10mg/mL) were prepared and marked as CSG, Tc1 and Tc10. The titanium plates were treated by sand-blasting and acid etching (SLA). Then the one-step EPD was applied to fabricated Tc-loaded CSG nanosphere coatings on the SLA titanium plates. After fabrication, the coatings were visualized by inverted fluorescence microscopy and scanning electron microscope (SEM). Zeta potential test of the electrophoretic solutions, as well as transmission electron microscope (TEM) observation the electrophoretic solution under different pH value and the socking solution of the coatings, were applied to explore the fabrication mechanism.Attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) analysis were used to analyse the chemical components and chemical bonds of the coatings. X-ray diffractometer (XRD) and micro-region XRD were used to analyse the crystal structure of the coatings. Mechanical tests were used to characterize the shear bond strength and tensile bond strength of the coatings to the titanium substrate. The swelling and degradation property of the coatings, as well as Tc entrapment and release profile were analysed by socking the coatings into phosphate buffered saline (PBS).For the cell culture experiment, pre-osteoblast MC3T3-E1 cell were seeded onto the coatings. Rhodamine Phalloidin staining was used to visualize the cell skeleton, DAPI nuclei staining and cell counting were used to reflect the proliferation of MC3T3-E1 cells. For the in vitro microbiology experiments, zone of inhibition tests, as well as the cell counting of the adherent and suspended bacteria cell were applied to characterize the antibacterial properties of the coating against Staphylococcus aureus and Escherichia coli. For the in vivo study, acute Staphylococcus aureus bacterial infection model in bone defect was used, where the bond defect was prepared in the distal femur condyle of New Zealand rabbit. In the surgery, Ti implant with systemic application of tetracycline, Ti implant with CSG coating, or Ti implant with Tc10 coating were implanted into the bone defect. White blood cell counting, bacteria cell counting of the homogenized bone tissue, fluorescence microscopy observation the un-decalcified histology section, and Goldner staining together with Gram staining the un-decalcified histology section were applied to evaluate the in vivo antibacterial properties and application prospects of the coatings.Results:By using the one-step EPD technique, CSG, Tc1 and Tc10 coatings were successfully prepared on SLA titanium substrates. Surface morphology observation of the coatings showed that the coating has a macro three-dimensional porous film-like structure, and micro nanosphere structure. Yellow-green fluorescence of Tc could be found in Tc1 coatings, and yellow-green crystals could be found on the surface of Tc10 coatings. For the mechanism study, Tc, chitosan, and gelatin were all positively charged in the electrophoretic solution, and repel each other. During EPD process, the nanosphere structure was formed during the pH value change near the cathode. Tc, chitosan, and gelatin were bonded under the influence of electrostatic interaction. Near the cathode, chitosan’s amino groups were deprotonated and became in-soluble. Then Tc, chitosan, gelatin, and hydrogen bubbles were co-deposited onto the SLA titanium substrate.ATR-FTIR results showed that the coating was composed by Tc, chitosan, and gelatin, new hydrogen bonds were formed between the peptide amino and carbonyl groups in the backbone of gelatin and amide group in benzene ring of Tc. XRD and micro-XRD results showed the typical gelatin crystalline peak in CSG coating, and after incorporation of Tc, this peak became higher and narrower, which indicated that the crosslink of gelatin with Tc would increase the crystallinity of the nanosphere coating. Mechanical test results showed that Tc10 coating had significantly higher shear and tensile bond strength than CSG and Te1 coatings. This significantly elevated mechanical property might result from the increased crystallinity of the Tc-incorporated CSG coating. Swelling and degradation results showed that the incorporation of Tc could reduce the swelling rate, and improve the degradation rate of the coatings. Tc and gelatin were firstly released from the coatings, then chitosan. Tc entrapment and release results showed that the Tc entrapment was positively correlated to the Tc concentration in the electrophoretic solutions. And most of Tc was released from the coatings in 3 hours.Immunofluorescence staining results of the MC3T3-E1 cell skeleton showed that SLA titanium, CSG and Tc coatings could support cell adhesion and extension, well-pronounced elongated filopodia could be found between cells. While the cells were round shaped and aggregated together on Tc10 coatings. Cell counting results showed that Tc1 coating could support cell proliferation, while Tc10 coatings exhibited cytotoxicity to MC3T3-E1 cells. Zone of inhibition results demonstrated that both Tc1 and Tc10 coatings have antibacterial ability against Staphylococcus aureus and Escherichia coli. And the bacteria cell counting results also showed that both Tc1 and Tc10 coatings have antibacterial ability against adherent and suspended bacteria in this study, especially for Tc10 coating. In vivo test results showed that Tc10 coating could be successfully applied in vivo, it could significantly reduce the white blood cell counting and bacteria cell counting of the homogenized bone tissue. The antibacterial property of Tc10 coating was better than systemic prophylaxis using Tc. Moreover, histology staining exhibited positive staining of Staphylococcus aureus in Ti+sys and CSG group, and micro-abscess formation in CSG group. While no sign of infection, but Tc deposition and osteoid formation in the edge of trabecular bone, could be found in Tc10 group.Conclusion:1. Tc-loaded CSG nanosphere coatings could be successfully prepared on the titanium substrate via EPD, the prepared coating had a macro three-dimensional porous film-like structure, and micro nanosphere structure.2. For the first time, the EPD fabrication mechanism of Tc-loaded CSG coating had been investigated. It could provide theoretical support for the further preparation of functionalized coating via EPD.3. Through analysing the physical-chemical properties of Tc-loaded CSG coatings, it was found that Tc, chitosan, and gelatin were co-deposited into the coating, and Tc was crosslinked with gelatin. Additionally, the incorporation of Tc could improve the crystallinity of the nanosphere coating, thus improve the shear and tensile bond strength of the coating.4. The incorporation of Tc could reduce the swelling rate, and improve the degradation rate of the coatings. And Tc was released with a burst release profile.5. Tc1 coating could support the adhesion, extension and proliferation of MC3T3-E1 cell, while Tc10 coating showed cytotoxicity to MC3T3-E1 cell.6. The antibacterial property of Tc was fully preserved in the coating. And Tc-loaded CSG nanosphere coatings showed excellent antibacterial property against both adherent and suspended bacteria cell.7. Tc-loaded CSG nanosphere coatings could be successfully applied in vivo, and showed better antibacterial property than systemic prophylaxis.8. As the first research study that incorporated functional agent into CSG nanosphere coating, this study would provide theoretical and experimental basis for the further loading of other functional agents, such as anticancer drugs, gene segments, protein molecule, metallic ions.