Titanium Surface Priming With Phase-transited Lysozyme To Established A Silver Nanoparticleloaded Antibacterial Multilayer Via Layer-by-layer Self-assembly

Objectives: Currently, dental implant technology has become an effective treatment method for dentition defect. Clinical long-term observations indicate that implant-associated infection remains the primary complication of implantation, leading to the implant loosen and even the failure of implant surgery. The formation of biofilm on the surfaces of implants following by bacteria adhesion is the primary cause of infections of the mucosa and bone adjacent to the implant. The biofilm makes the bacteria more invasive and it is very difficult to remove. Initial surface modification is usually required to incorporate antibacterial agents on titanium(Ti) surfaces to inhibit biofilm formation. However, simple and effective priming methods are still lacking for the development of an initial functional layer as a base for subsequent coatings on titanium surfaces. The purpose of our work was to establish a novel initial layer on Ti surfaces using phase-transited lysozyme(PTL), on which multilayer coatings can incorporate silver nanoparticles(Ag NP) using chitosan(CS) and hyaluronic acid(HA) via a layer-by-layer(Lb L) self-assembly technique.Methods: 1. In this study, the surfaces of Ti substrates were primed by dipping into a mixture of lysozyme and tris(2-carboxyethyl)phosphine(TCEP) to obtain PTL-functionalized Ti substrates. The subsequent alternating coatings of HA and chitosan loaded with Ag NP onto the precursor layer of PTL were carried out via Lb L self-assembly to construct multilayer coatings on Ti substrates. 2. The chemical constituents and surface morphology of the pristine and modified Ti were characterized by X-ray photoelectron spectroscopy(XPS), field-emission scanning electron microscopy(FE-SEM), energy dispersive X-ray detector(EDX) and the measurement of the contact angles. The size and morphology of the Ag nanoparticles was observed by transmission electron microscopy(TEM). 3. The serial dilutions and the spread plate method were used for antibacterial assay against Staphylococcus aureus. The growing condition andmorphology of bacteria adhered on the surfaces of samples were observed by SEM. Fluorescence staining observed by confocal laser scanning microscopy(CLSM) for live and dead bacteria was used to characterize the viability of adherent bacteria on the samples. 4. MC3T3-E1 murine preosteoblasts were used for biocompatibility tests of Ti surfaces incorporated Ag NP. The cytotoxicity of Ag NP to MC3T3 cells can be assessed by the activity of lactate dehydrogenase(LDH), alkaline phosphatase(ALP) activity, intracellular total protein and cell viability assay. The cell numbers were assessed by the cellular DNA analysis. CLSM was used to show the condition of adherent cells on the samples.Results: 1. The results of SEM and XPS indicated that the necklace-like PTL and self-assembled multilayer were successfully immobilized on the Ti substrates. The shape of the silver nanoparticles was a relatively uniform sphere averaging approximately 20-30 nm in size. 2. The multilayer coatings loaded with Ag NP can kill planktonic and adherent bacteria to 100% during the first 4 days. The antibacterial efficacy of the samples against planktonic and adherent bacteria achieved 65%-90% after 14 days. The sustained release of Ag over 14 days can prevent bacterial invasion until mucosa healing. 3. The CS/Ag samples exhibited cytotoxicity with the increase in the amount of incorporated Ag. The CS/Ag10 samples exhibited significantly lower cytotoxicity than the other samples.Conclusions: The PTL priming method provides a promising strategy for fabricating long-term antibacterial multilayer coatings on titanium surfaces via the Lb L self-assembly technique, which is effective in preventing implant-associated infections and guarantees normal early-stage wound healing. The toxicity can be reduced by controlling the Ag release rate and concentration.