Study On Antibacterial And Anti-Adhesion Function Of Acellular Tendon With Crosslinked Gallic Acid-Silver Nanoparticle

Objective: Infection and adhesion are the two main complications of tendon transplantation,especially infection,which can cause catastrophic results.The target of this study was to prepare a tendon with antibacterial and anti-adhesion functions.Methods: Decellularized tendons extracellular matrix(DT)was used as the substrate,and gallic acid silver nanoparticles was combined in the form of electrostatic adsorption and chemical crosslinking.Constructing a antibacterial scaffold(DT crosslinking GA-AgNPs,DT-Ag)which can slow release drug in the physical environment as well as sustain antibacterial ability,good biological compatibility and anti-adhesion.The following tests were performed on the prepared materials:(1)Zeta potential and infrared spectroscopy were used to analyze the changes in the charge of the drug-carrying scaffold on the formation of chemical bonds;XPS was used to analyze the valence state of elements on the surface of the scaffold;(2)In vitro bacterial co-culture was used to detect the killing ability and contact bactericidal ability of suspended bacteria;(3)In vitro DPPH assay,erythrocyte membrane stability assay and RT-qPCR assay were used to detect antioxidant capacity and anti-inflammatory capacity.(4)The anti-adhesion ability was analyzed by in vitro NIH3T3 co-culture and water contact Angle detection;(5)The antibacterial and anti-adhesion properties of the scaffolds were tested by rat tendon defect model in vivo.Results: Our results showed that:(1)DT has a strong positive charge,and can be loaded with negatively charged gallic acid silver nanoparticles by electrostatic adsorption,showing slowly release under agitation or ultrasonic shock;The amino group exposed by DT can be crosslinked with gallic acid silver nanoparticles in the form of amide bond.The positive charge of DT-Ag is significantly reduced,and it not only releases slowly,but also continuously under agitation or ultrasonic shock.(2)After co-culture with bacterial suspension,a small amount of supernatant was used for gradient dilution.Plate counting showed that DT-Ag had good bactericidal ability.Scanning electron microscope showed that the number of viable bacteria on DT-Ag surface decreased significantly.In the bacteriostatic zone experiment,only obvious bacteriostatic zone was observed in DT-Ag.(3)CCK-8 assay showed that the scaffold extract did not affect the proliferation of NIH3T3 cells.DPPH method shows that DT-Ag has excellent ability to clear oxygen radicals.The stability of erythrocyte membrane showed that the anti-inflammatory ability of DT-Ag was significantly improved compared with DT.RT-qPCR analysis showed that there was no statistical difference in the expression of M1 marker gene,and the increased expression of M2 marker was helpful to reduce inflammation,promote tissue repair and anti-infection.(4)Calcein fluorescence showed that the adhesion of NIH3T3 cells in DT-Ag decreased significantly;The water contact Angle experiment indicated that DT-Ag had improved hydrophilicity and played a lubricating role,which would help prevent the formation of adhesion.(5)Taking infected tendon defects in rats as a model,macro observation and adhesion score analysis showed that DT-Ag had the best anti-adhesion ability,and immunohistochemistry of CD68 and MPO staining showed that no inflammatory cell infiltration was observed in DT-Ag,which was not observed in the other two groups.Conclusion: In this study,a novel construction method of antibacterial scaffold was established,which has biocompatibility,anti-adhesion and continuous antibacterial ability.