Title

Covalent Tethering of Anti-Microbial Peptides onto Titanium Reduces Bacterial Colonization

Date of Award

2019

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Christopher Adams, PhD

Second Advisor

Denah Appelt, PhD

Third Advisor

Brian Balin, PhD

Abstract

INTRODUCTION: A major complication in the use of surgical prostheses is periprosthetic infection (PPI). PPI is associated with significant disability, morbidity, and mortality. Reducing the incidence of PPI can increase the quality of life for patients, as well as lower the overall societal economic burden. Once bacteria are able to attach, replicate, and begin biofilm production, they can avoid immune system detection as well as eradication by systemic antibiotics. Thus, we have focused on this initial colonization event as the critical stage of inhibiting PPL

OBJECTIVES: The objective of this project was to demonstrate that covalently tethering of the anti-microbial peptides (AMP), beta amyloid (A~) and LL-37, to the surface of titanium can reduce bacterial colonization.

METHODS: AMPs were tethered to the surface of titanium pins and mesh either with, or without an Fmoc-aminoethoxyethoxyacetate (Fmoc-AEEA) linker via a modified, previously-established synthesis protocol. Immunofluorescent detection was used to confirm successful tethering with confocal microscopy. The modified-titanium surfaces were then challenged against Staphylococcus aureus, a Gram-positive coccus, and Escherichia coli, a Gram-negative bacillus. Antimicrobial effect was determined by colony counting on 3MTM PetriFilms ™ and staining with a Bacterial Viability Kit for visualization by confocal microscopy.

RESULTS: Immunofluorescent analysis of the AMP-titanium surfaces demonstrated significant increase in fluorescence in comparison to control surfaces, which only showed minimal background fluorescence. Bacterial colonization, as measured by colony counts, was normalized to untethered control titanium. Incubation of bacteria in contact with both

AMP-titanium pins (both with linker and without) generated a statistically significant reduction in comparison to control surfaces. This reduction was similar to that seen for tethered vancomycin and tethered tetracycline, utilized as positive controls. Visualization of adherent bacteria using Live/DeadTM BacLight™ staining revealed a reduced colonization by both S. aureus and E. coli, similar to those seen with our positive control surfaces.

CONCLUSION: Aj3 and LL-37 can be covalently tethered to titanium surfaces. This tethered surface produces similar antimicrobial effects to tethered antibiotics previously described. Thus, this research provides an early proof of concept that tethered antimicrobial peptides might be capable of reducing PPI incidence on titanium prosthetic implants.

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