Synergistic Antimicrobial Components of Human Derived Composite Amnion-Chorion Membrane

Objectives: Barrier membranes are essential in guided tissue regeneration and guided bone regeneration to exclude fast growing epithelial and connective tissue cells and thereby, allow bone regeneration. We have previously demonstrated that a human derived amnion-chorion membrane (ACM) has endogenous antimicrobial properties, a unique property that may prevent colonization and survival of oral bacteria on exposed membranes. The current project is to decipher the mechanism by identifying the components of ACM that render the antibacterial property. In addition, antimicrobial efficacy of these identified components on Streptococcus gordonii was assessed.
Methods: Proteins in ACM were identified via mass spectrometry (MS). Those components with reported antimicrobial properties were then assessed for their efficacy in killing S. gordonii Challis. Log-phased bacterial cells (2 x 10⁵) were cultured with the commercially available peptides (identified in ACM with reported antimicrobial properties), either in combinations or individually, at different concentrations in Todd Hewitt Broth. After incubations of 8 or 24 hours, bacteria were stained with Live/Dead viability kit, component B and analyzed with confocal microscopy. In addition, viable cell count was carried out, as a parallel assessment.
Results: MS identified the presence of 4 antimicrobial proteins -- histone H2A/H2B, cathelicidin (LL-37), lactoferrin and lysozyme, in ACM. The combination of these proteins effectively killed S. gordonii Challis in a dose-dependent fashion, after 8h or 24h incubation. When each protein was tested individually, they killed S. gordonii Challis at a much lower efficacy, relative to the combinations. While bacteria cultured on the intact ACM, 0% of bacteria survived at 8h and 24h of incubations.
Conclusions:
There are at least 4 antimicrobial proteins present in ACM. The combination of these 4 proteins or the intact ACM membrane kill oral streptococci much more efficiently than any single protein, indicating a synergistic mechanism.