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Category: Research

BioXclude “Kills” S. Gordonii w/ Histone H2A/H2B, Cathelicidin, Lactoferrin and Lysozyme - New Poster Presentation, Update from AAP

I am pleased to share an exciting poster presentation on BioXclude® from the on-going annual American Academy of Periodontology meeting. Click to View the Poster.
Brummerhop et al. identified the presence of multiple antimicrobial proteins in Purion®-processed dehydrated human deepithelialized amnion-chorion membrane (ddACM) including histone H2A/H2B, cathelicidin (LL-37), lactoferrin, and lysozyme.
When tested in a quantitative in vitro assay, these factors showed antimicrobial properties with variable potencies against S. gordonii. When used together, however, these factors effectively killed the bacteria in culture as evidenced by 0% of the bacteria surviving the treatment, indicating that the synergistic activities of these factors are key to BioXclude’s success in preventing infections.
I love good science and research! It is one of Snoasis Medical’s core values. As such, I want to thank Dr. Brummerhop for conducting this exciting research and express my sincere appreciation to Dr. Bing and the periodontal program at Texas Health Sciences Center at Houston for the contribution in providing technically valid data on BioXclude to our customers.


Synergistic Antimicrobial Components of Human Derived Composite Amnion-Chorion Membrane
Alexandra Brummerhop, Chun-Teh Lee, Gena Tribble, Robin Weltman, Ransome Van Der Hoeven,
Jian-Ming Hong, Bing-Yan Wang
The University of Texas Health Science Center at Houston School of Dentistry

Background
The ultimate goal in periodontal therapy is regeneration, which is reproducing the original architecture and function of lost periodontal tissues around teeth. 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. One of the most common complications is membrane exposure that can be detrimental to regenerative outcomes. Therefore, a barrier product to isolate a defect and provide antimicrobial protection would be beneficial for these procedures. Previous studies 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.
Alexandra Brummerhop
DMD (Houston, TX)
Alexandra Brummerhop
DMD (Houston, TX)

Background
The ultimate goal in periodontal therapy is regeneration, which is reproducing the original architecture and function of lost periodontal tissues around teeth. 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. One of the most common complications is membrane exposure that can be detrimental to regenerative outcomes. Therefore, a barrier product to isolate a defect and provide antimicrobial protection would be beneficial for these procedures. Previous studies 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 individually or in combination 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 at 200x magnification. In addition, viable cell count was carried out as a parallel assessment. The mixtures of S. gordonii Challis remaining in the wells were added into Eppendorf tubes for serial titrations and plated onto mitas salivarius plates. After 24 to 48 hour incubation, the bacterial colonies were counted manually.

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 incubation.

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.

Figure 1. Antimicrobial components identified in human amnion-chorion membranes. A total of 1007 proteins were identified in three different membranes tested (represented as m1, m2 and m3) via mass spectrometry. 554 proteins (55%) shared amongst all the membranes. Proteins with antimicrobial properties were identified based on literature search.
Antimicrobial Proteins in ACM
Lactoferrin
Histone 2A
Histone 2B
Cathlecidin (LL-37)
Lysonzyme
Figure 1. Antimicrobial components identified in human amnion-chorion membranes. A total of 1007 proteins were identified in three different membranes tested (represented as m1, m2 and m3) via mass spectrometry. 554 proteins (55%) shared amongst all the membranes. Proteins with antimicrobial properties were identified based on literature search.
Antimicrobial Proteins in ACM
Lactoferrin
Histone 2A
Histone 2B
Cathlecidin (LL-37)
Lysonzyme



 8hr
Incubation
24hr
Incubation
Control
(THB)
2.54 x 107 ±
1.14 x 106
3.95 x 107 ±
4.67 x 105
Histones
(2?M)
1.52 x 107 ±
4.67 x 105
2.74 x 107 ±
5.26 x 106
Lactoferrin
(5mg/ml)
2.77 x 105 ±
3.06 x 104
2.66 x 105 ±
1.24 x 104
LL-37
(1mg/ml)
2.48 x 105 ±
1.20 x 105
5.50 x 102 ±
2.59 x 102
Lysonzyme
(10mg/ml)
2.81 x 105 ±
1.09 x 104
8.95 x 104 ±
5.50 x 104
Figure 2. Efficacy of single antimicrobial peptide in killing S. gordonii. S. gordonii cells were cultured with individual antimicrobial peptide for 8 or 24 hours, Live/Dead stained, and analyzed with confocal microscopy at 200x magnification. Green: live, Red: dead bacteria. In addition, viable cell count was carried out as a parallel assessment (Table). Lactoferrin, LL-37, and lysozyme at indicated concentrations significantly eliminated S. gordonii at both time points, relative to controls and histones (P 0.0001, ANOVA).
 8hr
Incubation
24hr
Incubation
Control
(THB)
2.54 x 107 ±
1.14 x 106
3.95 x 107 ±
4.67 x 105
Histones
(2?M)
1.52 x 107 ±
4.67 x 105
2.74 x 107 ±
5.26 x 106
Lactoferrin
(5mg/ml)
2.77 x 105 ±
3.06 x 104
2.66 x 105 ±
1.24 x 104
LL-37
(1mg/ml)
2.48 x 105 ±
1.20 x 105
5.50 x 102 ±
2.59 x 102
Lysonzyme
(10mg/ml)
2.81 x 105 ±
1.09 x 104
8.95 x 104 ±
5.50 x 104

 8hr
Incubation
24hr
Incubation
Combo0 ± 00 ± 0
X2 Dilution0 ± 00 ± 0
X5 Dilution2.86 x 103 ±
2.55 x 101
4.42 x 104 ±
1.50 x 104
Control
(THB)
2.94 x 101 ±
1.14 x 106
4.23 x 107 ±
1.37 x 106

Figure 3. Efficacy of antimicrobial peptide combination and ACM in killing S. gordonii. S. gordonii cells were cultured with mixtures of lactoferrin, LL-37, histones, and lysozyme at the same concentration as in individual assays, with x2 and x5 dilutions, and with 5mm diameter ACM for 8 or 24 hours, Live/Dead stained, and analyzed with confocal microscopy at 200x magnification. Green: live, Red: dead bacteria. Viable cell count was carried out as a parallel assessment (Table). ACM and the combinations were more efficacious in killing S. gordonii cells than any single peptide.
 8hr
Incubation
24hr
Incubation
Combo0 ± 00 ± 0
X2 Dilution0 ± 00 ± 0
X5 Dilution2.86 x 103 ±
2.55 x 101
4.42 x 104 ±
1.50 x 104
Control
(THB)
2.94 x 101 ±
1.14 x 106
4.23 x 107 ±
1.37 x 106



Warm Regards,
Snoasis Medical

Robert Tofe
President & Founder