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Silicon Nitride Bioceramics Induce Chemically Driven Lysis in Porphyromonas Gingivalis Giuseppe Pezzotti, Ryan M. Bock, Bryan J. McEntire, Erin Jones, Marco Boffelli, Wenliang Zhu, Greta Baggio, Francesco Boschetto, Leonardo Puppulin, Tetsuya Adachi, Toshiro Yamamoto, Narisato Kanamura, Yoshinori Marunaka, and B. Sonny Bal Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00393 • Publication Date (Web): 05 Mar 2016 Downloaded from http://pubs.acs.org on March 14, 2016
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Silicon Nitride Bioceramics Induce Chemically Driven Lysis in Porphyromonas Gingivalis Giuseppe Pezzotti, 1 ,2 * Ryan M. Bock, 3 Bryan J. McEntire, 3 Erin Jones, 3 Marco Boffelli, 1 ,4 Wenliang Zhu, 5 Greta Baggio, 1,4 Francesco Boschetto, 1 ,4 Leonardo Puppulin, 2 Tetsuya Adachi, 4 Toshiro Yamamoto, 4 Narisato Kanamura, 4 Yoshinori Marunaka, 2 and B. Sonny Bal 3 ,6 1
Ceramic Physics Laboratory, Kyoto Institute of Technology, Sakyo-ku, Matsugasaki, 606-8126 Kyoto, Japan
2
Department of Molecular Cell Physiology, Graduate School of Medical
Science, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan 3
Amedica Corporation, 1885 West 2100 South, Salt Lake City, UT 84119 4
Department of Dental Medicine, Graduate School of Medical Science,
Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan 5
Department of Medical Engineering for Treatment of Bone and Joint Disorders, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0854, Japan
6Department of Orthopaedic Surgery, University of Missouri, Columbia, MO 65212
*Corresponding author; e-mail:
[email protected] 1 ACS Paragon Plus Environment
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Abstract Organisms of gram-negative phylum bacteroidetes, Porphyromonas gingivalis, underwent lysis on polished surfaces of silicon nitride (Si3N4) bioceramics. The antibacterial activity of Si3N4 was mainly the result of chemically driven principles. The lytic activity, although not osmotic in nature, was related to the peculiar pHdependent surface chemistry of Si3N4.
A buffering effect via the formation of
ammonium ions (NH4+) (and their modifications) was experimentally observed by pH microscopy. Lysis was confirmed by conventional fluorescence spectroscopy; and the bacteria’s metabolism was traced with the aid of in situ Raman microprobe spectroscopy. This latter technique revealed the formation of peroxynitrite within the bacterium itself.
Degradation of the bacteria’s nucleic acid, drastic reduction in
phenilalanine, and reduction of lipid concentration were observed due to short-term exposure (6 days) to Si3N4.
Altering the surface chemistry of Si3N4 by either
chemical etching or thermal oxidation influenced peroxynitrite formation and affected bacteria metabolism in different ways. Exploiting the peculiar surface chemistry of Si3N4 bioceramics could be helpful in counteracting Porphyromonas gingivalis in an alkaline pH environment. Keywords: lysis, Porphyromonas gingivalis, silicon nitride, bioceramics
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1. Introduction One of the main challenges in modern dentistry is the development of new methods for maintaining an effective defense against periodontitis.1 In its early stages, this disease can be reversed by effective personal oral hygiene. However, the general susceptibility of the host and the local susceptibility of the teeth are both factors that are difficult to control and stabilize due to the evolving nature of the bacteria themselves.2, 3 Recent research reports have shown improved strategies for curing advanced periodontitis (e.g., root planing combined with local administration of a hydrogen peroxide gel using customized trays,4 the use of xylitol as a stoichiometrically unfavorable substance for extensive acid – especially lactic acid – fermentation,5 local drug delivery including the use of an ethylene vinyl acetate fiber that contains tetracycline,6–11 a gelatin chip that contains chlorhexidine,12 and a minocycline polymer formulation as adjuncts to scaling and root planning13). A detailed understanding of the molecular chemistry governing the metabolic deterioration of oral bacteria subjected to these therapeutic agents is central to their optimization. It is from these molecular chemistry arguments that a new potential method for counteracting periodontitis was discovered.
It involves the lysis of
Porphyromonas gingivalis (PG, the bacterium primarily responsible for periodontitis) using the peculiar chemical reactions which occur at the surface of Si3N4 bioceramics. Raman spectroscopy, which is widely applied to the study of bacteria,14–18 offers the possibility to monitor in situ bacterial metabolism. It can be used to observe specific time-dependent biochemical changes in the metabolic activity of bacteria and thereby assess mechanisms related to their lysis. In other words, the Raman spectrum of a bacterium at a particular point in time represents a “fingerprint” of its overall biochemical composition and health. However, detailed spectroscopic reports on PG 3 ACS Paragon Plus Environment
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are unavailable in the published literature. Withnall et al.19 reported the Raman spectrum of the PG bacterium, but only in the limited spectral region between 1000 and 1700 cm-1, thereby missing a number of crucial bands related to the lowfrequency vibrational aspects of its DNA/RNA structures. Moreover, no complete labeling of the detected Raman bands was given in that study. Their focus was solely placed on a discussion of the bacterium pigment and the mechanism of its formation. Conversely, Yamanaka et al.20 made significant progress in characterizing PG by quantitatively detecting it in tooth pockets and saliva.
They used a portable
electrochemical DNA sensor, which directly detected the polymerase chain reaction. However, a full understanding of PG metabolism by Raman spectroscopy and, more importantly, an approach to curing periodontitis represent as yet unachieved challenges. In this paper, the metabolism of PG was assessed in situ using Raman spectroscopy both before and after exposure to chemistry-modulated Si3N4 surfaces.
From a
spectroscopic viewpoint, the three following purposes were pursued: (i) Obtaining a comprehensive spectroscopic view and accurate spectral deconvolution/labeling of the PG Raman spectrum; (ii) Interpreting PG bacterial metabolism through the observed variations of its Raman spectrum after exposure to Si3N4 surfaces with different chemical characteristics; and, (iii) Clarifying the mechanism behind the bacteriostatic behavior of Si3N4 bioceramics at the molecular scale. For this latter purpose, we augment previous findings, which first reported the antibacterial properties of Si3N4 spinal fusion implants.21, 22 Substantiating a link between bacterial metabolism and the chemical reactions occurring at Si3N4 surfaces is considered a promising step forward in establishing advanced dental implants with improved functionality.
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2. Experimental procedures 2.1. Silicon nitride samples and their salient characterizations Polished Si3N4 bioceramic disks containing Y2O3 and Al2O3 sintering aids (AMEDICA Corporation, Salt Lake City, UT 84119, USA) were received and employed in this study.
Some manufacturing details of this bioceramic are as
follows: Si3N4 powder (Ube SN E-10, Ube City, Japan) was first mixed with Y2O3 (Grade
C,
H.
C.
Starck,
Munich,
Germany)
and
Al2O3
(SA8-DBM,
Baikowski/Malakoff, Charlotte, NC) sintering aids and cold-pressed at room temperature into disk-shaped samples. Then, the cold-pressed disks were sintered in an ambient nitrogen atmosphere at a temperature in excess of 1700°C to closed porosity and further densified by hot isostatic pressing at a temperature exceeding 1650°C and N2 gas pressure of >200 MPa. The disk surfaces were lapped using 6 µm diamond (Engis, Wheeling, IL) on a lapping machine (Lapmaster, Mt. Prospect, IL), and subsequently polished using colloidal silica (Leco, St. Joseph, MI). All samples were subjected to ultrasonic cleaning in deionized water of 17.5 MΩ cm resistivity (750II, Myron L Company, Carlsbad, CA) for 30 min to remove contaminants. To probe the effect of surface chemistry on bacterial response, the polished surfaces of some sintered Si3N4 disks were modified either by wet chemical etching or by oxidation. In the former treatment, hydrofluoric acid (HF) conspicuously removed the amphoteric SiO2 layer without significantly etching the underlying nitride. This treatment maximized the concentration of amine groups at the surface, while pushing the surface composition as far to the nitride end of the nitride-oxide spectrum as possible.
Polished samples were immersed in a 5 wt.% HF solution for 45 s,
transferred into a continuously refreshed DI water bath for 30 min, dried under a
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stream of filtered N2, and stored in a desiccator containing hygroscopic media (Indicating Drierite, W.A. Hammond DRIERITE Co., Xenia, OH) under partial vacuum (~100 Torr) to slow spontaneous re-oxidation. Conversely, the oxidation treatment yielded the maximum concentration of hydroxyl groups and pushed the surface composition as far to the oxide end of the nitride-oxide spectrum as possible. In this case, polished samples were kept for 7 h at 1070°C using an open-air kiln (Deltech, Denver, CO). X-ray photoelectron spectroscopy (XPS), shown in details of a previous study,23 revealed quite different atomic ratios of N/Si and O/Si in the variously treated Si3N4 samples. Indeed, the XPS analyses confirmed that amine-rich and hydroxyl-rich surfaces were obtained by wet-etching and thermal oxidation, respectively. The N/Si (O/Si) ratios were 1.22 (0.17), 1.05 (0.15), and 0.09 (1.98) for the as-sintered (polished), HF-etched, and oxidized samples, respectively. Wetting angle measurements were carried out in an optical comparator (2600 Series, S-T Industries, St. James, MN) with built-in goniometer functionality. Static contact angles were measured using deionized water droplets with a fixed volume (VWR Signature Variable Volume Pipette, VWR, Radnor, PA) of 25 µ L. Eight measurements were taken per each sample with angles being measured on both sides of the projected image of each droplet. Streaming zeta-potential (ζ-) measurements were performed using an electrokinetic analyzer (SurPASS, Anton-Paar USA, Ashland, VA). A background electrolyte of 1 mM HCl, which exhibited a natural pH of 5.5, was used throughout all experiments. Observed streaming potentials were converted into ζ -potentials using the HelmholzSmoluchowski equation.24 As explicitly shown in a previously published paper,23 the isoelectric points of the as-sintered, HF-etched, and thermally oxidized samples were 5.6, 5.4, and
