Lianrui Chu

Development of an antimicrobial resin—A pilot study

OBJECTIVES To demonstrate that silver nanoparticles (AgNPs) could be synthesized in situ in acrylic dental resins. METHODS Light-cure (LC; bisphenol A glycidyl methacrylate, tetraethyleneglycol dimethacrylate, bisphenol A ethoxylate dimethacrylate blend) and chemical-cure systems (CC; orthodontic denture resin) were used to synthesize AgNPs using different concentrations of Ag benzoate (AgBz). RESULTS Rockwell hardness for LC resins showed that resins could be cured with up to 0.15% AgBz, while the hardness of CC resins were unaffected in the concentrations tested. UV-Vis spectroscopy and transmission electron microscopy confirmed the presence of AgNPs in both LC and CC resins. Generally, CC resins had better distribution of and much smaller AgNPs as compared to LC resins overall. In some samples, especially in LC resins, nanoclusters were visible. An in vitro release study over four-weeks showed that CC resins released the most Ag(+) ions, with release detected in all samples. However, LC resins only released Ag(+) ions when AgBz concentration was greater than 0.1% (w/w). AgNP-loaded CC resins made with 0.2 and 0.5% (w/w) AgBz were tested for antibacterial activity in vitro against Streptococcus mutans, and results showed 52.4% and a 97.5% bacterial inhibition, respectively. Further work is now warranted to test mechanical properties and to optimize the initiator system to produce commercially useful dental and medical resins. SIGNIFICANCE Success in this work could lead to a series of antimicrobial medical and dental biomaterials that can prevent secondary caries and infection of implants.

Hydrogen sulfide induces apoptosis in human periodontium cells

BACKGROUND AND OBJECTIVE The existence of hydrogen sulfide (H(2)S) at high concentrations in periodontal pockets is a characteristic feature of periodontitis. Periodontal pathogens play a key role in the production of H(2)S under these etiology conditions. This study was designed to examine the cytotoxicity of H(2)S in periodontium cells, including human periodontal ligament (PDL) cells and human gingival fibroblasts (HGFs), as well as the role of H(2)S in apoptosis induction. MATERIAL AND METHODS Human PDL cells and HGFs were cultured in the presence of Na(2)S/HCl or in the presence of H(2)S produced enzymatically by the action of Treponema denticola cystalysin (l-cysteine desulfhydrase) on l-cysteine. Apoptosis was assessed morphologically after nuclear staining with DAPI or was quantified by flow cytometry after staining with annexin V. Caspase activation was measured by an enzymatic assay using DEVD-AMC, a synthetic caspase substrate. RESULTS Among the three products obtained following degradation of l-cysteine by T. denticola cystalysin, only H(2)S induced significant apoptosis in HGF cells. Hydrogen sulfide also induced typical apoptotic morphology in cultured PDL cells. The changes were dependent on the H(2)S dose and on the treatment time with H(2)S. Hydrogen sulfide-induced apoptosis was also confirmed by staining with annexin V and propidium iodide. In addition, treatment with H(2)S led to caspase activation in these cells. CONCLUSION These results showed that physiological concentrations of H(2)S can induce apoptosis of PDL cells and HGFs in periodontitis, suggesting that H(2)S may play an important role in periodontal tissue damage in periodontal diseases.

Antimicrobial acrylic materials with in situ generated silver nanoparticles

UNLABELLED Polymethyl methacrylate (PMMA) is widely used to treat traumatic head injuries (cranioplasty) and orthopedic injuries (bone cement), but there is a problem with implant-centered infections. With organisms such as Acinetobacter baumannii and methicillin-resistant staphylococcus aureus developing resistance to antibiotics, there is a need for novel antimicrobial delivery mechanisms without risk of developing resistant organisms. OBJECTIVES To develop a novel antimicrobial implant material by generating silver nanoparticles (AgNP) in situ in PMMA. RESULTS All PMMA samples with AgNPs (AgNP-PMMA) released Ag(+) ions in vitro for over 28 days. In vitro antimicrobial assays revealed that these samples (even samples with the slowest release rate) inhibited 99.9% of bacteria against four different strains of bacteria. Long-term antimicrobial assay showed a continued antibacterial effect past 28 days. Some AgNP-loaded PMMA groups had comparable Durometer-D hardness (a measure of degree of cure) and modulus to control PMMA, but all experimental groups had slightly lower ultimate transverse strengths. CONCLUSIONS AgNP-PMMA demonstrated a tremendously broad-spectrum and long-intermediate-term antimicrobial effect with comparable mechanical properties to control PMMA. Current efforts are focused on further improving mechanical properties by reducing AgNP loading and assessing fatigue properties.

Role of Glutathione Metabolism of Treponema denticola in Bacterial Growth and Virulence Expression

ABSTRACT Hydrogen sulfide (H2S) is a major metabolic end product detected in deep periodontal pockets that is produced by resident periodontopathic microbiota associated with the progression of periodontitis. Treponema denticola, a member of the subgingival biofilm at disease sites, produces cystalysin, an enzyme that catabolizes cysteine, releasing H2S. The metabolic pathway leading to H2S formation in periodontal pockets has not been determined. We used a variety of thiol compounds as substrates for T. denticola to produce H2S. Our results indicate that glutathione, a readily available thiol source in periodontal pockets, is a suitable substrate for H2S production by this microorganism. In addition to H2S, glutamate, glycine, ammonia, and pyruvate were metabolic end products of metabolism of glutathione. Cysteinyl glycine (Cys-Gly) was also catabolized by the bacteria, yielding glycine, H2S, ammonia, and pyruvate. However, purified cystalysin could not catalyze glutathione and Cys-Gly degradation in vitro. Moreover, the enzymatic activity(ies) in T. denticola responsible for glutathione breakdown was inactivated by trypsin or proteinase K, by heating (56°C) and freezing (−20°C), by sonication, and by exposure to Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK). These treatments had no effect on degradation of cysteine by the purified enzyme. In this study we delineated an enzymatic pathway for glutathione metabolism in the oral spirochete T. denticola; our results suggest that glutathione metabolism plays a role in bacterial nutrition and potential virulence expression.

A 52-kDa Leucyl Aminopeptidase from Treponema denticola Is a Cysteinylglycinase That Mediates the Second Step of Glutathione Metabolism

The metabolism of glutathione by the periodontal pathogen Treponema denticola produces hydrogen sulfide, which may play a role in the host tissue destruction seen in periodontitis. H2S production in this organism has been proposed to occur via a three enzyme pathway, γ-glutamyltransferase, cysteinylglycinase (CGase), and cystalysin. In this study, we describe the purification and characterization of T. denticola CGase. Standard approaches were used to purify a 52-kDa CGase activity from T. denticola, and high pressure liquid chromatography electrospray ionization tandem mass spectrometry analysis of this molecule showed that it matches the amino acid sequence of a predicted 52-kDa protein in the T. denticola genome data base. A recombinant version of this protein was overexpressed in and purified from Escherichia coli and shown to catalyze the hydrolysis of cysteinylglycine (Cys-Gly) with the same kinetics as the native protein. Surprisingly, because sequence homology indicates that this protein is a member of a family of metalloproteases called M17 leucine aminopeptidases, the preferred substrate for the T. denticola protein is Cys-Gly (kcat/Km of 8.2 μm–1 min–1) not l-Leu-p-NA (kcat/Km of 1.1 μm–1 min–1). The activity of CGase for Cys-Gly is optimum at pH 7.3 and is enhanced by Mn2+, Co2+, or Mg2+ but not by Zn2+ or Ca2+. Importantly, in combination with the two other previously purified T. denticola enzymes, γ-glutamyltransferase and cystalysin, CGase mediates the in vitro degradation of glutathione into the expected end products, including H2S. These results prove that T. denticola contains the entire three-step pathway to produce H2S from glutathione, which may be important for pathogenesis.

Systemic inflammatory responses in progressing periodontitis during pregnancy in a baboon model

This study tested the hypothesis that pregnant female baboons exhibit increased levels of various inflammatory mediators in serum resulting from ligature‐induced periodontitis, and that these profiles would relate to periodontal disease severity/extent in the animals. The animals were sampled at baseline (B), mid‐pregnancy (MP; two quadrants ligated) and at delivery (D; four quadrants ligated). All baboons developed increased plaque, gingival inflammation and bleeding, pocket depths and attachment loss following placement of the ligatures. By MP, both prostaglandin E2 (PGE2) and bactericidal permeability inducing factor (BPI) were greater than baseline, while increased levels of interleukin (IL)‐6 occurred in the experimental animals by the time of delivery. IL‐8, MCP‐1 and LBP all decreased from baseline through the ligation phase of the study. Stratification of the animals by baseline clinical presentation demonstrated that PGE2, LBP, IL‐8 and MCP‐1 levels were altered throughout the ligation interval, irrespective of baseline clinical values. IL‐6, IL‐8 and LBP were significantly lower in the subset of animals that demonstrated the least clinical response to ligation, indicative of progressing periodontal disease. PGE2, macrophage chemotactic protein (MCP)‐1, regulated upon activation, normal T cell expressed and secreted (RANTES) and LBP were decreased in the most diseased subset of animals at delivery. Systemic antibody responses to Fusobacterium nucleatum, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Campylobacter rectus were associated most frequently with variations in inflammatory mediator levels. These results provide a profile of systemic inflammatory mediators during ligature‐induced periodontitis in pregnant baboons. The relationship of the oral clinical parameters to systemic inflammatory responses is consistent with a contribution to adverse pregnancy outcomes in a subset of the animals.

Cystalysin, a 46-kDa L-Cysteine Desulfhydrase from Treponema denticola: Biochemical and Biophysical Characterization

A 46-kDa hemolytic protein referred to as cystalysin, from Treponema denticola ATCC 35404, was characterized and overexpressed in Escherichia coli LC-67. Cystalysin lysed erythrocytes, hemoxidized hemoglobin to sulfhemoglobin and methemoglobin, and removed the sulfhydryl and amino group from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. With L-cysteine as substrate, cystalysin obeys Michaelis-Menten kinetics. Cystathionine and s-aminoethyl-L-cysteine were also substrates. Several of the small alpha amino acids were found to be competitive inhibitors of cystalysin. The enzymatic activity was increased by beta-mercaptoethanol and was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme with the activity of an alphaC-N and betaC-S lyase (cystathionase). Since high amounts of H2S have been reported in deep periodontal pockets, this metabolic enzyme from T. denticola may also function in vivo as an important virulence molecule.

Role for Recombinant γ-Glutamyltransferase from Treponema denticola in Glutathione Metabolism

ABSTRACT Volatile sulfur compounds, including hydrogen sulfide (H2S), have been implicated in the development of periodontal disease. Glutathione is an important thiol source for H2S production in periodontal pockets. Our recent studies have delineated a pathway of glutathione metabolism in Treponema denticola that releases H2S. In this pathway, γ-glutamyltransferase (GGT) has been proposed to catalyze the first step of glutathione degradation. We have cloned the gene of GGT from T. denticola, which contains an open reading frame of 726 bp encoding a protein of 241 amino acids. Transformation of this gene into Escherichia coli led to the expression of a recombinant protein. After purification by chromatography, the recombinant protein showed enzymatic activity typical of GGT, catalyzing the degradation of Na-γ-glutamyl-4-nitroaniline (GNA) and the hydrolysis of glutathione, releasing glutamic acid or glutamine and cysteinylglycine. l-Cysteine is not a substrate of GGT. Importantly, GNA, when added to T. denticola, was able to compete with glutathione and inhibit the production of H2S, ammonia, and pyruvate. This was accompanied by the suppression of hemoxidative and hemolytic activities of the bacteria. Purified GGT was inactivated by TLCK (Nα-p-tosyl-l-lysine chloromethyl ketone) and proteinase K treatment. However, higher enzymatic activity was demonstrated in the presence of 2-mercaptoethanol and dithiothreitol. Our further experiments showed that the addition of recombinant GGT to Porphyromonas gingivalis, a bacterium without significant glutathione-metabolizing capacity, drastically increased the utilization of glutathione by the bacterium, producing H2S, ammonia, and pyruvate. This was again accompanied by enhanced bacterial hemoxidative and hemolytic activities. Together, the results suggest an important role for GGT in glutathione metabolism in oral bacteria.

Novel mechanism for conditional aerobic growth of the anaerobic bacterium Treponema denticola.

ABSTRACT Treponema denticola, a periodontal pathogen, has recently been shown to exhibit properties of a facultative anaerobic spirochete, in contrast to its previous recognition as an obligate anaerobic bacterium. In this study, the capacity and possible mechanism of T. denticola survival and growth under aerobic conditions were investigated. Factors detrimental to the growth of T. denticola ATCC 33405, such as oxygen concentration and hydrogen sulfide (H2S) levels as well as the enzyme activities of gamma-glutamyltransferase, cysteinylglycinase, and cystalysin associated with the cells were monitored. The results demonstrated that T. denticola grew only at deeper levels of broth (≥3 ml in a 10-ml tube), high inoculation ratios (≥20% of culture in medium), and short cultivation times (≤4 days for one passage) and in media containing l-cysteine or glutathione as the substrate for H2S production during aerobic growth. The determination of the factors showed that oxygen levels were always lower (0 to 0.6%) with significantly higher concentrations of H2S and higher activities of the three enzymes in all cultures grown aerobically. Further data revealed that H2S production from the T. denticola enzymes plus their substrates resulted in removal of dissolved O2 in the growth cultures in a dose-dependent manner. These results demonstrated that T. denticola was able to generate microanaerobic environments in growth media for its survival and growth under aerobic conditions. Furthermore, the organism can be defined as a true obligate anaerobic spirochete. These findings suggest that spirochetes may play a significant role in maintaining the anaerobic environment at diseased sites in periodontitis.

Impact of Mucosal Inflammation on Oral Simian Immunodeficiency Virus Transmission

ABSTRACT Mucosal tissues are the primary route of transmission for most respiratory and sexually transmitted diseases, including human immunodeficiency virus (HIV). There is epidemiological evidence that genital mucosal inflammation leads to enhanced HIV type 1 (HIV-1) transmission. The objective of this study was to assess the influence of periodontal inflammation on oral HIV transmission using a nonhuman primate model of teeth ligature-induced periodontitis. Simian immunodeficiency virus (SIV) was nontraumatically applied to the gingiva after moderate gingivitis was identified through clinical and immunologic analyses (presence of inflammatory cytokines). Overall oral SIV infection rates were similar in the gingivitis-induced and control groups (5 infections following 12 SIV administrations for each), although more macaques were infected with multiple viral variants in the gingivitis group. SIV infection also affected the levels of antiviral and inflammatory cytokines in the gingival crevicular fluid, and a synergistic effect was observed, with alpha interferon and interferon-inducible protein 10 undergoing significant elevations following SIV infection in macaques with gingivitis compared to controls. These increases in antiviral and inflammatory immune modulators in the SIV-infected gingivitis macaques could also be observed in blood plasma, although the effects at both compartments were generally restricted to the acute phase of the infection. In conclusion, while moderate gingivitis was not associated with increased susceptibility to oral SIV infection, it resulted in elevated levels of cytokines in the oral mucosa and plasma of the SIV-infected macaques. These findings suggest a synergy between mucosal inflammation and SIV infection, creating an immune milieu that impacts the early stages of the SIV infection with potential implications for long-term pathogenesis.