Regulation of matrix metalloproteinase secretion by green tea catechins in a three-dimensional co-culture model of macrophages and gingival fibroblasts.

OBJECTIVES: Elevated levels of matrix metalloproteinases (MMPs) have been associated with the active phases of tissue and bone destruction in periodontitis, an inflammatory disease characterized by a significant breakdown of tooth support. In the present study, we used a three-dimensional (3D) co-culture model of macrophages and gingival fibroblasts to investigate the ability of a green tea extract and its major constituent epigallocatechin-3-gallate (EGCG) to regulate the secretion of MMP-3, -8, and -9. METHODS: The 3D co-culture model was composed of gingival fibroblasts embedded in a type I collagen matrix overlaid with macrophages. Two arbitrary ratios were tested. The ratio composed of 1 macrophage to 10 fibroblasts was used to mimic a slightly inflamed periodontal site while the ratio composed of 10 macrophages to 1 fibroblast was used to mimic a severely inflamed periodontal site. The 3D co-culture model was pre-treated for 2h with either the green tea extract or EGCG. It was then stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS). The model was also first stimulated with LPS for 2h and then incubated with the green tea extract or EGCG. The concentrations of secreted MMP-3, -8, and -9 were quantified by enzyme-linked immunoassays. RESULTS: When the 3D co-culture model was stimulated with A. actinomycetemcomitans LPS, the 10:1 ratio of macrophages to gingival fibroblasts was associated with a highest secretion of MMP-3 and -9 and, to a lesser extent, MMP-8, than the 1:10 ratio. Non-cytotoxic concentrations of the green tea extract or EGCG reduced the basal secretion levels of all three MMPs. A 2-h treatment with the green tea extract or EGCG prior to the stimulation with LPS resulted in a dose-dependent decrease in MMP secretion, with MMP-9 showing the most significant decrease. A decrease in MMP secretion was also observed when the green tea extract or EGCG was added following a 2-h stimulation with LPS. CONCLUSIONS: Our results suggested that green tea catechins, and more specifically EGCG, offer promising prospects for the development of a novel adjunctive treatment for periodontitis because of their ability to decrease the secretion of MMPs, which are important tissue-destructive enzymes produced by mucosal and immune cells.

Ultrastructural changes in bacterial membranes induced by nano-assemblies ß-cyclodextrin chlorhexidine: SEM, AFM, and TEM evaluation.

Chemical hosts bind their guests by the same physical mechanisms as biomolecules and often display similarly subtle structure activity relationships. The cyclodextrins have found increasing application as inert, nontoxic carriers of active compounds in drug formulations. The present study was conducted to prepare inclusion complexes of chlorhexidine:ß-cyclodextrin (Cx:ß-cd), and evaluate their interactions with bacterial membrane through: scanning electron microscopy (SEM) and transmission electron microscopy (TEM); and measuring morphology alterations, roughness values, and cell weights by atomic force microscopy (AFM). It was found that the antimicrobial activity was significantly enhanced by cyclodextrin encapsulation. SEM analysis images demonstrated recognizable cell membrane structural changes and ultrastructural membrane swelling. By TEM, cellular alterations such as vacuolization, cellular leakage, and membrane defects were observed; these effects were enhanced at 1:3 and 1:4 Cx:ß-cd. In addition, AFM analysis at these ratios showed substantially more membrane disruption and large aggregates mixing with microorganism remains. In conclusion, nanoaggregates formed by cyclodextrin inclusion compounds create cluster-like structures with the cell membrane, possibly due to a hydrogen rich bonding interaction system with increasing surface roughness and possibly increasing the electrostatic interaction between cationic chlorhexidine with the lipopolysaccharides of Gram negative bacteria.

Protective effects of grape seed proanthocyanidins against oxidative stress induced by lipopolysaccharides of periodontopathogens.

BACKGROUND: During phagocytosis or stimulation with bacterial components, macrophages activate various cell processes, including the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are critical for successful defense against invading organisms. Increased levels of ROS/RNS create oxidative stress that results in tissue and bone destruction. Grape seed proanthocyanidins have been reported to possess a wide range of biologic properties against oxidative stress. In the present study, we investigated the effects of a grape seed proanthocyanidin extract (GSE) and commercial polyphenols on the production of ROS and RNS and on the protein expression of inducible nitric oxide synthase (iNOS) by murine macrophages stimulated with lipopolysaccharides (LPS) of periodontopathogens. METHODS: Macrophages (RAW 264.7) were treated with non-toxic concentrations of either GSE or commercial polyphenols (gallic acid [GA] and [-]-epigallocatechin-3-gallate [EGCG]) and stimulated with LPS of Actinobacillus actinomycetemcomitans or Fusobacterium nucleatum, and iNOS expression was evaluated by immunoblotting. Nitric oxide (NO) production was quantified using the colorimetric Griess assay, whereas ROS production was measured with the fluorescent 123-dihydrorhodamine dye. RESULTS: GSE strongly decreased NO and ROS production and iNOS expression by LPS-stimulated macrophages. GA also revealed a strong inhibitory effect on NO production without affecting iNOS expression but slightly increasing ROS production. EGCG showed an inhibitory effect on NO and ROS production and on iNOS expression by macrophages. CONCLUSION: Our findings demonstrate that proanthocyanidins have potent antioxidant properties and should be considered a potential agent in the prevention of periodontal diseases.