Alternative plasticizer, 4-cyclohexene-1,2-dicarboxylic acid dinonyl ester, for blood containers with protective effects on red blood cells and improved cold resistance.

Di (2-ethylhexyl) phthalate (DEHP), a typical plasticizer used for polyvinyl chloride (PVC), is eluted from PVC-made blood containers and protects against red blood cell (RBC) hemolysis. However, concerns have arisen regarding the reproductive and developmental risks of DEHP in humans, and the use of alternative plasticizers for medical devices has been recommended worldwide. In this study, we propose that the use of a novel plasticizer, 4-cyclohexene-1,2-dicarboxylic acid dinonyl ester (DL9TH), could help produce more useful and safe blood containers. PVC sheet containing DL9TH and di (2-ethylhexyl) 4-cyclohexene-1,2-dicarboxylate (DOTH) provides comparable or superior protective effects to RBCs relative to PVC sheet containing DEHP or di-isononyl-cyclohexane-1,2-dicarboxylate (DINCH® , an alternative plasticizer that has been used in PVC sheets for blood containers). The total amount of plasticizer eluted from DOTH/DL9TH-PVC sheets is nearly the same as that eluted from DEHP-PVC sheets. In addition, DOTH/DL9TH-PVC has better cold resistance than DEHP- and DINCH® -PVC sheets. In vitro and in vivo tests for biological safety based on International Organization for Standardization guidelines (10993 series) suggest that the DOTH/DL9TH-PVC sheet can be used safely. Subchronic toxicity testing of DL9TH in male rats in accordance with the principles of Organisation for Economic Co-operation and Development Test Guideline 408 showed that DL9TH did not induce adverse effects up to the highest dose level tested (717 mg/kg body weight/day). There were no effects on testicular histopathology and sperm counts, and no indications of endocrine effects: testosterone, thyroid-stimulating hormone, follicle-stimulating hormone, and 17ß-estradiol were unchanged by the treatment, compared with the control group. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1052-1063, 2018.

Antibacterial Action of a Condensed Tannin Extracted from Astringent Persimmon as a Component of Food Addictive Pancil PS-M on Oral Polymicrobial Biofilms.

The purpose of this study was to evaluate the antibacterial activity against polymicrobial (PM) biofilms of a condensed tannin extracted from astringent persimmon (PS-M), which is contained in refreshing beverages commercially available in Japan. Salivary PM biofilms were formed anaerobically on glass coverslips for 24 and 72 h and were treated for 5 min with sterilized deionized water (DW), 0.05 and 0.2 wt% chlorhexidine digluconate (CHX), and 0.5-4.0 wt% PS-M solution. The colony forming units (CFU/mL) were determined and morphological changes of the biofilms were observed by scanning electron microscopy (SEM). The CFUs were lower in all PS-M and CHX groups compared to the DW group. PS-M exerted a dose-dependent effect. PS-M (1.53 × 10(7)) at a dose of 4.0 wt% had the same effect as 0.2 wt% CHX (2.03 × 10(7)), regardless of the culture period. SEM revealed the biofilm structures were considerably destroyed in the 4.0 wt% PS-M and 0.2 wt% CHX. These findings indicate that the antibacterial effects of PS-M, a naturally derived substance, are comparable to those of CHX. PS-M may keep the oral cavity clean and prevent dental caries and periodontal disease related to dental plaque, as well as systemic disease such as aspiration pneumonitis.

Formation of subsurface dentin lesions using a polymicrobial biofilm model.

PURPOSE: To simulate an oral demineralization environment by multiple species of bacteria by inducing subsurface dentin lesions with a polymicrobial biofilm model. METHODS: Polymicrobial biofilms consisting of multiple species of bacteria were generated from stimulated saliva using a high-throughput active attachment model. Biofilms were grown on dentin specimens in McBain medium containing 0, 0.2 or 2.5 ppm F and on glass without fluoride for 192 hours. The medium was refreshed twice daily, after 10 and 14 hours, until 72 hours, followed by treatment for 5 minutes with 400 ppm fluoride. Specimens were recovered after 192 hours. The number of colony forming units (CFU) was measured, and integrated mineral loss (IML) was determined by transversal microradiography. RESULTS: Mineral profiles in specimens grown with 0.2F and 2.5F revealed surface layers and initial lesions distinct from those grown with 0F. IML was significantly lower with 0.2F and 2.5F than with 0F (P < 0.05), although CFUs were similar. CFUs of biofilms grown on dentin in medium containing 0F were 10-fold higher than on glass (P < 0.05). Subsurface lesions on dentin formed consistently, with their growth progression inhibited by application of fluoride. To our knowledge, this is the first report describing the induction of subsurface dentin lesions by a polymicrobial biofilm model, and this model may be useful for studies of demineralization supporting in situ and in vivo models.

Antimicrobial effects of carbamide peroxide against a polymicrobial biofilm model.

PURPOSE: To investigate the in vitro antimicrobial effects of carbamide peroxide (CP) and CP-based home bleaching agents against polymicrobial (PM) biofilms. METHODS: Using a high-throughput active attachment model, PM biofilms were cultured on glass coverslips by diluting the stimulated saliva of one healthy adult. All experiments were performed anaerobically in McBain medium, which was refreshed twice daily. After biofilm formation for 24 or 72 hours, the biofilms were treated with 0.5%, 2.5%, 5%, or 10% CP, 20-fold dilutions of HiLite Shade Up (HS) or Opalescence Regular (OR), 0.2% chlorhexidine digluconate (CHX), 0.2% NaF, or deionized water (n = 10 each). Biofilms were dispersed and the number of colony forming units (CFU) was measured on tryptic soy agar blood plates. Coverslips containing 72-hour biofilms treated with 0.5% and 10% CP and deionized water were stained and scanned by confocal laser scanning microscopy (CLSM). RESULTS: Treatment of 24- and 72-hour biofilms with HS, OR and CH yielded significantly fewer colonies than treatment with water or 0.2% NaF. No growing colonies were observed after treatment with 10% CP. CLSM showed that the percentage of dead bacteria increased as the concentration of CP increased.

Peptidoglycan of Actinomyces naeslundii induces inflammatory cytokine production and stimulates osteoclastogenesis in alveolar bone resorption.

OBJECTIVE: Actinomyces naeslundii, plays an important role in forming dental biofilms and causes gingival inflammation. Although peptidoglycan, the major cell wall component of Gram-positive bacteria, has been demonstrated to induce inflammatory cytokines, little is known about the association of peptidoglycan with alveolar bone resorption. This study investigated the involvement of peptidoglycan from A. naeslundii in osteoclast formation and bone resorption. DESIGN: Osteoclast formation and function induced by peptidoglycan of A. naeslundii T14V were examined using the co-culture system of MCTC3/PA6 cells and BALB/c mouse bone marrow cells. Osteoclast formation was evaluated to count TRAP-positive multi-nuclei cells as osteoclasts. The function of osteoclasts was assessed by measuring the areas of pits absorbed. Inflammatory cytokine genes expressions, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α, were examined by RT-PCR analysis using murine peritoneal macrophages. Experimental periodontitis was performed in Sprague-Dawley rats orally infected with A. naeslundii. RESULTS: TRAP-positive multi-nuclei cells and the areas of pits induced by peptidoglycan were significantly greater than controls (p<0.01). Gene expression levels of IL-1ß, IL-6, and TNF-α induced by A. naeslundii PGN were stronger than controls. In experimental periodontitis, bone loss of A. naeslundii-infected rats was comparable to that of rats induced by Porphyromonas gingivalis, which has been reported to be a periodontal pathogenic agent, being significantly greater than that of the sham group (p<0.01). CONCLUSIONS: These results suggest that peptidoglycan of A. naeslundii is an important virulence factor in the development of periodontitis.

Purification and characterization of a novel secondary fimbrial protein from Porphyromonas gulae.

BACKGROUND: Porphyromonas gulae are black-pigmented anaerobic bacteria isolated from the gingival sulcus of various animal hosts and are distinct from Porphyromonas gingivalis originating in humans. We previously reported the antigenic similarities of 41-kDa fimbriae between P. gulae ATCC 51700 and P. gingivalis ATCC 33277. In this study, to clarify the presence of another type of fimbriae of P. gulae, we have purified and characterized the secondary fimbrial protein from P. gulae ATCC 51700. METHODS: The secondary fimbrial protein was purified from P. gulae ATCC 51700 using an immunoaffinity column coupling with antibodies against the 41-kDa fimbrial protein. The expression of fimbriae on the cell surface of P. gulae ATCC 51700 was investigated by transmission electron microscopy. The N-terminal amino acid sequence was determined by an amino acid sequencer system. RESULTS: The molecular mass of this protein was approximately 53-kDa, as estimated by SDS-PAGE. The polyclonal antibodies against the 53-kDa protein did not react with the 41-kDa fimbrial protein of P. gulae ATCC 51700. Immunogold electron microscopy revealed that anti-53-kDa fimbrial serum bound to fimbria on the cell surface of P. gulae ATCC 51700. The amino acid sequence of the N-terminal 15 residues of the 53-kDa fimbrial protein showed only 1 of 15 residues identical to the 41-kDa fimbrial protein. CONCLUSION: The 53-kDa fimbriae are different in molecular weight and antigenicity from the 41-kDa fimbrial protein of P. gulae ATCC 51700. These results clearly suggest that the 41-kDa and the 53-kDa fimbriae are distinct types of fimbriae expressed simultaneously by this organism.

Quest for anti-inflammatory substances using IL-1ß-stimulated gingival fibroblasts.

BACKGROUND: We have previously reported that azulene-related compounds, and alkaline extract of Sasa senanensis Rehder potently inhibited nitric oxide (NO) production by lipopolysaccharide (LPS)-stimulated mouse macrophages. We investigated here whether they can inhibit pro-inflammatory cytokine production, by activated human gingival fibroblast (HGF). MATERIALS AND METHODS: HGF was established from the periodontal tissues of extracted tooth. Viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Production of Prostaglandin E(2) (PGE(2)) and cytokines was determined by enzyme immunoassay, and enzyme-linked immunosorbent assay, respectively. RESULTS: Interleukin (IL)-1ß did not inhibit, but rather slightly stimulated the growth of HGF cells. IL-1ß stimulated the production of PGE(2), IL-6, IL-8 and monocyte chemotactic protein-1 very potently, but not that of nitric oxide and tumor necrosis factor-α. Native LPS and synthetic lipid A from E. coli and P. gingivalis was much less stimulatory. Dexamethasone, not indomethacin, was an efficient inhibitor of IL-8 production. Among five azulene-related compounds, benzo[b]cyclohepta[e][1,4]thiazine most potently inhibited the IL-8 production by HGF cells, as well as NO production by activated RAW264.7 cells. The alkaline extract of Sasa senanensis Rehder significantly inhibited IL-8 production, without affecting the cell viability. CONCLUSION: The present system may be applicable for use in the search for anti-gingivitis substances.

Prevotella falsenii sp. nov., a Prevotella intermedia-like organism isolated from monkey dental plaque.

Eight anaerobic, pigmented, non-spore-forming, Gram-negative, rod-shaped strains isolated from monkey oral cavities were characterized phenotypically and chemotaxonomically and their phylogenetic positions were determined using 16S rRNA gene sequence analysis. The 16S rRNA gene sequence analysis showed that these isolates represent a single species of the genus Prevotella. These strains were most closely related to Prevotella intermedia ATCC 25611(T), with 95.0 % 16S rRNA gene sequence similarity. The next most closely related species were Prevotella pallens and Prevotella nigrescens (92.7 and 92.1 % similarity to the respective type strains). The phenotypic and biochemical characteristics of the isolates were the same as those of P. intermedia JCM 12248(T) and P. nigrescens JCM 12250(T). The isolates could be differentiated from P. pallens JCM 11140(T) on the basis of mannose fermentation and alpha-fucosidase activity. The isolates could not be distinguished from P. intermedia or P. nigrescens using conventional biochemical tests. DNA-DNA hybridization experiments revealed the genomic distinctiveness of these eight strains with respect to P. pallens JCM 11140(T), P. intermedia JCM 12248(T) and P. nigrescens JCM 12250(T). On the basis of these data, strains 04013, 04021, 04043, 04052(T), 0406, 04113, 04111 and 04161 represent a novel Prevotella species, for which the name Prevotella falsenii sp. nov. is proposed. The type strain is 04052(T) (=JCM 15124(T) =CCUG 56137(T)).

Molecular and antigenic similarities of the fimbrial major components between Porphyromonas gulae and P. gingivalis.

Porphyromonas gulae is black-pigmented anaerobic bacteria associated with canine periodontitis. There is little information available about the specific identify and relative occurrence of pigmented anaerobes in companion animals. Our aim was to clarify the factor involved in the adherence and colonization of the organism in the oral cavity. Fimbrial protein was purified from P. gulae ATCC 51700. The molecular mass of this protein was approximately 41kDa as estimated by SDS-PAGE. An antibody against 41-kDa fimbrial protein from P. gingivalis ATCC 33277 reacted with fimbrillin of P. gulae ATCC 51700. Immunogold electron microscopy revealed that the anti-41kDa fimbrial serum bound to fimbria on the cell surface of P. gulae ATCC 51700. Thus, fimbrial protein of P. gulae ATCC 51700 had the same size and antigenicity as 41-kDa fimbriae of P. gingivalis ATCC 33277. The nucleotide sequence of the fimA gene from P. gulae ATCC 51700 showed 94% homology with that of P. gingivalis ATCC 33277. Moreover, the deduced amino acid sequences have 96.8% identity. P. gulae has adherent ability to gingival epithelial cells. The properties of P. gulae fimbriae are similar to those of P. gingivalis fimbriae. We suggest that the surface structure of P. gulae may play a role in the colonization of this organism in periodontal pockets in companion animals.

Antifungal effect of acrylic resin containing apatite-coated TiO2 photocatalyst.

The purpose of this study was to develop an acrylic resin with antifungal properties by leveraging the photocatalytic activity of apatite-coated titanium dioxide (Ap-TiO2). Candida albicans was used for antifungal activity assay of the specimen plates under ultraviolet A (UVA) with a black light source. Statistically significant decreases in cell viability in acrylic resins containing 5 wt% and 10 wt% Ap-TiO2 were observed after irradiation for two, four, and six hours (P<0.01), when compared to the control. As for the flexural strength and modulus values of acrylic resins mixed with Ap-TiO2 and TiO2 particles, they varied before and after irradiation. Among the tested specimens, a 5 wt% content of Ap-TiO2 in acrylic resin exceeded the requirements of ISO 1567. It was thus suggested that acrylic resin containing 5 wt% Ap-TiO2 could exert antifungal effects on C. albicans, while at the same time maintain adequate mechanical properties for clinical use.

Serum antibodies of periodontitis patients compared to the lipopolysaccharides of Porphyromonas gingivalis and Fusobacterium nucleatum.

Serum antibody titers against the lipopolysaccharides (LPSs) of Porphyromonas gingivalis and Fusobacterium nucleatum were compared between 9 periodontitis patients and 24 healthy persons. The IgG titers against the LPSs of P. gingivalis ATCC 33277(T) and W50 were clearly higher in the patients than in the healthy persons. However, IgM titers against the LPSs of P. gingivalis strains were relatively low, and no significant difference was observed between the patients and healthy persons. On the other hand, IgG and IgM titers against the LPS of Fusobacterium nucleatum JCM 8532(T) in some patients were significantly higher than those in the healthy persons, although the difference in IgG titers was not large compared to that of the LPS of P. gingivalis. These results suggest that the antibody measurement of patients' sera against the LPS of periodontal bacteria can be applied for the diagnosis of periodontitis.

Endotoxic properties of free lipid A from Porphyromonas gingivalis.

The relationship between chemical structure and biological activity of the lipid A from Porphyromonas gingivalis, which we recently isolated and whose complete chemical structure was determined [Kumada et al. (1995). J Bacteriol 177, 2098-2106], was studied. The lipid A exhibited endotoxic activity in all the assay systems tested: Limulus gelation activity, lethal toxicity in galactosamine-sensitized mice, mitogenicity in mouse spleen cells and induction of nitric oxide (NO) and tumour necrosis factor alpha (TNF) release from both mouse peritoneal macrophages and the J774-1 mouse macrophage-like cell line. The activity was, however, about 100-fold less than that of Salmonella minnesota LPS used as a control. The moderate activity of the lipid A may be partially explained by its unique fatty acid composition and the lack of a phosphate group in position 4. In contrast, the lipid A as well as whole LPS of P. gingivalis unexpectedly exhibited an even stronger induction of TNF from the human monocytic THP-1 cell line than control LPS when measured by the minimum stimulatory dose. The difference in sensitivity of human and mouse cells to P. gingivalis lipid A suggests that the recognition mechanism, including that for the receptor for endotoxin, may be regulated in different ways in the two cells.