Anti-inflammatory effect of luteoloside against methylglyoxal induced human dental pulp cells.

PURPOSE: The aim of this study was to investigate whether luteoloside, a flavonoid, could protect human dental pulp cells (HDPCs) against inflammation and oxidative stress induced by methylglyoxal (MGO), one of the advanced glycated end products (AGE) substances. METHODS: HDPCs were stimulated with MGO and treated with luteoloside. MTT assay was used to determine cell viability. Protein expression was measured via western blotting. Reactive oxygen species (ROS) were measured with a Muse Cell Analyzer. Alkaline phosphatase activity (ALP) and Alizarin red staining were used for mineralization assay. RESULTS: Luteoloside down-regulated the expression of inflammatory molecules such as ICAM-1, VCAM-1, TNF-α, IL-1ß, MMP-2, MMP-9, and COX-2 in MGO-induced HDPCs without showing any cytotoxicity. It attenuated ROS formation and enhanced osteogenic differentiation such as ALP activity and Alizarin red staining in MGO-induced HDPCs. Overall, luteoloside showed protective actions against inflammation and oxidative stress in HDPCs induced by MGO through its anti-inflammatory, anti-oxidative, and osteogenic activities by down-regulating p-JNK in the MAPK pathway. CONCLUSION: These results suggest that luteoloside might be a potential adjunctive therapeutic agent for treating pulpal pathological conditions in patients with diabetes mellitus.

Nanoparticle mediated PPARγ gene delivery on dental implants improves osseointegration via mitochondrial biogenesis in diabetes mellitus rat model.

Diabetes mellitus (DM) has a detrimental effect on osseointegration, stability and longevity of implants due to osteoporosis. In this study, PPARγ-loaded dental implants were investigated for the improvement of osseointegration and peri-implantitis. Chitosan gold nanoparticles conjugated with PPARγ cDNA were introduced on titanium mini-implant surfaces for PPARγ release to rat mandibular. DM-induced rat mandible showed structural changes such as decreased bone mass and increased inflammatory molecules, and diminution of PPARγ expression and bone formation molecules compared to normal rats. PPARγ induced bone formation via reduction of inflammatory molecules even under glucose oxidative stress. Furthermore, PPARγ strongly activated mitochondrial biogenesis and cell viability via p-AMK and Wnt/ß-catenin signaling. Consequently, PPARγ gene delivery on regional dental implants contributed osseointegration, new bone formation and mineralization in DM-induced rats. This study demonstrates that PPARγ can be used as a therapeutic gene with dental implantation in diabetic patients since regional PPARγ expression enhances osseointegration and implant longevity.

PPARγ delivered by Ch-GNPs onto titanium surfaces inhibits implant-induced inflammation and induces bone mineralization of MC-3T3E1 osteoblast-like cells.

OBJECTIVES: To deliver the efficacy and safety of Ch-GNPs (Chitosan gold nanoparticles) conjugated anti-inflammatory molecules peroxisome proliferator activated receptor gamma (PPARγ) on implant surface titanium (Ti) to reduce implant-induced inflammation. MATERIALS AND METHODS: The Ch-GNPs were conjugated with the PPARγ cDNA through a coacervation process. Conjugation was cast over Ti surfaces by dipping, and cells were seeded on different sizes (6 × 6 × 0.1 cm and 1 × 1 × 0.1 cm; n = 3) of Ti surfaces. The size of Ch-GNPs and surface characterization of Ti was performed using UV-vis spectroscopy, TEM (Transmission electron microscopy) and EDX (energy-dispersive X-ray). The DNA conjugation and transfection capacity of Ch-GNPs were simultaneously confirmed by agarose gel electrophoresis, ß-galactosidase staining, and immunoblotting. RESULTS: The Ch-GNPs were well dispersed and spherical in shape, with average size around 10-20 nm. Ti surfaces coated with Ch-GNPs/LacZ, as transfection efficacy molecule, showed strong ß-galactosidase staining in MC-3T3 E1 cells. Cells cultured on Ch-GNPs/PPARγ-coated Ti surfaces were able to inhibit implant-induced inflammation by simultaneously suppressing the expression of tumor necrosis factor- alpha (TNF-α), interleukin-1 beta (IL-1ß), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and matrix metalloproteinase-2 (MMP-2). The inhibition mechanism of Ch-GNPs/PPARγ was due to inhibition of both reactive oxygen species (ROS) and nitric oxide (NO) secretion (n = 3; P < 0.05). In addition, Ch-GNPs/PPARγ was able to increase expression of bone morphogenetic protein (BMP-7) and runt-related transcription factor-2 (RUNX-2). Furthermore, alkaline phosphatase activity (ALP) was also increased than that in control (n = 3; P < 0.01). Whereas, expression of receptor activator of NF-κB ligand (RANKL) was decreased. CONCLUSIONS: The novel gene delivery materials, like Ch-GNPs, can carry the PPARγ cDNA into the required areas of the implant surfaces, thus aiding to inhibit inflammation and promote osteoblast function. Thus, the PPARγ on implant surfaces may promote its clinical application on peri-implantitis or periodontitis like diseases.

Nasal obstruction promotes alveolar bone destruction in the juvenile rat model.

BACKGROUND/PURPOSE: Nasal obstruction leads to oral breathing and consequently hypoxia. The purpose of this study was to determine the influence of hypoxia on inflammatory response and the effect on alveolar bone development in a rat model in which mouth breathing was induced by nasal obstruction. MATERIALS AND METHODS: Unilateral nasal obstruction was performed by injecting a Merocel sponge into the nasal cavity of 8-week-old Sprague Dawley (SD) rats. After 3 and 6 weeks of nasal obstruction, rats were sacrificed, the organs were weighed, and the changes in mandibular bone quality were examined by micro-computed tomography (µ-CT). The stereomicroscope was used for the morphological analysis of alveolar bone loss in response to nasal obstruction. Hematoxylin and Eosin (H&E) and immunohistochemical staining were employed to examine inflammation and bone remodeling induced by hypoxia. RESULTS: Nasal obstruction led to a delay in overall growth and organ development. The bone mineral density (BMD) and bone volume/total volume (BV/TV) of the mandible were reduced due to nasal obstruction, and the loss of the alveolar bone was confirmed morphologically. Our nasal obstruction method was observed to be successful in inducing hypoxia along with an increase in hypoxia-inducible factor 1-alpha (HIF-α). Oral hypoxia induced by nasal obstruction increased inflammatory response, and increased expression of receptor activator of nuclear factor kappa-Β ligand (RANKL) led to bone destruction. CONCLUSION: This study demonstrated that nasal obstruction induced mouth breathing led to hypoxia in a rat model. Under hypoxic conditions, an increase in osteoclast differentiation induced by activation of the inflammatory pathway causes destructive changes in the alveolar bone.

Insulin growth factor binding protein-3 enhances dental implant osseointegration against methylglyoxal-induced bone deterioration in a rat model.

PURPOSE: The aim of this study was to determine the effect of insulin growth factor binding protein-3 (IGFBP-3) on the inhibition of glucose oxidative stress and promotion of bone formation near the implant site in a rat model of methylglyoxal (MGO)-induced bone loss. METHODS: An in vitro study was performed in MC3T3 E1 cells treated with chitosan gold nanoparticles (Ch-GNPs) conjugated with IGFBP-3 cDNA followed by MGO. An in vivo study was conducted in a rat model induced by MGO administration after the insertion of a dental implant coated with IGFBP-3. RESULTS: MGO treatment downregulated molecules involved in osteogenic differentiation and bone formation in MC3T3 E1 cells and influenced the bone mineral density and bone volume of the femur and alveolar bone. In contrast, IGFBP-3 inhibited oxidative stress and inflammation and enhanced osteogenesis in MGO-treated MC3T3 E1 cells. In addition, IGFBP-3 promoted bone formation by reducing inflammatory proteins in MGO-administered rats. The application of Ch-GNPs conjugated with IGFBP-3 as a coating of titanium implants enhanced osteogenesis and the osseointegration of dental implants. CONCLUSIONS: This study demonstrated that IGFBP-3 could be applied as a therapeutic component in dental implants to promote the osseointegration of dental implants in patients with diabetes, which affects MGO levels.

Mussel adhesive protein blended with gelatin loaded into nanotube titanium dental implants enhances osseointegration.

The purpose of this study was to investigate whether mussel adhesive protein (MAP) blended with gelatin loaded into nanotube titanium (Ti) dental implants enhances osseointegration and supports bone formation. Cell viability, crystal violet staining, Western blot analysis, alizarin red S staining, alkaline phosphatase (ALP) activity, micro-computed tomography (µ-CT), hematoxylin and eosin (H&E), and immunohistochemistry (IHC) staining were employed to test the biocompatibility of MAP blended with gelatin (MAP/Gel). MC3T3 E1 cells were used for in vitro and Sprague-Dawley rats for in vivo models in this study. MC3T3 E1 cells cultured in MAP/Gel loaded into nanotube Ti surface demonstrated activation of FAK-PI3K-MAPKs-Wnt/ß-catenin signaling pathway and enhanced osteogenic differentiation. µ-CT, H&E, and IHC staining confirmed that MAP/Gel dental implants promoted bone regeneration around the nanotube Ti implants by upregulation of Runx-2, BMP-2/7, Osterix, and OPG in rat mandible model. MAP/Gel supports osseointegration of dental implant after implantation. It is hypothesized that MAP/Gel loaded into nanotube Ti dental implants may be applicable as a potential treatment for bone formation and proper integration of dental implants with alveolar bone. Graphical abstract.

Phelligridin D maintains the function of periodontal ligament cells through autophagy in glucose-induced oxidative stress.

PURPOSE: The objective of this study was to investigate whether phelligridin D could reduce glucose-induced oxidative stress, attenuate the resulting inflammatory response, and restore the function of human periodontal ligament cells (HPDLCs). METHODS: Primary HPDLCs were isolated from healthy human teeth and cultured. To investigate the effect of phelligridin D on glucose-induced oxidative stress, HPDLCs were treated with phelligridin D, various concentrations of glucose, and glucose oxidase. Glucose-induced oxidative stress, inflammatory molecules, osteoblast differentiation, and mineralization of the HPDLCs were measured by hydrogen peroxide (H2O2) generation, cellular viability, alkaline phosphatase (ALP) activity, alizarin red staining, and western blot analyses. RESULTS: Glucose-induced oxidative stress led to increased production of H2O2, with negative impacts on cellular viability, ALP activity, and calcium deposition in HPDLCs. Furthermore, HPDLCs under glucose-induced oxidative stress showed induction of inflammatory molecules (intercellular adhesion molecule-1, vascular cell adhesion protein-1, tumor necrosis factor-alpha, interleukin-1-beta) and disturbances of osteogenic differentiation (bone morphogenetic protein-2, and -7, runt-related transcription factor-2), cementogenesis (cementum protein-1), and autophagy-related molecules (autophagy related 5, light chain 3 I/II, beclin-1). Phelligridin D restored all these molecules and maintained the function of HPDLCs even under glucose-induced oxidative stress. CONCLUSIONS: This study suggests that phelligridin D reduces the inflammation that results from glucose-induced oxidative stress and restores the function of HPDLCs (e.g., osteoblast differentiation) by upregulating autophagy.

Terrein reduces pulpal inflammation in human dental pulp cells.

Terrein is a bioactive fungal metabolite whose anti-inflammatory properties are virtually unknown. The purpose of this study was to determine the effects of terrein on lipopolysaccharide (LPS)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human dental pulp cells and to determine the mechanism of the observed effects. The LPS-induced expression of ICAM-1 and VCAM-1 was inhibited by terrein in both a time- and dose-dependent manner. LPS-stimulated translocation of nuclear factor kappa B (NF-kappaB) into the nucleus, which was blocked by inhibitors of amino kinase terminal (AKT, LY294002), extracellular signal regulated kinase 1/2 (ERK 1/2, PD98059), p38 (SB203580), and c-jun NH2-terminal kinase (JNK, SP600125) or terrein. In addition, these inhibitors and terrein also reduced the level of ICAM-1 and VCAM-1 expression in LPS-induced inflammation of pulp cells. Terrein suppressed NF-kappaB activation by blocking the activation of Akt. These results strongly suggest the potential role of terrein as an anti-inflammatory modulator in pulpal inflammation.

Schisandrin C enhances odontoblastic differentiation through autophagy and mitochondrial biogenesis in human dental pulp cells.

OBJECTIVE: To investigate the role of Schisandrin C in odontoblastic differentiation, and its relations between autophagy and mitochondrial biogenesis in human dental pulp cells (HPDCs). DESIGN: Fresh third molars were used, and cultured for HDPCs. Western blotting technique, Alizarin red S staining, alkaline phosphatase (ALP) activity, and confocal microscopy were used to detect autophagy, mitochondrial biogenesis, and odontoblastic differentiation. To understand the mechanism of Schisandrin C, the HDPCs were treated with lipopolysaccharide (LPS), autophagy and heme oxygenase-1 (HO-1) inhibitors: 3-Methyladenine (3-MA) and Zinc protoporphyrin IX (ZnPP), respectively. RESULTS: LPS decreased the expression of autophagy molecules [autophagy protein 5 (ATG-5), beclin-1, and microtubule-associated protein 1A/1B light chain 3 (LC3-I/II)] and mitochondrial biogenesis molecules [heme oxygenase-1 (HO-1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)], and disrupted odontoblastic differentiation. The down-regulation of autophagy and mitochondrial biogenesis with 3-MA and ZnPP inhibited odontoblastic differentiation. However, Schisandrin C restored the expression of all the above molecules, even with LPS and inhibitor treatment. This result demonstrates that autophagy and mitochondrial biogenesis plays an essential role in odontoblastic differentiation, and Schisandrin C activates these systems to promote odontoblastic differentiation of HDPCs. CONCLUSION: Schisandrin C has potential characters to regulate odontoblastic differentiation, and may be recommended for use as a compound for pulp homeostasis.

Chitosan-gold nanoparticles mediated gene delivery of c-myb facilitates osseointegration of dental implants in ovariectomized rat.

Osseointegration of dental implants is affected by osteoporosis. The purpose of this study was overcome the implant failure and facilitate the osseointegration of dental implants by c-myb in ovariectomized (OVX)-induced osteoporosis. c-myb is a transcription factor and supports bone formation. Plasmid DNA/c-myb conjugated with chitosan-gold nanoparticles (Ch-GNPs/c-myb) promoted osteogenesis and inhibited osteoclastogenesis in MC-3T3 E1 cells. Ch-GNPs/c-myb involved the reduction of the nuclear factor of activated T-cells 1, c-Fos, and tartrate-resistant acid phosphatase-positive multinucleated osteoclasts in receptor activator of nuclear factor-κB ligand (RANKL) stimulated bone marrow macrophages. In vivo results of rat mandibles demonstrated Ch-GNP/c-myb-coated titanium (Ti) implants increased the volume and density of newly formed bone and the osseointegration of dental implant with bone by micro computed tomography examination after OVX-induced osteoporosis. Immunohistochemical analysis showed increased c-myb expression and upregulation of bone morphogenic proteins, osteoprotegerin and EphB4, as well as the downregulation of RANKL by Ch-GNP/c-myb-coated Ti implants. Hematoxylin and Eosin staining expressed new bone formation by Ch-GNP/c-myb-coated Ti implants. Our findings indicated that c-myb delivered by Ch-GNPs supports osseointegration of dental implant even in osteoporotic condition. c-myb may be applicable to support dental implant integration and treatment in age-dependent bone destruction disease.

Phelligridin D-loaded oral nanotube titanium implant enhances osseointegration and prevents osteolysis in rat mandible.

Poor bone quality and osteolysis are the major causes of implant failure in dentistry. Here, this study tested the effect of phelligridin D-loaded nanotubes titanium (Ti) for bone formation around the dental implants. The purpose of this study was to enhance osseointegration of phelligridin D-loaded implant into the bone for bone formation and prevention of osteolysis. Cell viability, crystal violet staining, Western blot, alizarin red S staining, alkaline phosphatase activity, tartrate-resistant acid phosphatase staining, micro-computed tromography (µ-CT), hematoxylin and eosin (H&E) and immunohistochemical staining were used in vitro and in vivo to test the biocompatibility of phelligridin D. Phelligridin D enhanced osteoblast differentiation and mineralization by increasing bone morphogenic protein-2/7 (BMP-2/7), Osterix, Runx-2, osteoprotegerin (OPG), alkaline phosphatase and inhibited osteoclast differentiation by decreasing receptor activator of nuclear factor kappa-B ligand (RANKL) in MC-3T3 E1 cells. Further, phelligridin D promoted bone regeneration around nanotube Ti implant surface by increasing the levels of BMP-2/7 and OPG in a rat model. Phelligridin D also inhibited osteolysis by suppressing the expression of RANKL. These findings strongly suggest that phelligridin D is a new compound representing a potential therapeutic candidate for implant failure caused by osteolysis and poor bone quality of teeth.

Enhancing of Osseointegration with Propolis-Loaded TiO2 Nanotubes in Rat Mandible for Dental Implants.

TiO2 nanotubes (TNT) formation is beneficial for improving bone cell-material interaction and drug delivery for Ti dental implants. Among the natural drugs to be installed in TNT, selected propolis has antibacterial and anti-inflammatory properties. It is a resinous natural product which is collected by the honeybees from the various types of plants with their salivary enzymes. This study concludes that TNT loaded with a propolis (PL-TNT-Ti) dental implant has the ability to improve osseointegration. The propolis particles were embedded within the TNT or adhered to the top. In a cytotoxicity test using osteoblast, PL-TNT-Ti group exhibited an increased cell proliferation and differentiation. A Sprague Dawley rat mandibular model was used to evaluate the osseointegration and bone bonding of TNT or PL-TNT-Ti. From the µ-CT and hematoxylin and eosin (HE) histological results after implantation at 1 and 4 weeks to rat mandibular, an increase in the extent of new bone formation and mineral density around the PL-TNT-Ti implant was confirmed. The Masson's trichrome staining showed the expression of well-formed collagenous for bone formation on the PL-TNT-Ti. Immunohistochemistry staining indicate that bone morphogenetic proteins (BMP-2 and BMP-7) around the PL-TNT-Ti increased the expression of collagen fibers and of osteogenic differentiation whereas the expression of inflammatory cytokine such as interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) is decreased.

In vitro effects of a fraction separated from Polygonum cuspidatum root on the viability, in suspension and biofilms, and biofilm formation of mutans streptococci.

Polygonum cuspidatum (Polygonaceae) has traditionally been used in folk medicine to control oral diseases. Nevertheless, there are no reports related to its possible effect on the diseases, particularly on biofilm-related diseases such as dental caries. In this study, we evaluated in vitro effects of a fraction separated from Polygonum cuspidatum root on the viability, in both suspension and biofilms, and the biofilm formation of mutans streptococci. The separated fraction (F1) showed bacteriostatic and bactericidal activity against mutans streptococci in suspension, with minimum inhibitory concentration (MIC) range of 31.3-250 microg/ml and minimum bactericidal concentration (MBC) range of 0.5-1 mg/ml. At a concentration of 1.5 mg/ml, F1 killed approximately 2 log(10)CFU/ml of Streptococcus mutans and Streptococcus sobrinus after 2h of exposure. In biofilms, F1 also inhibited the viability of Streptococcus mutans and Streptococcus sobrinus, dependent on the biofilm age, the concentration of F1, and the treatment time. Four hours of exposure to 1.5 mg/ml F1 reduced the viable counts of Streptococcus mutans and Streptococcus sobrinus by greater than 2 log(10)CFU/disc. Furthermore, at sub-MIC levels, F1 inhibited biofilm formation by Streptococcus mutans and Streptococcus sobrinus in a dose-dependent fashion. Based on the preliminary phytochemical analysis, the activity of F1 may be related to the presence of anthraquinones, cardiac glycosides, terpenoids, and phenolics. These results indicate that F1 is probably useful in the control of oral biofilms and subsequent dental caries development.

Effect of Ca-P compound formed by hydrothermal treatment on biodegradation and biocompatibility of Mg-3Al-1Zn-1.5Ca alloy; in vitro and in vivo evaluation.

Chemical combinations of Ca-P produced via plasma electrolytic oxidation (PEO) and a hydrothermal treatment were fabricated to improve the initial corrosion resistance and biocompatibility of a biodegradable Mg-3Al-1Zn-1.5Ca alloy. For the formation of an amorphous calcium phosphate composite layer on the surface of a magnesium alloy, a PEO layer composed of MgO and Mg3(PO4)2 was formed by PEO in electrolytes containing preliminary phosphate ions. During the second stage, a thick and dense Ca layer was formed by Ca electrodeposition after PEO. Finally, a hydrothermal treatment was carried out for chemical incorporation of P ions in the PEO layer and Ca ions in the electrodeposition layer. The amorphous calcium phosphate composite layer formed by the hydrothermal treatment enhanced osteoblast activity and reduced H2O2 production, which is a known stress indicator for cells. As a result of co-culturing osteoblast cells and RAW 264.7 cells, the formation of amorphous calcium phosphate increased osteoblast cell differentiation and decreased osteoclast cell differentiation. Implanting the alloy, which had an amorphous calcium phosphate composite layer that had been added through hydrothermal treatment, in the tibia of rats led to a reduction in initial biodegradation and promoted new bone formation.

Existence of ATP sensitive potassium currents on human periodontal ligament cells.

OBJECTIVE: Potassium channels of the ATP-sensitive family (KATP channel) are inhibited by increase in intracellular ATP. Electrophysiological studies have demonstrated that the kinetics and pharmacological properties of KATP channels vary among different tissues, suggesting structurally and functionally distinct types. There are studies showing human periodontal ligament (PDL) cells respond to mechanical stress by increasing ATP release, which participates in bone resorption or bone homeostasis. So, in this study we investigated the existence of KATP channel subunit and their single channel properties in human periodontal ligaments. MATERIALS & METHOD: The human PDL cells were isolated from healthy erupted third molar. For patch-clamp experiments, human PDL fibroblasts were seeded on 3.5cm plastic dishes. The inside-out patch clamp recordings were performed under voltage clamp mode. Reverse transcriptase polymerase chain reaction (RT-PCR) was conducted to identify the channel subunits. All pair-wise comparisons were performed by Paired t-test. A P value <0.05 was considered significant. RESULTS: We observed mRNA transcripts for Kir6.1, Kir6.2 and Sur2B subuits in the human PDL cells. In inside-out patch mode, the single channel conductance was 163pS at symmetrical K+ concentration of 140mM and inward rectification was seen in ATP-free bath solution. The reversal potential of the currents was found to be 0mV at symmetrical concentration (140mM) of K+ in bath solution. The single channel currents were almost blocked by adding 5mM ATP in the bath solution. However, the currents were not blocked by 100µM glibenclamide, a subunit specific KATP channel blocker. CONCLUSIONS: These results indicate that human PDL cells express KATP channels subunit including Sur2B and Kir6.1 and Kir6.2 which are sensitive to ATP but insensitive to glibenclamide.

Hydrophilic nanofibrous structure of polylactide; fabrication and cell affinity.

Microstructure and architecture of the scaffolds along with the surface chemistry exert profound effect on biological activity (cell distribution, proliferation, and differentiation). For the biological activity, scaffolds in tissue engineering have been widely designed. The objective of this study was to develop hydrophilic nanofibrous structure of polylactides (PLLA) polymer in the form of nonwoven mat by electrospinning technique, and further evaluate the fibroblast NIH3T3 cell proliferation, morphology, and cell-matrix interaction. Hydrophilicity of the PLLA fibers was improved by adding small fraction of low molecular weight polyethylene glycol (PEG) into the electrospinning solution. Four different ratio types (100/0, 80/20, 70/30, and 50/50) of PLLA/PEG electrospun matrices were fabricated, and the pore characteristics, tensile properties, contact angle, and hydrolytic degradation were observed. Furthermore, scanning electron microscope (SEM) and fluorescence actin staining images were used for micro-observation of cell-matrix interaction and cell morphology. It was found that the electrospun mat of PLLA/PEG (80/20), composed of fibers with diameters in the range 540-850 nm, majority of pore diameter less than 100 microm, tensile strength 8 MPa, elongation 150%, porosity more than 90%, and improved hydrophilicity with slow hydrolytic degradation, is favorable for biological activity of NIH3T3 fibroblast cell. Based on these results, the correct composition of PLLA and PEG in the porous electrospun matrix (i.e., PLLA/PEG (80/20)) will be a better candidate rather than other compositions of PLLA/PEG as well as hydrophobic PLLA for application in tissue engineering.

Novel block copolymer (PPDO/PLLA-b-PEG): enhancement of DNA uptake and cell transfection.

The cationic lipid mediated uptake of plasmid DNA by cells in monolayer culture was significantly enhanced with an aqueous solution of the block copolymer poly(p-dioxanone-co-L-lactide)-b-poly(ethylene glycol) (PPDO/PLLA-b-PEG). Plasmid uptake studies with DNA encoding the beta-galactosidase gene and cytotoxicity evaluations were performed on MCF-7, NIH 3T3 and CT-26 cell lines. Transfection yields and time courses for maximum release of FITC labeled DNA in MCF-7 cells were observed and quantified by beta-galactosidase assay and spectrofluorometry, respectively. The reported results suggest that the studied block copolymer might be useful for the enhancement of polycation mediated transfection and could find application in gene therapy.

In vitro inhibitory effects of Polygonum cuspidatum on bacterial viability and virulence factors of Streptococcus mutans and Streptococcus sobrinus.

OBJECTIVES: Polygonum cuspidatum has been used in Korean folk medicine to improve oral hygiene. This study was performed to evaluate the effects of methanol extract from root of P. cuspidatum (MEP) on bacterial viability and the virulence factors of Streptococcus mutans and Streptococcus sobrinus. METHODS: To test the effects of MEP on bacterial viability, we determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against 20 bacterial strains, including S. mutans and S. sobrinus, using a micro-dilution assay. In case of S. mutans and S. sobrinus, the assays for time-kill and bacterial growth rate at sub-MIC concentrations were also performed. To determine effects of the extract on the virulence factors of S. mutans and S. sobrinus, the assays for sucrose-dependent adherence, water-insoluble glucan formation, glycolytic acid production, and acid tolerance were performed at sub-MIC levels. Phytochemical analysis for constituents of MEP was carried out. RESULTS: MEP showed a broad antibacterial range (MIC 0.5-4 mg/ml). The MBC was two to four times higher than the MIC. The time-kill curves showed S. mutans and S. sobrinus were significantly killed after 1h of incubation. At sub-MIC levels, doubling times of S. mutans and S. sobrinus dose-dependently increased up to 211% and 123%, respectively. At sub-MIC levels, MEP also showed inhibitory effects on the virulence factors of S. mutans and S. sobrinus in a dose-dependent fashion. Phytochemical analysis revealed the presence of alkaloids, sterol/terpenes, tannins, flavonoids, and carbohydrates. CONCLUSION: These data indicate that MEP has inhibitory effects on bacterial viability at higher concentrations (> or =MIC) and the virulence factors of S. mutans and S. sobrinus at sub-MIC concentrations, suggesting that it might be useful for the control of dental plaque formation and subsequent dental caries formation.

In vitro anti-cariogenic activity of dichloromethane fraction from Rheum undulatum L. root.

This study aimed to evaluate in vitro effects of Rheum undulatum L. root on the development of dental caries, especially its effects on viability, dental plaque formation, and glycolytic acid production of Streptococcus mutans and Streptococcus sobrinus. Methanol extract of Rheum undulatum L. root and its fractions were prepared and tested. Among the test extract and fractions, dichloromethane fraction (DF) showed the most active antibacterial activity (inhibition zone: 13-17 mm) against S. mutans and S. sobrinus in a disc diffusion method. Minimal inhibitory concentrations (MICs) of DF against these bacteria ranged from 0.25 to 0.5 mg/mL. Furthermore, DF significantly inhibited the caries-inducing factors of these bacteria. At sub-MIC levels, DF inhibited in vitro dental plaque formation by S. mutans and S. sobrinus (IC50= 0.079 and 0.142 mg/mL, respectively), which was caused, in part, by the inhibitory effect on the activity of glucosyltransferases. A significant reduction of glycolytic acid production was found at the concentration as low as 0.032 mg/mL for S. mutans and 0.063 mg/mL for S. sobrinus. The possible bioactive compounds that are inducing in vitro anti-cariogenic activity of DF are unknown. Based on the preliminary phytochemical analysis, the activity of DF may be related to the presence of anthraquinones, cardiac glycosides, coumarines, sterols/terpenes, and phenolics. These results indicate that DF is probably useful for the control of dental plaque formation and subsequent dental caries development.

Functional Relationship between Sucrose and a Cariogenic Biofilm Formation.

Sucrose is an important dietary factor in cariogenic biofilm formation and subsequent initiation of dental caries. This study investigated the functional relationships between sucrose concentration and Streptococcus mutans adherence and biofilm formation. Changes in morphological characteristics of the biofilms with increasing sucrose concentration were also evaluated. S. mutans biofilms were formed on saliva-coated hydroxyapatite discs in culture medium containing 0, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, or 40% (w/v) sucrose. The adherence (in 4-hour biofilms) and biofilm composition (in 46-hour biofilms) of the biofilms were analyzed using microbiological, biochemical, laser scanning confocal fluorescence microscopic, and scanning electron microscopic methods. To determine the relationships, 2nd order polynomial curve fitting was performed. In this study, the influence of sucrose on bacterial adhesion, biofilm composition (dry weight, bacterial counts, and water-insoluble extracellular polysaccharide (EPS) content), and acidogenicity followed a 2nd order polynomial curve with concentration dependence, and the maximum effective concentrations (MECs) of sucrose ranged from 0.45 to 2.4%. The bacterial and EPS bio-volume and thickness in the biofilms also gradually increased and then decreased as sucrose concentration increased. Furthermore, the size and shape of the micro-colonies of the biofilms depended on the sucrose concentration. Around the MECs, the micro-colonies were bigger and more homogeneous than those at 0 and 40%, and were surrounded by enough EPSs to support their structure. These results suggest that the relationship between sucrose concentration and cariogenic biofilm formation in the oral cavity could be described by a functional relationship.