Release and MMP-9 Inhibition Assessment of Dental Adhesive Modified with EGCG-Encapsulated Halloysite Nanotubes.

Degradation of the collagen fibrils at the dentin-resin interface by the enzymatic activity of matrix metalloproteinases (MMPs) has been known to permit some dental restoration complications, such as microleakage, secondary caries, and, ultimately, restoration failures. This study aimed to evaluate a modified adhesive by adding an MMP inhibitor from green tea extract with and without nanotube encapsulation to sustain the drug release. Epigallocatechin-3-gallate (EGCG) and Halloysite nanotubes (HNTs) were prepared to produce three variant combinations of modified adhesive (EGCG, EGCG-encapsulated HNT, and EGCG-free HNT). The drug loading efficiency and EGCG release over time were evaluated using UV-vis spectrometry. MMP-mediated ß-casein (BCN) cleavage rate assays were used to determine the ability of the EGCG in eluates of the adhesive to inhibit MMP-9 activities. For up to 8 weeks, HNT encapsulation reduced release to a statistically significant level. MMP-mediated ß-casein cleavage rate assays showed a significant decrease for the EGCG groups compared to the non-EGCG adhesive groups. Furthermore, the use of HNT for EGCG encapsulation to modify a dental adhesive helped slow down the rate of EGCG release without impacting its MMP inhibitory capabilities, which may help to maintain the dentin-resin interface's integrity over the long term after dental restoration placement.

Effect of a modified adhesive system with encapsulated arginine and calcium carbonate on dentin permeability.

To modify an adhesive system with halloysite clay nanotubes (HNTs) containing arginine and calcium carbonate and to evaluate their cytocompatibility, viscosity and efficacy in reducing dentin permeability. HNTs containing arginine and calcium carbonate were incorporated into the primer and adhesive of a three-step adhesive system (SBMP), and their viscosity was measured. Discs (n = 4/group) were prepared: SBMP (control), HNT-PR (modified primer), HNT-ADH (modified adhesive) and HNT-PR + ADH (modified primer and adhesive) were evaluated regarding cell death and viability. Dentin discs were prepared and randomly assigned into the following treatments (n = 10): NC (no treatment), SBMP, HNT-PR, HNT-ADH, HNT-PR + ADH and COL (Colgate® Sensitive Pro-relief™ prophylaxis paste). After, they were submitted to an erosive-abrasive cycling. Dentin permeability (hydraulic conductance) was evaluated at baseline, 24 h after treatment and after cycling. Both the modified primer and adhesive showed significantly higher viscosity than their controls. Group HNT-PR resulted in significantly higher cytotoxicity when compared to SBMP and HNT-PR + ADH groups. Group HNT-ADH resulted in the highest cell viability compared to all other groups. All groups showed significantly lower dentin permeability when compared to the NC group. Post-cycling, SBMP and HNT-ADH groups showed significantly lower permeability when compared to COL group. The addition of encapsulated arginine and calcium carbonate did not affect the cytocompatibility of the materials nor their ability to reduce dentin permeability.

An in vitro comparison of four antibacterial agents with and without nicotine and their effects on human gingival fibroblasts.

BACKGROUND: The purpose of the study was to compare anti-bacterial activity of 0.12% chlorhexidine (CHX), 10% povidone iodine (PVD), Vega oral care gel (VEGA), and antioxidant gel (AO) on Streptococcus mutans, Streptococcus sanguis, Fusobacterium nucleatum, and Porphyromonas gingivalis with and without nicotine and to evaluate their effects on human gingival fibroblasts (HGFs). METHODS: S. mutans, S. sanguis, P. gingivalis, and F. nucleatum were incubated with serial dilutions (1/4, 1/8, 1/16, 1/32, and 1/64) of anti-bacterial agents in media (with and without nicotine). Minimum inhibitory and minimum bactericidal concentrations (MIC/MBC) were measured, and confocal microscopy was performed.  HGFs were exposed to serial dilutions (1/10, 1/100, 1/1000, and 1/10,000) of antibacterial agents with media. Water-soluble tetrazolium-1 (WST-1) assay and lactate dehydrogenase (LDH) assay were used to assess proliferation and cytotoxicity towards HGFs. RESULTS: CHX and PVD significantly inhibited growth of all bacterial species (P < 0.0001) at all dilutions. AO and VEGA inhibited growth of all bacterial species up to only the 1/4 dilution. CHX and PVD decreased HGF proliferation at 1/10 and 1/100 dilution, whereas AO at all dilutions (P < 0.05). CHX and AO were cytotoxic at all dilutions (P < 0.05). VEGA was not cytotoxic to HGFs and did not affect HGF proliferation at any dilution (P > 0.05). An increased bacterial growth was seen for all species except P. gingivalis with addition of nicotine. CONCLUSION: CHX and PVD demonstrate superior antibacterial properties, but significantly reduce HGF proliferation. AO is bacteriostatic at lower dilutions but is highly toxic to HGFs. VEGA was bacteriostatic and demonstrated no detrimental effects on HGF's.

Effects of platelet-rich fibrin on human gingival and periodontal ligament fibroblast proliferation from chronic periodontitis versus periodontally healthy subjects.

BACKGROUND: Platelet-rich fibrin (PRF), an autogenous blood concentrate, contains multiple growth factors and is used as an adjunct in the periodontal regeneration and implant site development procedures to stimulate wound healing. Patient-related factors such as chronic periodontitis may affect the quality of PRF. OBJECTIVES: This study aimed to investigate and compare PRF's effects from patients diagnosed with generalized moderate or severe chronic periodontitis to patients who presented with intact periodontium on human gingival fibroblast (HGF) and human periodontal ligament fibroblast (HPLF) proliferation. MATERIALS AND METHODS: A total of 33 ml of whole intravenous blood was collected from each subject and centrifuged at 2700 rpm for 12 min in three 10 ml tubes, and 3 ml of blood was used for Complete Blood Count analysis. Three PRF clots were compressed to produce the membranes and liquid exudate. PRF membrane and 10% liquid exudate were exposed to 20,000 HPLFs/well or 25,000 HGFs/well in triplets from each subject in a 48 cell well plate. After 72 h of incubation, the conditioned media were evaluated by Water Soluble Tetrazolium-1 assays to determine fibroblast proliferation. Controls included cells alone and media without cells. Complete blood counts were measured. RESULTS: Subjects in both groups were age and gender-matched (intact 46.7 ± 11.4 years and periodontitis 54.8 ± 10.4 years, p-value = 0.1344). Body Mass Index and White Blood Corpuscles in the periodontitis group was significantly higher than the intact group (p = 0.0176 and p = 0.0038) whereas no differences were seen for Red Blood Corpuscles (p = 0.2020), Hemoglobin (p = 0.2290) and Platelets (p = 4,094). There were no significant differences in the HGF and HPLF proliferation with PRF exudates and membranes between intact periodontium and periodontitis groups (all p > 0.05). However, PRF exudates in both groups induced significant more cell proliferation when compared to PRF membranes. CONCLUSIONS: PRF exudates induced significant proliferation of fibroblasts and can play a vital role in wound healing. The current study concluded that PRF membranes, in combination with PRF exudates, can be utilized for their therapeutic and wound healing potential, not affected by the periodontal condition of the patient.

Combined effects of soft drinks and nicotine on Streptococcus mutans metabolic activity and biofilm formation.

The purpose of this study was to explore the effects of nicotine on the activity of Streptococcus mutans (S. mutans) in soft drinks. Regular soft drinks contain large proportions of high-fructose corn syrup (HFCS), which increases the activity of S. mutans resulting in high-caries risk compared with sugar-free soft drinks. Nicotine use exhibits a strong correlation with increased S. mutans biofilm formation. The soft drinks chosen were (Coca-Cola Classic, Diet Coke, Coca-Cola Zero Sugar, Caffeine-Free Coca-Cola, Caffeine-Free Diet Coke, Caffeine-Free Coca-Cola Zero Sugar). S. mutans was grown overnight in tryptic soy broth; nicotine was diluted in tryptic soy broth supplemented with 1.0% sucrose followed by soft drinks in dilution of 1:3. Total growth absorbance and biofilm growth were determined by spectrophotometry, absorbance measured to determine biofilm formation, and metabolic activity quantified. One-way ANOVA showed a considerable effect for HFCS and caffeine in the presence of nicotine and their interaction in all measures. Results showed sugar-free caffeinated colas demonstrated significant effect in inhibiting S. mutans biofilm formation and metabolic activity with nicotine. Nicotine-induced S. mutans increased biofilm formation and metabolic activity in the presence of HFCS and caffeine in soft drinks. In conclusion, smokers should consider sugar-free caffeinated versions to minimize the chance of developing dental caries dut to the reduction of biofilm formation.

Levels of growth factors from platelet-rich fibrin from chronic periodontitis versus periodontally healthy subjects: a pilot study.

OBJECTIVES: This study aimed to (1) compare the amounts of growth factors from platelet-rich fibrin (PRF) between chronic periodontitis and periodontally healthy subjects and (2) evaluate the relationships between the amounts of growth factors from PRF with complete blood counts (white blood cell (WBC) and platelet counts) and the serum concentrations of IL-1ß, IL-6, and tumor necrosis factor-α (TNF-α). MATERIALS AND METHODS: Venous blood was collected from chronic periodontitis (test) and periodontally healthy subjects (control). PRF and serum were collected from the centrifuged blood. Liquid exudates from the compression of PRF were collected. The compressed PRF membranes were incubated in saline, and eluted aliquots were collected at 1, 24, and 72 h, and the membranes were then digested with trypsin. Epidermal growth factor, insulin-like growth factor-1, platelet-derived growth factor-BB, transforming growth factor-ß1, and vascular endothelial growth factor in the exudates and eluents were quantified by ELISA. Serum was used for IL-1ß, IL-6, and TNF-α quantifications. Complete blood counts were measured. RESULTS: There were no significant differences in the amounts of growth factors from PRF exudates and membranes measured between groups (all p > 0.05). The test group had significantly higher WBC (p < 0.05). However, there was no significant correlation between the WBC and the amounts of the growth factors from PRF (all p > 0.05). CONCLUSIONS: PRF can be utilized as an autologous source of growth factors not affected by periodontal condition and WBC level. CLINICAL RELEVANCE: The amounts of growth factors from PRF were not affected by the periodontal condition of the patient.

Nicotine Upregulates Coaggregation of Candida albicans and Streptococcus mutans.

PURPOSE: Denture stomatitis is a condition of painless inflammation of denture-bearing mucosa. Reports indicate that nicotine, the major psychoactive ingredient in tobacco, increases growth of Streptococcus mutans and Candida albicans in denture biofilm. The purpose of this study was to determine the in vitro effects of nicotine on coaggregation of C. albicans with S. mutans. MATERIAL AND METHODS: C. albicans strain ATCC 10231, S. mutans strain UA159 (ATTC 700610), and nicotine dilutions (ranging from 0 to 32 mg/ml) were used for this study. Both microorganisms were grown for 24 hours in dilutions of nicotine (0 to 32 mg/ml) made in tryptic soy broth (TSB) or TSB supplemented with 1% sucrose (TSBS; S. mutans) or yeast peptone dextrose broth (YPD; C. albicans). Suspensions of the nicotine-treated cells were prepared, mixed together and incubated for up to 24 hours to determine if there was an increase in coaggregation of nicotine-treated cells compared to the no nicotine control cells. Qualitative analysis of coaggregation was performed using a visual aggregation assay and light microscopic observation. A spectrophotometric assay was used to provide a quantitative analysis of the coaggregation. RESULTS: The visual aggregation assay indicated a significant increase in coaggregation between C. albicans and S. mutans with increasing incubation time (0 to 24 hours) and nicotine concentrations (0 to 4 mg/ml). Microbial growth in nicotine at 4 mg/ml demonstrated a significant increase in coaggregation after 24 hours of incubation. The numbers of coaggregated S. mutans/C. albicans cells exhibited a significant increase with incubation time and nicotine concentrations when the samples were examined microscopically. More coaggregation of S. mutans and C. albicans was observed with incubation time and increased nicotine compared to the 0 mg/ml nicotine group. There was a noticeable increase of coaggregation when cells were grown in TSBS compared to TSB. Absorbance of nicotine-treated cells (0.25 to 4 mg/ml) exhibited a decrease in values compared to 0 mg/ml at 0 hours of incubation, confirming increased coaggregation. CONCLUSION: These results demonstrated the effect of nicotine in increasing the coaggregation of S. mutans with C. albicans. Coaggregation increased with incubation time and nicotine concentration. Coaggregation was increased with S. mutans grown in TSBS compared to TSB, suggesting that growth in sucrose media leads to an increase in receptors responsible for coaggregation.

Doxycycline-loaded nanotube-modified adhesives inhibit MMP in a dose-dependent fashion.

OBJECTIVES: This article evaluated the drug loading, release kinetics, and matrix metalloproteinase (MMP) inhibition of doxycycline (DOX) released from DOX-loaded nanotube-modified adhesives. DOX was chosen as the model drug, since it is the only MMP inhibitor approved by the U.S. Food and Drug Administration. MATERIALS AND METHODS: Drug loading into the nanotubes was accomplished using DOX solution at distinct concentrations. Increased concentrations of DOX significantly improved the amount of loaded DOX. The modified adhesives were fabricated by incorporating DOX-loaded nanotubes into the adhesive resin of a commercial product. The degree of conversion (DC), Knoop microhardness, DOX release kinetics, antimicrobial, cytocompatibility, and anti-MMP activity of the modified adhesives were investigated. RESULTS: Incorporation of DOX-loaded nanotubes did not compromise DC, Knoop microhardness, or cell compatibility. Higher concentrations of DOX led to an increase in DOX release in a concentration-dependent manner from the modified adhesives. DOX released from the modified adhesives did not inhibit the growth of caries-related bacteria, but more importantly, it did inhibit MMP-1 activity. CONCLUSIONS: The loading of DOX into the nanotube-modified adhesives did not compromise the physicochemical properties of the adhesives and the released levels of DOX were able to inhibit MMP activity without cytotoxicity. CLINICAL SIGNIFICANCE: Doxycycline released from the nanotube-modified adhesives inhibited MMP activity in a concentration-dependent fashion. Therefore, the proposed nanotube-modified adhesive may hold clinical potential as a strategy to preserve resin/dentin bond stability.

In Vitro Effects of Plantago Major Extract, Aucubin, and Baicalein on Candida albicans Biofilm Formation, Metabolic Activity, and Cell Surface Hydrophobicity.

PURPOSE: To determine the in vitro effectiveness of Plantago major extract, along with two of its active components, aucubin and baicalein, on the inhibition of Candida albicans growth, biofilm formation, metabolic activity, and cell surface hydrophobicity. MATERIALS AND METHODS: Twofold dilutions of P. major, aucubin, and baicalein were used to determine the minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and the minimum biofilm inhibitory concentration (MBIC) of each solution. Separately, twofold dilutions of P. major, aucubin, and baicalein were used to determine the metabolic activity of established C. albicans biofilm using a 2,3-bis (2- methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-carboxanilide reduction assay. Twofold dilutions of P. major, aucubin, and baicalein were used to determine the cell surface hydrophobicity of treated C. albicans biofilm by a two-phase assay using hexadecane. The hydrophobicity percentage of the cell surface was then calculated. A mixed-model ANOVA test was used for intergroup comparisons. RESULTS: The MICs of P. major extract (diluted 1:2 to 1:8), aucubin (61 to 244 µg/ml), and baicalein (0.0063 to 100 µg/ml) on the total growth of C. albicans were noticeable at their highest concentrations, and the inhibition was dose dependent. The MFC was evaluated after 48 hours of incubation, and aucubin (244 µg/ml) exhibited a strong fungicidal activity at its highest concentration against C. albicans growth. The MBIC indicated no growth or reduced growth of C. albicans biofilm at the highest concentrations of aucubin (61 to 244 µg/ml) and baicalein (25 to 100 µg/ml). Similarly, the effects of these reagents on C. albicans biofilm metabolic activity and hydrophobicity demonstrated high effectiveness at their highest concentrations. CONCLUSION: P. major extract, aucubin, and baicalein caused a dose-dependent reduction on the total growth, biofilm formation, metabolic activity, and cell surface hydrophobicity of C. albicans. This demonstrates their effectiveness as antifungals and suggests their promising potential use as solutions for C. albicans biofilm-related infections.

Green tea catechin inhibits the activity and neutrophil release of Matrix Metalloproteinase-9.

Green tea (Camellia sinensis; lǜ chá) extracts have been shown to possess anti-oxidant and anti-inflammatory effects in various cell types. Green tea extract (GTX) has been shown to significantly inhibit the activity of collagenase-3 (matrix metalloproteinase-13 (MMP-13)) in vitro. MMPs, such as MMP-9, are known to be involved in many inflammatory diseases including periodontal disease. GTX and a major catechin, epigallocatechin-gallate (EGCG), were examined for their ability to inhibit purified MMP-9 activity and its release from stimulated neutrophils. Methanol extract of Green tea and commercially purchased EGCG (>95 % purity) were tested in vitro for their ability to inhibit MMP-9 activity and/or its release from neutrophils using a ß-casein cleavage assay and gelatin zymography, respectively. Statistical analysis was performed by Student's t-test. GTX and EGCG at 0.1% (w/v) completely inhibited the activity of MMP-9. In addition, GTX and EGCG (0.1 %) significantly inhibited (p < 0.001) the release of MMP-9 from formyl-Met-Leu-Phe (FMLP)-stimulated human neutrophils by 62.01% ± 6.717 and 79.63% ± 1.308, respectively. The inhibitory effects of GTX and EGCG occurred in unstimulated neutrophils (52.42% ± 3.443 and 62.33% ± 5.809, respectively). When the inhibitory effect of EGCG was further characterized, it significantly inhibited the release of MMP-9 from the FMLP-stimulated human neutrophils in a dose-dependent manner. The effects of GTX and EGCG on MMPs could be extrapolated to clinical/in vivo studies for the development of oral care products to prevent or treat chronic inflammatory diseases including periodontal diseases.

The effects of radicular dentine treated with double antibiotic paste and ethylenediaminetetraacetic acid on the attachment and proliferation of dental pulp stem cells.

AIM: This study explored the effects of dentine treated with two concentrations of double antibiotic paste (DAP) and ethylenediaminetetraacetic acid (EDTA) on the attachment and proliferation of dental pulp stem cells (DPSCs). MATERIALS AND METHODS: Radicular dentine samples were prepared with identical dimensions and randomized into six groups (n = 4). Four groups were treated with double antibiotic paste (DAP) at concentrations of 500 mg ml(-1) or 1 mg ml(-1) with or without EDTA. The other two groups were treated with EDTA only or received no treatment. DPSCs were seeded on each dentine sample (10 000 cells per sample). Lactate dehydrogenase activity assays were used to calculate the attached DPSCs after 1 day of incubation. Water soluble tetrazolium assays were performed to investigate DPSCs proliferation on the treated dentine samples after three additional days of incubation. Two-way anova followed by Tukey-Kramer tests was used for statistical analyses (α = 0.05). RESULTS: Dentine treated with 1 or 500 mg ml(-1) of DAP followed by EDTA caused significant increases in DPSCs attachment compared to the dentine treated with the DAP alone. The 500 mg ml(-1) of DAP with or without EDTA caused significant reductions in DPSCs proliferation. However, the treatment of dentine with 1 mg ml(-1) of DAP did not have significant negative effects on DPSCs proliferation regardless of the use of EDTA. CONCLUSION: The use of 1 mg ml(-1) of DAP followed by 10 min of irrigation with EDTA in endodontic regeneration procedure may have no negative effects on the attachment and proliferation of DPSCs.

Increasing Tobacco Intervention Strategies by Oral Health Practitioners in Indiana.

PURPOSE: To implement and assess an evidence-based 7-hour continuing education program for Indiana oral health care practitioners on tobacco use, dependence and treatment using a team-based approach. Program effectiveness was assessed by participants' reported increase in knowledge and the extent to which they implemented course concepts and strategies into dental practice. METHODS: Course attendees' study participation was based on agreeing to provide their contact information and to complete two surveys (an 18 item post-session and 14 item 3-month follow-up) which captured their self-reported knowledge and application of course concepts. Surveys included open-ended and multiple choice (dichotomous or 5-point Likert scale) items. Follow-up surveys were mailed / delivered electronically to participants; non-responders were sent two reminders. De-identified data were analyzed in an aggregate using descriptive statistics, percentages and counts. RESULTS: Eleven programs were attended by 626 practitioners. Initial survey response rate was 91% (565); hygienists (70%), dentists (25%); unidentified (5%). Most indicated the program enhanced their knowledge of most course concepts; 98% (522) planned to use learned communication strategies. Of dentists, 90% (113) planned to refer to the Indiana quitline and 60% (71) planned to provide patient cessation materials. Follow-up response rate was 40% (250); 79% (184) reported implementing cessation communication strategies. One-third of respondents reported referring patients to the quitline for counseling. CONCLUSION: Continuing education for oral health providers in understanding tobacco use, dependence and treatment may be beneficial to enhance their capacity and willingness to integrate tobacco cessation interventions into oral healthcare settings. However, this does not necessarily assure that they will change their practice behaviors by utilizing the learned concepts and skills with patients.

A novel three-dimensional scaffold for regenerative endodontics: materials and biological characterizations.

An electrospun nanocomposite fibrous material holds promise as a scaffold, as well as a drug-delivery device to aid in root maturogenesis and the regeneration of the pulp-dentine complex. A novel three-dimensional (3D) nanocomposite scaffold composed of polydioxanone (PDS II®) and halloysite nanotubes (HNTs) was designed and fabricated by electrospinning. Morphology, structure, mechanical properties and cell compatibility studies were carried out to evaluate the effects of HNTs incorporation (0.5-10 wt% relative to PDS w/w). Overall, a 3D porous network was seen in the different fabricated electrospun scaffolds, regardless of the HNT content. The incorporation of HNTs at 10 wt% led to a significant (p < 0.0001) fibre diameter increase and a reduction in scaffold strength. Moreover, PDS-HNTs scaffolds supported the attachment and proliferation of human-derived pulp fibroblast cells. Quantitative proliferation assay performed with human dental pulp-derived cells as a function of nanotubes concentration indicated that the HNTs exhibit a high level of biocompatibility, rendering them good candidates for the potential encapsulation of distinct bioactive molecules. Collectively, the reported data support the conclusion that PDS-HNTs nanocomposite fibrous structures hold potential in the development of a bioactive scaffold for regenerative endodontics.

The direct cytotoxic effects of medicaments used in endodontic regeneration on human dental pulp cells.

AIM: The purpose of this in vitro study was to evaluate the effects of intracanal medicaments commonly used in endodontic regeneration on the survival of human dental pulp cells (DPCs). METHODS: DPCs were cultured and exposed to either no medicament treatment or low concentrations (0.3-5 mg ml(-1) ) of calcium hydroxide [Ca(OH)2 ], triple antibiotic paste (TAP), or double antibiotic paste (DAP) for 3 days. After that, toxicity to the DPCs was determined by lactate dehydrogenase activity assays (LDH) and cell proliferation was measured by colorimetric assays (WST-1). Two-way anova followed by Fisher's protected least significant differences was used for statistical analyses (α = 0.05). RESULTS: The group-by-concentration interactions were significant for the LDH and WST-1 assays (P < 0.0001). For the LDH assays, only the highest tested concentration (5 mg ml(-1) ) of Ca(OH)2 and TAP caused significant toxicity to the DPCs compared with the untreated control, while four tested concentrations of DAP (0.5, 1, 2.5, and 5 mg ml(-1) ) caused significant toxicity to the DPCs compared with the untreated control. For the WST-1 assays, the highest concentrations that did not negatively affect the proliferation rate of DAP, TAP and Ca(OH)2 were 0.3, 2, and 2.5 mg ml(-1) , respectively. CONCLUSION: The low concentrations of intracanal medicaments tested in this study were not cytotoxic in cultured cells. However, these concentrations are much lower than the concentrations that have been advocated in endodontic regeneration. Furthermore, the negative effects of TAP on DPCs were detected at lower concentrations by using the WST-1 assays than by measuring the LDH release.

Effects of interim acrylic resins on the expression of cytokines from epithelial cells and on collagen degradation.

STATEMENT OF PROBLEM: Interim acrylic resins release agents that alter cytokine expression in the surrounding tissues, which could alter extracellular matrix degradation. PURPOSE: The purpose of the study was to evaluate the responses of human epidermal keratinocytes to eluates of interim acrylic resins in regards to cytokine expression and cell-mediated collagen degradation. MATERIAL AND METHODS: Specimens of 4 different interim acrylic resins (HI-I, Jet Acrylic, SNAP acrylic, and Protemp Plus) were placed in Epilife medium for 48 hours and the eluates collected. The cells were incubated for 72 hours in nontoxic concentrations of the eluates. Cytotoxicity was evaluated with lactate dehydrogenase assays and cytokine expression with cytokine antibody arrays. Collagen degradation was determined with a collagen type I assay. The experiments were performed 3 times. Data were analyzed with 1-way and mixed-model ANOVA (α=.05). RESULTS: None of the eluates were cytotoxic. Cytokine expression from the heat-activated polymethyl methacrylate resin group was significantly less for interleukin-3, but significantly greater for interlukin-7. Expression for the chemically activated polymethyl methacrylate resin group was significantly less for growth-regulated oncogene-α, interleukin-1α, and interleukin-3. Expression for the chemically activated polyethyl methacrylate resin group was significantly less for interleukin-1α and interleukin-3, but significantly greater for interleukin-13 and monocytes chemoattractant protein-3. The cytokine expression induced by chemically activated bis-acryl composite resin was significantly greater for granulocyte-macrophage colony stimulating factor, interleukin-7, and monocytes chemoattractant protein-3, but significantly less for growth-regulated oncogene-α. Collagen degradation was not significantly different in any of the groups. CONCLUSIONS: The eluates used were not cytotoxic and did not induce cell-mediated collagen degradation. Some significant changes in cytokine expression were noted.

Nanotube-modified dentin adhesive--physicochemical and dentin bonding characterizations.

OBJECTIVE: The aim of this study was to investigate the effect of aluminosilicate clay nanotubes (Halloysite, HNT) incorporated into the adhesive resin of a commercially available three-step etch and rinse bonding system (Adper Scotchbond Multi-Purpose/SBMP) on dentin bond strength, as well as the effect on several key physicochemical properties of the modified adhesive. METHODS: Experimental adhesives were prepared by adding five distinct HNT amounts (5-30 wt.%) into the adhesive resin (w/v) of the SBMP dentin bonding system. Bond strength to human dentin, microhardness, and degree of conversion (DC) of the modified adhesives were assessed. RESULTS: From the shear bond strength data, it was determined that HNT incorporation at a concentration of 30 wt.% resulted in the highest bond strength to dentin that was statistically significant (p=0.025) when compared to the control. Even though a significant increase in microhardness (p<0.001) was seen for the 30 wt.% HNT-incorporated group, a significantly lower DC (p<0.001) was recorded when compared to the control. SIGNIFICANCE: It was concluded that HNT can be incorporated up to 20 wt.% without jeopardizing important physicochemical properties of the adhesive. The modification of the SBMP dentin bonding agent with 20 wt.% HNT appears to hold great potential toward contributing to a durable dentin bond; not only from the possibility of strengthening the bond interface, but also due to HNT intrinsic capability of encapsulating therapeutic agents such as matrix metalloproteinase (MMP) inhibitors.

Effects of resolvin D1 on cell survival and cytokine expression of human gingival fibroblasts.

BACKGROUND: Tissue breakdown in periodontitis is initiated by bacteria, such as Porphyromonas gingivalis, and is caused largely by host responses. Resolvins protect the host against acute inflammation by blocking the migration of polymorphonuclear neutrophils to initiate resolution. The effects of resolvins on human gingival fibroblasts (HGFs) are unknown. This study examines the effects of resolvin D1 on HGF survival and cytokine expression when treated with or without P. gingivalis supernatant. METHODS: Cytotoxicity of resolvin D1 on HGFs with or without a toxic level of P. gingivalis supernatant was measured with lactate dehydrogenase assays. Cytokine arrays were performed on HGF-conditioned media treated with or without resolvin D1 and with or without P. gingivalis supernatant. RESULTS: Resolvin D1 had no cytotoxic effects on HGFs at concentrations between 1 and 1,000 nM (all P > 0.05). Resolvin D1 (1,000 nM) significantly inhibited the toxic effects of 13.5% (v/v) P. gingivalis supernatant on HGFs (P = 0.002). Resolvin D1 significantly reduced the expression of interleukin (IL)-6 (P = 0.010) and monocyte chemoattractant protein (MCP)-1 (P = 0.04) in untreated fibroblasts. P. gingivalis (10%) supernatant significantly increased the expression levels of granulocyte-macrophage colony-stimulating factor (CSF), granulocyte CSF, growth-regulated oncogene (GRO), IL-5, IL-6, IL-7, IL-8, IL-10, MCP-1, MCP-2, MCP-3, and monokine induced by γ-interferon. Resolvin D1 significantly reduced the expression of GRO (P = 0.04), marginally reduced the levels of MCP-1 (P = 0.10), and marginally increased the levels of transforming growth factor (TGF)-ß1 (P = 0.07) from HGFs treated with P. gingivalis supernatant. CONCLUSIONS: Resolvin D1 altered the cytotoxicity of P. gingivalis supernatant on HGFs. Resolvin D1 significantly reduced GRO, marginally reduced MCP-1, and marginally increased TGF-ß1 from P. gingivalis-treated HGFs, which could alter the ability of P. gingivalis to induce inflammation.

The Effects of Plantago major on the Activation of the Neutrophil Respiratory Burst.

Plantago major is a common plant that grows worldwide in temperate zones and is found in fields, lawns, and on the roadsides. Its leaves and seeds have been used in almost all parts of the world for centuries as a wound healer, analgesic, antioxidant, and antibiotic, as well as an immune system modulator, antiviral, antifungal, and anti-inflammatory agent. Baicalein and aucubin are the two most biologically active components of P. major, and both have been shown to have antioxidant, anti-inflammatory, and anticancer properties. Neutrophils have a pivotal role in wound healing and inflammation. Their principal mechanism of host defense is the killing of pathogens via the production of reactive oxygen species (ROS). The aim of the present study was to determine the in vitro effects of P. major extract, baicalein, and aucubin on human neutrophil respiratory burst activity. The cytotoxicity of the agents was assessed by lactate dehydrogenase (LDH) assays. A standard luminol-dependent chemiluminescence (CL) assay was utilized to monitor the respiratory burst of the neutrophils after exposure to P. major extract and its two active ingredients, baicalein and aucubin. Three replicates per group were included in each of the three runs of the experiments and analysis of variance (ANOVA) was used for statistical analysis. P. major and baicalein were not toxic to the cells at any of the concentrations examined. Aucubin was toxic to the cells only at the highest concentration tested (P = 0.0081). However, genistein was toxic to the cells at all of the concentrations examined except for the lowest concentration of 16.9 µg/ml (P = 0.985). P. major (-0.10 ± 0.11), aucubin (0.06 ± 0.16), baicalein (-0.10 ± 0.11), and genistein (-0.18 ± 0.07) all significantly (P < 0.0001) inhibited ROS production from the neutrophils. P. major extract inhibited neutrophil ROS production, as did aucubin and baicalein. Therefore, these components should be investigated further with relation to the regulation of destructive ROS production in conditions such as periodontal disease.

Effects of Calendula officinalis on human gingival fibroblasts.

BACKGROUND: Calendula officinalis is commonly called the marigold. It is a staple topical remedy in homeopathic medicine. It is rich in quercetin, carotenoids, lutein, lycopene, rutin, ubiquinone, xanthophylls, and other anti-oxidants. It has anti-inflammatory properties. Quercetin, one of the active components in Calendula, has been shown to inhibit recombinant human matrix metalloproteinase (MMP) activity and decrease the expression of tumor necrosis factor-α, interleukin-1ß (IL), IL-6 and IL-8 in phorbol 12-myristate 13-acetate and calcium ionophore-stimulated human mast cells. OBJECTIVES: To examine the effects of Calendula on human gingival fibroblast (HGF) mediated collagen degradation and MMP activity. MATERIAL AND METHODS: Lactate dehydrogenate assays were performed to determine the non-toxic concentrations of Calendula, doxycycline and quercetin. Cell-mediated collagen degradation assays were performed to examine the inhibitory effect on cell-mediated collagen degradation. Gelatin zymography was performed to examine their effects on MMP-2 activity. The experiments were repeated three times and ANOVA used for statistical analyses. RESULTS: Calendula at 2-3% completely inhibited the MMP-2 activity in the zymograms. Doxycycline inhibited HGF-mediated collagen degradation at 0.005, 0.01, 0.02 and 0.05%, and MMP-2 activity completely at 0.05%. Quercetin inhibited HGF-mediated collagen degradation at 0.005, 0.01 and 0.02%, and MMP-2 activity in a dose-dependent manner. Calendula inhibited HGF-mediated collagen degradation and MMP-2 activity more than the same correlated concentration of pure quercetin. CONCLUSION: Calendula inhibits HGF-mediated collagen degradation and MMP-2 activity more than the corresponding concentration of quercetin. This may be attributed to additional components in Calendula other than quercetin.

Effects of alendronate on human osteoblast-like MG63 cells and matrix metalloproteinases.

OBJECTIVE: The objective of this study was to examine the effects of alendronate on the expression and activity of matrix metalloproteinases (MMPs) and the expression of the tissue inhibitors of MMPs (TIMPs) from human osteoblast-like MG63 cells. MATERIALS AND METHODS: MG63 cells were exposed to various concentrations of alendronate. Cell proliferation and cytotoxicity were evaluated by water-soluble tetrazolium-1 and lactate dehydrogenase, respectively. MG63-mediated collagen degradation was assessed utilising Type I collagen assays. Conditioned media and membrane extracts were collected for Western blot analyses of select MMPs and TIMPs. Gelatin zymography gels were incubated with alendronate to assess its effects on MMP-2 activity. RESULTS: Alendronate affected MG63 proliferation and cytotoxicity at concentrations equal to/or greater than 10(-5) M (all p < 0.05). There were no significant differences in the collagen degrading ability of treated cells at non-toxic levels vs. untreated cells. Alendronate had no effects on the expression of MMP-2 or MT1-MMP (membrane type-1 MMP) in the conditioned media or membrane extracts, and of MMP-1 or TIMP-2 in the conditioned media. TIMP-2 in the membrane extracts was not detectable. MMP-2 activity in the zymograms was inhibited by 10(-3) and 10(-2) M alendronate. CONCLUSION: Alendronate at 10(-5) M or higher was toxic to the cells. Alendronate at 10(-8) to 10(-6) M did not alter the expression of MMP-1, MMP-2, MT1-MMP or TIMP-2, as well as did not alter collagen degradation. Alendronate inhibited MMP-2 activity at 10(-3) and 10(-2) M in the zymograms. In conclusion, non-toxic levels of alendronate (10(-8) to 10(-6) M) did not alter MMP expression in MG63 cells or inhibit MMP-2 activity.