Juliana Santos

Determination of Matrix Metalloproteinases in Human Radicular Dentin

Matrix metalloproteinases (MMPs) are present in sound coronal dentin and may play a role in collagen network degradation in bonded restorations. We investigated whether these enzymes can also be detected in root dentin. Crown and root sections of human teeth were powderized, and dentin proteins were extracted by using guanidine-HCl and EDTA. Extracts were analyzed by zymography and Western blotting for matrix metalloproteinases detection. Zymography revealed gelatinolytic activities in both crown and root dentin samples, corresponding to MMP-2 and MMP-9. MMP-2 was more evident in demineralized root dentin matrix, whereas MMP-9 was mostly extracted from the mineralized compartment of dentin and presented overall lower levels. Western blot analysis detected MMP-8 equally distributed in crown and root dentin. Because MMPs are also present in radicular dentin, their contribution to the degradation of resin-dentin bonds should be addressed in the development of restorative strategies for the root substrate.

Long‐term sealing ability of resin‐based root canal fillings

AIM To evaluate the ability of two resin-based filling materials to provide immediate and long-term sealing of the root canal. METHODOLOGY A total of eighty-two human roots were instrumented and filled with AH Plus/gutta-percha or Epiphany/Resilon. Root filled teeth were sealed coronally either with Coltosol or Clearfil SE Bond/Filtek Z250 or were left unsealed. The quality of root canal sealing was assessed by a fluid filtration method performed at immediate and 180-day time intervals. Mean fluid filtration rates were analyzed by three-way repeated measures anova and Tukey post hoc test. RESULTS Specimens filled with Epiphany/Resilon exhibited higher leakage than specimens filled with AH Plus/gutta-percha (P < 0.05), regardless of the coronal sealing condition and period of evaluation. No difference was detected between coronal restorative materials (P > 0.05), whilst leakage in teeth without any coronal restoration was significantly higher (P < 0.05). After storage, a significant decrease in leakage (P < 0.05) was observed in all experimental groups. CONCLUSIONS AH Plus/gutta-percha provided superior root canal sealing at both immediate and 180-day time periods. The presence of a coronal seal reduced leakage significantly. Storage of root filled specimens did not disturb the sealing ability of the tested materials.

Cranberry Proanthocyanidins: Natural Weapons against Periodontal Diseases

Cranberry ( Vaccinium macrocarpon ) is known to have a beneficial effect on several aspects of human health. Proanthocyanidins (PACs), the most abundant flavonoids extracted from red cranberry fruits, have been reported to possess antimicrobial, antiadhesion, antioxidant, and anti-inflammatory properties. Recent in vitro studies have shown that cranberry PACs may be potential therapeutic agents for the prevention and management of periodontitis, an inflammatory disease of bacterial origin affecting tooth-supporting tissues. After presenting an overview of cranberry phytochemicals and their potential for human health benefits, this review will focus on the effects of cranberry PACs on connective tissue breakdown and alveolar bone destruction, as well as their potential for controlling periodontal diseases. Possible mechanisms of action of cranberry PACs include the inhibition of (i) bacterial and host-derived proteolytic enzymes, (ii) host inflammatory response, and (iii) osteoclast differentiation and activity. Given that cranberry PACs have shown interesting properties in in vitro studies, clinical trials are warranted to better evaluate the potential of these molecules for controlling periodontal diseases.

Activity of matrix metalloproteinases in bovine versus human dentine.

Metalloproteinases (MMPs) have been implicated with metabolism of collagen in physiological and pathological processes in human dentine. As bovine teeth have been used as a substitute for human teeth in laboratory analysis, this study evaluated the activity of MMP-2 and -9 in bovine versus human dentine. Bovine and human dentine fragments, from crowns and roots, were powderized. Protein extraction was performed by two protocols: a neutral extraction with guanidine-HCl/EDTA (pH 7.4) and an acidic extraction with citric acid (pH 2.3). Gelatinolytic activities of extracts were revealed by zymography. MMP-2 and -9 were detected in crown and root dentine from bovine and human teeth. Total activities of MMP-2 were 11.4 ± 2.2, 14.6 ± 2.0, 9.7 ± 1.2 and 12.4 ± 0.9 ng/ml for bovine root, human root, bovine crown and human crown dentine, respectively. Corresponding activities for MMP-9 were 14.9 ± 2.0, 15.3 ± 1.3, 15.4 ± 1.3 and 15.5 ± 1.3 ng/ml, respectively. Bovine dentine was found to be a reliable substrate for studies involving the activity of MMP-2 and -9.

A-Type Cranberry Proanthocyanidins Inhibit the RANKL-Dependent Differentiation and Function of Human Osteoclasts

This study investigated the effect of A-type cranberry proanthocyanidins (AC-PACs) on osteoclast formation and bone resorption activity. The differentiation of human pre-osteoclastic cells was assessed by tartrate-resistant acid phosphatase (TRAP) staining, while the secretion of interleukin-8 (IL-8) and matrix metalloproteinases (MMPs) was measured by ELISA. Bone resorption activity was investigated by using a human bone plate coupled with an immunoassay that detected the release of collagen helical peptides. AC-PACs up to 100 µg/mL were atoxic for osteoclastic cells. TRAP staining evidenced a dose-dependent inhibition of osteoclastogenesis. More specifically, AC-PACs at 50 µg/mL caused a 95% inhibition of RANKL-dependent osteoclast differentiation. This concentration of AC-PACs also significantly increased the secretion of IL-8 (6-fold) and inhibited the secretion of both MMP-2 and MMP-9. Lastly, AC-PACs (10, 25, 50 and 100 µg/ml) affected bone degradation mediated by mature osteoclasts by significantly decreasing the release of collagen helical peptides. This study suggests that AC-PACs can interfere with osteoclastic cell maturation and physiology as well as prevent bone resorption. These compounds may be considered as therapeutic agents for the prevention and treatment of periodontitis.

Comparative evaluation of two structurally related flavonoids, isoliquiritigenin and liquiritigenin, for their oral infection therapeutic potential.

Isoliquiritigenin (1) and liquiritigenin (2) are structurally related flavonoids found in a variety of plants. The purpose of this study was to perform a comparative analysis of biological properties of these compounds in regard to their therapeutic potential for oral infections. Compound 1 demonstrated significant antibacterial activity against three major periodontopathogens, Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia. In contrast, 2 exerted less pronounced effects on the above bacterial species. Neither compound was effective against cariogenic bacteria (Streptococcus mutans and Streptococcus sobrinus). Furthermore, 1 exhibited a stronger inhibitory activity than 2 toward P. gingivalis collagenase and human matrix metalloproteinase 9. Finally, the capacity of 1 to attenuate the inflammatory response of macrophages induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) was much higher when compared to 2. The activation of transcriptional factors nuclear factor-κB (NF-κB) p65 and activator protein-1 (AP-1) associated with the LPS-induced inflammatory response in macrophages was inhibited strongly by 1, but less affected by 2.

Inhibition of host- and bacteria-derived proteinases by natural anthocyanins

BACKGROUND AND OBJECTIVES Host- and bacteria-derived proteinases are considered to play critical roles in periodontitis progression. This study investigated the ability of a blackcurrant extract and its major anthocyanins (cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside and delphinidin-3-O-rutinoside) to inhibit the activity of matrix metalloproteinases (MMPs), neutrophil elastase and periodontopathogen (Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola) proteinases. MATERIAL AND METHODS Enzyme inhibition was detected using fluorometric and colorimetric assays after incubating blackcurrant extract and its major anthocyanins (at concentrations of 6.25, 12.5, 25 and 50 μg/mL) with MMPs, elastase or bacterial proteinases, along with their specific substrates. Substrate degradation was recorded every hour for up to 4 h. RESULTS The blackcurrant extract (50 μg/mL) inhibited all proteinases tested. MMP-1 and MMP-9 were significantly inhibited by pure anthocyanins at concentrations ranging from 6.25 to 50 μg/mL. Elastase activity was inhibited by cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside in the range of 6.25-50 μg/mL and by delphinidin-3-O-rutinoside at 50 μg/mL. P. gingivalis, T. forsythia and T. denticola proteinases were also significantly inhibited by pure anthocyanins. In all cases, enzyme inhibition was time-dependent. CONCLUSION Our study showed that a blackcurrant extract and its major anthocyanins were able to inhibit the activity of host- and bacteria-derived proteinases. This suggests that such natural compounds may represent promising agents for use in adjunctive treatments for periodontitis.

Technical quality of root canal treatment performed by undergraduate students using hand instrumentation: a meta-analysis

The technical quality of root canal treatment (RCT) may impact on the outcome. The quality of education received during undergraduate school may be linked to the quality of treatment provided in general dental practice. In this context, the aim of this systematic review was to answer the following focused questions: (i) What is the frequency of acceptable technical quality of root fillings, assessed radiographically, performed by undergraduate students? (ii) What are the most common errors assessed radiographically and reported in these treatments? For this purpose, articles that evaluated the quality of root fillings performed by undergraduate students were selected. Data were collected based on predetermined criteria. The key features from the included studies were extracted. GRADE-tool assessed the quality of the evidence. MAStARI evaluated the methodological quality, and a meta-analysis on all studies was conducted. At the end of the screening, 24 articles were identified. Overall frequency of acceptable technical quality of root fillings was 48%. From this total, 52% related to anterior teeth, 49% to premolars and 26% to molars. The main procedural errors reported were ledge formation, furcation perforation, apical transportation and apical perforation. The heterogeneity amongst the studies was high (84-99%). Five studies had a high risk of bias, eight had a moderate risk, and 11 had low risk. The overall quality of evidence identified was very low. The conclusion was that technical quality of root fillings performed by undergraduate students is low, which may reveal that endodontic education has limited achievement at undergraduate level. A plan to improve the quality of root fillings, and by extrapolation the overall quality of root canal treatment, should be discussed by the staff responsible for endodontic education and training.

Collinin Reduces Porphyromonas gingivalis Growth and Collagenase Activity and Inhibits the Lipopolysaccharide-Induced Macrophage Inflammatory Response and Osteoclast Differentiation and Function

BACKGROUND Collinin is a secondary plant metabolite belonging to the class of geranyloxycoumarins. We explored the potential beneficial impact of collinin on periodontal health by investigating its effect on Porphyromonas gingivalis (P. gingivalis), lipopolysaccharide (LPS)-induced inflammatory response of macrophages, and osteoclastogenesis. METHODS Collinin was synthesized from pyrogallol and propiolic acid. A microdilution assay was used to determine antibacterial activity of collinin. The effect of collinin on collagenase activity of P. gingivalis was determined using fluorescent collagen. Macrophages were treated with collinin before being stimulated with LPS. The secretion of interleukin-6, chemokine (C-C motif) ligand 5, and prostaglandin E2 was assessed by enzyme-linked immunosorbent assays (ELISA). The inhibitory effect of collinin on differentiation of human preosteoclastic cells was assessed by tartrate-resistant acid phosphatase staining, whereas the secretion of matrix metalloproteinase-9 (MMP-9) was measured by ELISA. Bone resorption activity was investigated by using a human bone plate coupled with an immunoassay that detected the release of collagen fragments. RESULTS Collinin inhibited the growth of P. gingivalis. The effect was more pronounced under iron-restricted conditions. Collinin dose dependently inhibited the degradation of type I collagen by P. gingivalis. It was also a potent inhibitor of the LPS-induced inflammatory response in macrophages and completely inhibited receptor activator of nuclear factor κB ligand-dependent osteoclast differentiation and MMP-9 secretion. Last, collinin affected bone degradation mediated by mature osteoclasts by significantly decreasing the release of collagen helical peptides. CONCLUSION Although clinical trials are required, data from these in vitro analyses support the potential of collinin as a therapeutic agent for treating inflammatory periodontitis associated with bone breakdown.