Expression analysis of matrix metalloproteinase-9 in epithelialized and nonepithelialized apical periodontitis lesions

Objective. The objective of this study was to determine the expression of matrix metalloproteinase-9 (MMP-9) in apical periodontitis lesions. Study design. Nineteen epithelialized and 18 nonepithelialized apical periodontitis lesions were collected after periapical surgery. After histological processing, serial sectioning, H&E staining, and microscopic analysis, 10 epithelialized and 10 nonepithelialized lesions were selected for immunohistochemical analysis for MMP-9 and CD 68. At least one third of each specimen collected was frozen at -70 degrees C for further mRNA isolation and reverse transcription into cDNA for real-time-PCR procedures. Geometric averaging of multiple housekeeping genes normalized MMP-9 mRNA expression level. Results. Polymorphonuclear neutrophils, macrophages and lymphocytes presented MMP-9 positive immunostaining in both types of lesions. When present, epithelial cells were also stained. The number and the ratio of MMP-9(+)/total cells were greater in nonepithelialized than epithelialized lesions (P = .0001) presenting a positive correlation to CD68(+)/total cells (P = .045). Both types of lesions presented increased MMP-9 expression (P < .0001) when compared to healthy periapical ligaments. However, no significant differences were observed for MMP-9 mRNA expression between ephithelized and nonephithelized lesions. Conclusion. The present data suggest the participation of several inflammatory cells, mainly CD68(+) cells, in the MMP-9 expression in apical periodontitis lesions. MMP-9 could be actively enrolled in the extracellular matrix degradation in apical periodontitis lesions. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 107: 127-132)

Expression of matrix metalloproteinases-2 and-9 and RECK during alveolar bone regeneration in rat

MMPs are endopeptidases that play a pivotal role in ECM turnover. RECK is a single membrane-anchored MMP-regulator. Here, we evaluated the temporal and spatial expression of MMP-2, MMP-9, and RECK during alveolar bone regeneration. The maxillary central incisor of Wistar rats was extracted and the animals were killed at 1, 3, 7, 10, 14, 21, 28, and 42 days post-operatively (n = 3/period). The hemimaxillae were collected, demineralized and embedded in paraffin. Immunohistochemical analysis was performed by the immunoperoxidase technique with polyclonal antibodies. On day 1, polymorphonuclear cells in the blood clot presented mild immunolabeling for MMPs. During bone remodeling, osteoblasts facing new bone showed positive staining for gelatinases and RECK in all experimental periods. MMPs were also found in the connective tissue and endothelial cells. Our results show for the first time that inactive and/or active forms of MMP-2, MMP-9 and RECK are differentially expressed by osteogenic and connective cells during several events of alveolar bone regeneration. This may be important for the replacement of the blood clot by connective tissue, and in the formation, maturation and remodeling of new bone.

The anti-MMP activity of benzalkonium chloride

Objective: This study evaluated the ability of benzalkonium chloride (BAC) to bind to dentine and to inhibit soluble recombinant MMPs and bound dentine matrix metalloproteinases (MMPs). Methods: Dentine powder was prepared from extracted human molars. Half was left mineralized; the other half was completely demineralized. The binding of BAG to dentine powder was followed by measuring changes in the supernatant concentration using UV spectrometry. The inhibitory effects of BAC on rhMMP-2, -8 and -9 were followed using a commercially available in vitro proteolytic assay. Matrix-bound endogenous MMP-activity was evaluated in completely demineralized beams. Each beam was either dipped into BAG and then dropped into 1 mL of a complete medium (CM) or they were placed in 1 mL of CM containing BAG for 30 days. After 30 days, changes in the dry mass of the beams or in the hydroxyproline (HYP) content of hydrolysates of the media were quantitated as indirect measures of matrix collagen hydrolysis by MMPs. Results: Demineralized dentine powder took up 10-times more BAG than did mineralized powder. Water rinsing removed about 50% of the bound BAC, whilst rinsing with 0.5 M NaCl removed more than 90% of the bound BAG. BAG concentrations 0.5 wt% produced 100% inhibition of soluble recombinant MMP-2, -8 or -9, and inhibited matrix-bound MMPs between 55 and 66% when measured as mass loss or 76-81% when measured as solubilization of collagen peptide fragments. Conclusions: BAC is effective at inhibiting both soluble recombinant MMPs and matrix-bound dentine MMPs in the absence of resins. (C) 2010 Elsevier Ltd. All rights reserved.

Effect of oxalate desensitizers and dentin moisture during total-etch bonding

Purpose: To evaluate the effect of oxalate during total-etch bonding, under different dentin moisture conditions, over time. The null hypothesis tested was that microtensile bond strength (mu TBS) was not affected by oxalate treatment and dentin moisture during two evaluation periods. Methods: Extracted human third molars had their mid-coronal dentin exposed flat and polished with 600-grit SiC paper. The surfaces were etched with 35% phosphoric acid for 15 seconds, washed and blot dried. After etching, a 3% potassium oxalate gel was applied for 120 seconds, except for the control group (no desensitizer). The surface was then washed and left moist (Wet bonding) or air-dried for 30 seconds (Dry bonding). The surfaces were bonded with: (I) two 2-step etch-and-rinse adhesives: Single Bond (SB); Prime & Bond NT (PBNT) and (2) one 3-step etch-and-rinse adhesive: Scotchbond Multi Purpose (SBMP). Composite buildups were constructed incrementally with Tetric Ceram resin composite. Each increment was cured for 40 seconds. After storage in water for 24 hours or 1 year at 37 C, the specimens were prepared for mu TBS testing with a cross-sectional area of approximately 1 mm(2). They were then tested in tension in an Instron machine at 0.5 mm/minute. Data were analyzed by ANOVA and Student-Newman-Keuls at alpha = 0.05. Results: Application of potassium oxalate had no significant effect on the bond strengths of SBMP and PBNT, regardless of the surface moisture condition (P > 0.05). Conversely, reduced bond strengths were observed after oxalate treatment for SB in both moisture conditions, that being significantly lower when using a dry-bonding procedure (P < 0.05). Lower bond strength was obtained for PBNT when a dry-bonding technique was used, regardless of the oxalate treatment (P < 0.05). After aging the specimens for 1 year, bond strengths decreased. Smaller reductions were observed for SBMP, regardless of moisture conditions. For the WB technique, smaller reductions after 1 year were observed without oxalate treatment for SB and after oxalate treatment for PBNT. (Am J Dent 2010;23:137-141).

State of the art etch-and-rinse adhesives

Objectives: The aim of this study was to explore the therapeutic opportunities of each step of 3-step etch-and-rinse adhesives. Methods: Etch-and-rinse adhesive systems are the oldest of the multi-generation evolution of resin bonding systems. In the 3-step version, they involve acid-etching, priming and application of a separate adhesive. Each step can accomplish multiple goals. Acid-etching, using 32-37% phosphoric acid (pH 0.1-0.4) not only simultaneously etches enamel and dentin, but the low pH kills many residual bacteria. Results: Some etchants include anti-microbial compounds such as benzalkonium chloride that also inhibits matrix metalloproteinases (MMPs) in dentin. Primers are usually water and HEMA-rich solutions that ensure complete expansion of the collagen fibril meshwork and wet the collagen with hydrophilic monomers. However, water alone can re-expand dried dentin and can also serve as a vehicle for protease inhibitors or protein cross-linking agents that may increase the durability of resin-dentin bonds. In the future, ethanol or other water-free solvents may serve as dehydrating primers that may also contain antibacterial quaternary ammonium methacrylates to inhibit dentin MMPs and increase the durability of resin-dentin bonds. The complete evaporation of solvents is nearly impossible. Significance: Manufacturers may need to optimize solvent concentrations. Solvent-free adhesives can seal resin-dentin interfaces with hydrophobic resins that may also contain fluoride and antimicrobial compounds. Etch-and-rinse adhesives produce higher resin-dentin bonds that are more durable than most 1 and 2-step adhesives. Incorporation of protease inhibitors in etchants and/or cross-linking agents in primers may increase the durability of resin-dentin bonds. The therapeutic potential of etch-and-rinse adhesives has yet to be fully exploited. (C) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Mechanical stability of adhesives under water storage

Objectives. To evaluate the effects of storage condition (wet or dry) and storage time (24 h and 3 months) on the ultimate tensile strength (UTS) of Single Bond (SB), 3M-ESPE; Opti Bond Solo Plus (OB), Kerr; One Step (OS), Bisco, and Prime & Bond NT (PB), Dentsply adhesive resins. Methods. Hourglass-shaped specimens were obtained from a metallic matrix. Each adhesive was dispensed to fill the molds completely and left undisturbed in a dark chamber for 4 min at 37 degrees C for solvent evaporation. They were individually light-cured for 80 s at 500 mW/cm(2) and randomly divided into three groups: 24 h of water storage; 3 months of water storage; 3 months of dry storage. The specimens were tested in tension at 0.5 mm/min using the microtensile method and data were analyzed by two-way ANOVA and SNK tests for each material. Results. Water storage for 3 months did not cause significant changes in the UTS of any of the adhesives (p-value). Values for water storage ranged from 25.9 MPa for Single Bond at 24 h to 32.7 MPa for Prime & Bond NT after 3 months. Dry storage for 3 months yielded significantly higher UTS for most adhesives, which ranged from approximately 20% for Opti Bond to 160% higher values for Single Bond compared to their 3 months wet storage values. Conclusion. The effects of storage condition and time on the UTS of adhesives were material-dependent. (C) 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

The requirement of zinc and calcium ions for functional MMP activity in demineralized dentin matrices

The progressive degradation of resin-dentin bonds is due, in part, to the slow degradation of collagen fibrils in the hybrid layer by endogenous matrix metalloproteinases (MMPs) of the dentin matrix. In in vitro durability studies, the storage medium composition might be important because the optimum activity of MMPs requires both zinc and calcium. Objective. This study evaluated the effect of different storage media on changes in matrix stiffness, loss of dry weight or solubilization of collagen from demineralized dentin beams incubated in vitro for up to 60 days. Methods. Dentin beams (1 mm x 2 mm x 6 mm) were completely demineralized in 10% phosphoric acid. After baseline measurements of dry mass and elastic modulus (E) (3-point bending, 15% strain) the beams were divided into 5 groups (n = 11/group) and incubated at 37 degrees C in either media containing both zinc and calcium designated as complete medium (CM), calcium-free medium, zinc-free medium, a doubled-zinc medium or water. Beams were retested at 3, 7, 14, 30, and 60 days of incubation. The incubation media was hydrolyzed with HCl for the quantitation of hydroxyproline (HOP) as an index of solubilization of collagen by MMPs. Data were analyzed using repeated measures of ANOVA. Results. Both the storage medium and the storage time showed significant effects on E, mass loss and HOP release (p < 0.05). The incubation in CM resulted in relatively rapid and significant (p < 0.05) decreases in stiffness, and increasing amounts of mass loss. The HOP content of the experimental media also increased with incubation time but was significantly lower (p < 0.05) than in the control CM medium, the recommended storage medium. Conclusions. The storage solutions used to age resin-dentin bonds should be buffered solutions that contain both calcium and zinc. The common use of water as an aging medium may underestimate the hydrolytic activity of endogenous dentin MMPs. (c) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Influences of surface and solvent on retention of HEMA/mixture components after evaporation

Objectives: This study examined the retention of solvents within experimental HEMA/solvent primers after two conditions for solvent evaporation: from a free surface or from dentine surface. Methods: Experimental primers were prepared by mixing 35% HEMA with 65% water, methanol, ethanol or acetone (v/v). Aliquots of each primer (50 mu l) were placed on glass wells or they were applied to the surface of acid-etched dentine cubes (2 mm x 2 mm x 2 mm) (n = 5). For both conditions (i.e. from free surface or dentine cubes), change in primers mass due to solvent evaporation was gravimetrically measured for 10 min at 51% RH and 21 degrees C. The rate of solvent evaporation was calculated as a function of loss of primers mass (%) over time. Data were analysed by two-way ANOVA and Student-Newman-Keuls (p < 0.05). Results: There were significant differences between solvent retention(%) and evaporation rate (%/min) depending on the solvent present in the primer and the condition for evaporation (from free surface or dentine cubes) (p < 0.05). For both conditions, the greatest amount of retained solvent was observed for HEMA/water primer. The rate of solvent evaporation for HEMA/acetone primer was almost 2- to 10-times higher than for HEMA/water primer depending whether evaporation occurred, respectively, from a free surface or dentine cubes. The rate of solvent evaporation varied with time, being in general highest at the earliest periods. Conclusions: The rate of solvent evaporation and its retention into HEMA/solvent primers was influenced by the type of the solvent and condition allowed for their evaporation. (C) 2009 Elsevier Ltd. All rights reserved.

The influence of storage condition and duration on the resistance to fracture of different fiber post systems

Purpose: To evaluate the effects of storage condition and duration on the resistance to fracture of different fiber post systems (and to morphologically assess the post structure before and after storage. Methods: Three types of fiber posts (DT Light Post, GC Post, FRC Postect Plus) were divided in different groups (n=12) according to the storage condition (dry at 37 degrees C; saline water at 37 degrees C; mineral oil at 37 degrees C and storage inside the roots of extracted human teeth immersed in saline water at 37 degrees C and duration (6, 12 months). A universal testing machine loading at a 90 degrees angle was employed for the three-point bending test. The test was carried out until fracture of the post. A 3-way ANOVA and Tukey`s test (alpha= 0.05) were used to compare the effect of the experimental factors on the fracture strength. Two posts of each group were observed before and after the storage using a scanning electron microscope. Results: Storage condition and post type had a significant effect on post fracture strength (P< 0.05). The interaction between these factors was significant (P< 0.05). Water storage significantly decreased the fracture strength, regardless of the post type and the storage duration. Storage inside roots, in oil, and at dry conditions did not significantly affect post fracture strength. SEM micrographs revealed voids between fibers and resin matrix for posts stored in water. Posts stored under the other conditions showed a compact matrix without porosities. (Am J Dent 2009;22:366-370).

Cytotoxic effects and pulpal response caused by a mineral trioxide aggregate formulation and calcium hydroxide

Purpose: To evaluate: the in vivo pulpal response after pulpotomy with different capping agents. In addition, the in vitro cytotoxic effects of both materials were assessed by applying them on culture of pulp cells. Methods: For the in vivo test, the coronal pulp of 28 teeth of dogs was mechanically removed and the root pulps were capped with the following dental materials: Group 1: Pro-Root NITA (PRMTA); and Group 2 (control): calcium hydroxide saline paste (CH). After 60 days, the animals were sacrificed and the teeth processed for histological analysis. In the in vitro test, experimental extracts obtained from both capping agents were applied on the cultured MDPC-23 odontoblast-like cells. Results: In the root pulps capped with PRMTA or CH, coagulation necrosis partially replaced by dystrophic calcification as well as tubular dentin matrix laid down by elongated pulp cells was observed. None or mild inflammatory response occurred beneath the capped pulpal wound. Regarding the pulpal response, PRMTA and CH presented no statistical difference. However, the teeth capped CH presented greater healthy pulp loss which resulted in convex shape of the hard barrier than PRMTA. When applied on the cultured cells, it was demonstrated that PRMTA and CH solutions decreased the cell metabolic activity by 9.9% and 29.4%, respectively. CH caused higher cytotoxic effects to the MDPC-23 cells as well as deeper healthy pulp tissue loss than PRMTA. However, similar sequence of healing occurred after pulpotomy with both dental materials.

In vitro osteogenesis induced by cells derived from sites submitted to sinus grafting with anorganic bovine bone

Objectives: This study evaluated key parameters of the in vitro osteogenesis induced by osteoblastic cells obtained from sites submitted to sinus grafting with anorganic bovine bone (ABB) in comparison with cells derived from bone sites of the same patients. Materials and methods: In three patients, the augmentation of maxillary sinus was carried out using ABB (Bio-Oss (R)). After at least 6 months, during the surgical intervention for titanium implants placement, biopsies were taken from these areas using trephine burs (grafted group). Bone fragments, of the same patients, from sites that had not received graft were also obtained with trephine burs and used as a control group. Osteoblastic cells were obtained from grafted and control groups by enzymatic digestion and cultured under standard osteogenic condition until subconfluence. First passaged cells were cultured in 24-well culture plates. Cell adhesion was evaluated at 24 h. For proliferation and viability assay, cells were cultured for 1, 3, 7, and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 3, 7, 10, 14, 17, and 21 days. Cultures were stained with Alizarin red S at 21 days, for detection of mineralized matrix. Data were compared by Student`s t-test. Results: Cell adhesion and viability were not affected by cell source (P>0.05). Total protein content was greater (P<0.05) for grafted group. Cell proliferation, ALP activity, and bone-like nodule formation were all greater (P<0.05) for the control group. Conclusions: Taken together, these results indicate that the in vivo long-term contact of cells with ABB downregulates the expression of osteoblast phenotype and consequently the in vitro osteogenesis.

Pyrrolidine dithiocarbamate down-regulates vascular matrix metalloproteinases and ameliorates vascular dysfunction and remodelling in renovascular hypertension

BACKGROUND AND PURPOSE Mounting evidence implicates matrix metalloproteinase (MMP) in the vascular dysfunction and remodelling associated with hypertension. We tested the hypothesis that treatment with pyrrolidine dithiocarbamate (PDTC), which interferes with NF-kappa B-induced MMPs gene transcription, could exert antihypertensive effects, prevent MMP-2 and MMP-9 up-regulation, and protect against the functional alterations and vascular remodelling of two-kidney, one clip (2K1C) hypertension. EXPERIMENTAL APPROACH Sham-operated or hypertensive rats were treated with vehicle or PDTC (100 mg.Kg(-1).day(-1)) by gavage for 8 weeks. Systolic blood pressure (SBP) was monitored weekly. Aortic rings were isolated to assess endothelium-dependent relaxations. Quantitative morphometry of structural alterations of the aortic wall was carried out in haematoxylin/eosin sections. Formation of vascular reactive oxygen species (ROS), and inducible (i) NOS and phosphorylated-p65 NF-kappa B subunit expression were measured in the aortas. MMP-2 and MMP-9 aortic levels and gelatinolytic activity were determined by gelatin and in situ zymography and by immunofluorescence. KEY RESULTS Treatment with PDTC attenuated the increases in SBP and prevented the endothelial dysfunction associated with 2K1C hypertension. Moreover, PDTC reversed the vascular aortic remodelling, the increases in aortic ROS levels and in iNOS and phosphorylated-p65 NF-kappa B expression found in 2K1C rats. These effects were associated with attenuation of 2K1C up-regulation of aortic MMP-2 and MMP-9 levels and gelatinolytic activity. CONCLUSION AND IMPLICATIONS These findings suggest that PDTC down-regulates vascular MMPs and ameliorates vascular dysfunction and remodelling in renovascular hypertension, thus providing evidence supporting the suggestion that PDTC is probably a good candidate to be used to treat hypertension.

Comparative study on antioxidant effects and vascular matrix metalloproteinase-2 downregulation by dihydropyridines in renovascular hypertension

The vascular remodeling associated with hypertension involves oxidative stress and enhanced matrix metalloproteinases (MMPs) expression/activity, especially MMP-2. While previous work showed that lercanidipine, a third-generation dihydropyridine calcium channel blocker (CCB), attenuated the oxidative stress and increased MMP-2 expression/activity in two-kidney, one-clip (2K1C) hypertension, no previous study has examined whether first- or second-generation dihydropyridines produce similar effects. We compared the effects of nifedipine, nimodipine, and amlodipine on 2K1C hypertension-induced changes in systolic blood pressure (SBP), vascular remodeling, oxidative stress, and MMPs levels/activity. Sham-operated and 2K1C rats were treated with water, nifedipine 10 mg/kg/day, nimodipine 15 mg/kg/day, or amlodipine 10 mg/kg/day by gavage, starting 3 weeks after hypertension was induced. SBP was monitored weekly. After 6 weeks of treatment, quantitative morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin-stained sections. Aortic and systemic reactive oxygen species levels were measured by using dihydroethidine and thiobarbituric acid-reactive substances (TBARs), respectively. Aortic MMP-2 levels and activity were determined by gelatin zymography, in situ zymography, and immunofluorescence. Nifedipine, nimodipine, or amlodipine attenuated the increases in SBP in hypertensive rats by approximately 17% (P<0.05) and prevented vascular hypertrophy (P<0.05). These CCBs blunted 2K1C-induced increases in vascular oxidative stress and plasma TBARs concentrations (P<0.05). All dihydropyridines attenuated the increases in aortic MMP-2 levels and activity associated with 2K1C hypertension. These findings suggest lack of superiority of one particular dihydropyridine, at least with respect to antioxidant effects, MMPs downregulation, and inhibition of vascular remodeling in hypertension.

Spironolactone and hydrochlorothiazide exert antioxidant effects and reduce vascular matrix metalloproteinase-2 activity and expression in a model of renovascular hypertension

Background and purpose: Increased oxidative stress and up-regulation of matrix metalloproteinases (MMPs) may cause structural and functional vascular changes in renovascular hypertension. We examined whether treatment with spironolactone (SPRL), hydrochlorothiazide (HCTZ) or both drugs together modified hypertension-induced changes in arterial blood pressure, aortic remodelling, vascular reactivity, oxidative stress and MMP levels and activity, in a model of renovascular hypertension. Experimental approach: We used the two-kidney,one-clip (2K1C) model of hypertension in Wistar rats. Sham-operated or hypertensive rats were treated with vehicle, SPRL (25 mg center dot kg-1 center dot day-1), HCTZ (20 mg center dot kg-1 center dot day-1) or a combination for 8 weeks. Systolic blood pressure was monitored weekly. Aortic rings were isolated to assess endothelium-dependent and -independent relaxations. Morphometry of the vascular wall was carried out in sections of aorta. Aortic NADPH oxidase activity and superoxide production were evaluated. Formation of reactive oxygen species was measured in plasma as thiobarbituric acid-reactive substances. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry and immunohistochemistry. Key results: Treatment with SPRL, HCTZ or the combination attenuated 2K1C-induced hypertension, and reversed the endothelial dysfunction in 2K1C rats. Both drugs or the combination reversed vascular aortic remodelling induced by hypertension, attenuated hypertension-induced increases in oxidative stress and reduced MMP-2 levels and activity. Conclusions and implications: SPRL or HCTZ, alone or combined, exerted antioxidant effects, and decreased renovascular hypertension-induced MMP-2 up-regulation, thus improving the vascular dysfunction and remodelling found in this model of hypertension.

Imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases in hypertensive vascular remodeling

Structural vascular changes in two-kidney, one-clip (2K-1C) hypertension may result from increased matrix metalloproteinase (MMP)-2 activity. MMP-2 activation is regulated by other MMPs, including transmembrane-MMPs, and by tissue inhibitors of MMPs (TIMPs). We have investigated the localization of MMP-2, -9, -14, and TIMPs 1-4 in hypertensive aortas and measured their levels by zymography/Western blotting and immunohistochemistry. Gelatinolytic activity was assayed in tissues by in situ zymography. Sham-operated and 2K-1C hypertensive rats were treated with doxycycline (or vehicle) for 8 weeks, and the systolic blood pressure was monitored weekly. Doxycycline attenuated 2K-1C hypertension (165 +/- 11.7 mmHg versus 213 +/- 7.9 mm Hg in hypertensive controls, P<0.01), and completely prevented increase in the thicknesses of the media and the intima in 2K-1C animals (P<0.01). Increased amounts of MMP-2, -9, and -14 were found in hypertensive aortas, as well as enhanced gelatinolytic activity. A gradient in the localization of MMP-2, -9, and -14 was found, with increased amounts detected in the intima, at sites with higher gelatinolytic activity. Doxycycline attenuated hypertension induced increases in all the 3 investigated MMPs in both the media and the intima (all P<0.05). but it did not change the amounts of TIMPs 1-4 (P>0.05). Therefore, an imbalance between increased amounts of MMPs at the tissue level without a corresponding increase in the quantities of TIMPs, particularly in the intima and inner media layers, appears to account for the increased proteolytic activity found in 2K-1C hypertension-induced maladaptive vascular remodeling. (C) 2009 Elsevier B.V. All rights reserved.