Immunocytochemical detection of dentine matrix protein 1 in experimentally induced reactionary and reparative dentine in rat incisors

Objective: Although the general mechanisms of dentinogenesis are understood, several aspects regarding tertiary dentine formation still deserve investigation, especially regarding the presence and distribution of some noncollagenous matrix proteins. As dentine matrix protein 1 (DMP 1) is present in primary dentine, it is possible that this protein may also be present in the dentine matrix secreted after injury, but there are no immunocytochemical studies attempting its detection in tertiary dentine. The aim of this study was to examine the ultrastructural immunolocalization of DMP 1 in the tertiary dentine after extrusion of the rat incisor. Study design: Upper incisors were extruded 3 mm and then repositioned into their sockets. After several periods, the incisors were fixed and processed for transmission electron microscopy and for immunocytochemistry for DMP 1. Results: Extrusion yielded both types of tertiary dentine, which varied in aspect and related cells. DMP 1 was found in the mineralized matrix of all types of dentine, presenting high affinity for collagen, but rare colloidal gold particles over predentine. DMP 1 was evident in the supranuclear region and inside the nucleus of some odontoblast-like cells. Conclusion: The observed association between DMP 1 and collagen seem to be essential for reactionary and reparative dentine formation. (C) 2010 Elsevier Ltd. All rights reserved.

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. Copyright (C) 2011 S. Karger AG, Basel

Effect of Iron on Matrix Metalloproteinase Inhibition and on the Prevention of Dentine Erosion

It is known that some metal salts can inhibit matrix metalloproteinase (MMP) activity, but the effect of iron has not been tested yet. On the other hand, it has recently been suggested that MMP inhibition might influence dentine erosion. Based on this, the aims of this study were: (1) to test in vitro the effect of FeSO(4) on MMP-2 and -9 activity, and (2) to evaluate in situ the effect of FeSO(4) gel on dentine erosion. MMP-2 and -9 activities were analysed zymographically in buffers containing FeSO(4) in concentrations ranging between 0.05 and 1.5 mmol/l or not. Volunteers (n = 10) wore devices containing bovine dentine blocks (n = 60) previously treated with the following gel treatments: FeSO(4) (1 mmol/l FeSO(4)), F (NaF 1.23%; positive control) and placebo (negative control). The gels were applied once and removed after 1 min. Erosion was performed extraorally with Coca-Cola 4 times per day for 5 min over 5 days. Dentine wear was evaluated by profilometry. The data were analysed by Kruskal-Wallis and Dunn`s tests (p < 0.05). FeSO(4) inhibited both MMP-2 (IC(50) = 0.75 mmol/l) and MMP-9 (IC(50) = 0.50 mmol/l) activities. In the in situ experiment, the mean wear (+/- SD) found for the F gel (0.79 8 +/- 0.08 mu m) was significantly reduced in more than 50% when compared to the placebo gel (1.77 +/- 0.33 mu m), but the FeSO(4) gel completely inhibited the wear (0.05 +/- 0.02 mu m). Since FeSO(4) was able to inhibit MMP in vitro, it is possible that the prevention of dentine wear by the FeSO(4) gel in situ might be due to MMP inhibition, which should be investigated in further studies. Copyright (C) 2010 S. Karger AG, Basel

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.

Functional polymorphisms in the promoter of the matrix metalloproteinase-9 (MMP-9) gene are not linked with significant plasma MMP-9 variations in healthy subjects

Background: Matrix metalloproteinase-9 (MMP-9) is involved in the degradation of the extracellular matrix during physiological and pathological processes. Two functional polymorphisms [C(-1562)T and microsatellite (CA)(13-25)] in the promoter region of the MMP-9 gene have been associated with several diseases. The aim of this study was to examine whether these MMP-9 polymorphisms and haplotypes are linked with plasma MMP-9 variations in healthy subjects. Methods: We studied 177 healthy male white volunteers (age range 20-55 years) who were non-smokers and not taking any medication. Genomic DNA was extracted from whole blood and genotypes for the C(-1562)T and the microsatellite (CA)(n) polymorphisms were determined. MMP-9 levels were measured in plasma samples by gelatin zymography. Results: The frequency of the alleles C and T for the C(-1562)T polymorphism were 90% and 10%, respectively. The frequency of the alleles with less than 21 CA repeats Q and with 21 repeats or higher (H) were 47% and 53%, respectively. We found no differences in plasma MMP-9 levels among the genotype groups or among different haplotypes (all p > 0.05). Conclusions: These findings suggest that functional polymorphisms in the promoter of the MMP-9 gene are not linked with significant plasma MMP-9 variations in healthy subjects.

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.

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.

Evidence for the involvement of matrix metalloproteinases in the cardiovascular effects produced by nicotine

Nicotine plays a role in smoking-associated cardiovascular diseases, and may upregulate matrix metalloproteinase (MMP)-2 and MMP-9. We examined whether nicotine induces the release of MMP-2 and MMP-9 by rat smooth muscle cells (SMC), and whether doxycycline (non-selective MMP inhibitor) inhibits the vascular effects produced by nicotine. SMC were incubated with nicotine 0, 50, and 150 nM for 48 h. MMP-2 and MMP-9 levels in the cell supernatants were determined by gelatin zymography. The acute changes in mean arterial pressure caused by nicotine 2 mu mol/kg (or saline) were assessed in rats pretreated with doxycycline (or saline). We also examined whether doxcycline (30 mg/Kg, i.p., daily) modifies the effects of nicotine (10 mg/kg/day; 4 weeks) on the endothelium-dependent relaxations of rat aortic rings. Aortic MMP-2 levels were assessed by gelatin zymography. Aortic gelatinolytic activity was assessed using a gelatinolytic activity kit. MMP-2 and MMP-9 levels increased in the supernatant of SMC cells incubated with nicotine 150 nM (P<0.05) but not with 50 nM. Nicotine (2 mu mol/kg) produced lower increases in the mean arterial pressure in rats pretreated with doxycycline than those found in rats pretreated with saline (26 +/- 4 vs. 37 +/- 4 mmHg, respectively; P<0.05). Nicotine impaired of the endothelium-dependent responses to acetylcholine, and treatment with doxycycline increased the potency (pD2) by approximately 25% (P<0.05). While we found no significant differences in aortic MMP-2 levels, nicotine significantly increased gelatinolytic activity (P<0.05). These findings suggest that nicotine produces cardiovascular effects involving MMPs. It is possible that MMPs inhibition may counteract the effects produced by nicotine. (C) 2009 Elsevier B.V. All rights reserved.

A genetic polymorphism of matrix metalloproteinase 9 (MMP-9) affects the changes in circulating MMP-9 levels induced by highly active antiretroviral therapy in HIV patients

We examined whether two functional polymorphisms (g.-1562C>T and g.-90(CA)14-24) in the matrix metalloproteinase (MMP)-9 gene or MMP-9 haplotypes affect the circulating levels of pro-MMP-9 and pro-MMP-9/TIMP-1 (tissue inhibitor of metalloproteinase-1) ratios in AIDS patients, and modulate alterations in these biomarkers after highly active antiretroviral therapy (HAART). We studied 82 patients commencing HAART. Higher pro-MMP-9 concentrations and pro-MMP-9/TIMP-1 ratios were found in CT/TT patients compared with CC patients. HAART decreased pro-MMP-9 levels and pro-MMP-9/TIMP-1 ratios in CT/TT patients, it did not modify pro-MMP-9 levels and it increased pro-MMP-9/TIMP-1 ratios in CC patients. The g.-90(CA)14-24 polymorphism, however, produced no significant effects. Moreover, we found no significant differences in HAART-induced changes in plasma pro-MMP-9, TIMP-1 and pro-MMP-9/TIMP-1 ratios when different MMP-9 haplotypes were compared. These findings suggest that the g.-1562C>T polymorphism affects pro-MMP-9 levels in patients with AIDS and modulates the alterations in pro-MMP-9 levels caused by HAART, thus possibly affecting the risk of cardiovascular complications. The Pharmacogenomics Journal (2009) 9, 265-273; doi: 10.1038/tpj.2009.13; published online 21 April 2009

Assessment of matrix metalloproteinase (MMP)-2, MMP-8, MMP-9, and their inhibitors, the tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in obese children and adolescents

Objectives: To compare the circulating levels of matrix metalloproteinase (MMP)-8, pro-MMP-2, pro-MMP-9, and total MMP-9, their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2, and the MMP-8/TIMP-1, MMP-9/TIMP-1, and MMP-2/TIMP-2 ratios in normotensive obese children and adolescents with those found in non obese children and adolescents. Design and methods: We studied 40 obese and 40 non obese (controls) children and adolescents in this cross-sectional study. MMP and TIMP concentrations were measured in plasma samples by gelatin zymography and ELISA. Results: Obese children and adolescents had higher circulating MMP-8 concentrations, lower plasma TIMP-1 concentrations, and higher MMP-8/TIMP-1 ratios than non obese controls (P < 0.05). We found no differences in pro-MMP-9 or total MMP-9 levels, or in MMP-9/TIMP-1 ratios between groups (P > 0.05). While we found no significant differences in pro-MMP-2 levels (P > 0.05) obese Subjects had higher TIMP-2 concentrations and lower pro-MMP-2/TIMP-2 ratios (P < 0.05) than non obese controls. Conclusions: In conclusion, we found evidence indicating higher net MMP-8 (but not MMP-9 and MMP-2) activity in childhood obesity. The increased MMP-8 levels found in obese children suggest a possibly relevant pathophysiological mechanism that may be involved in the increase of cardiovascular risk associated with childhood obesity. (c) 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Increased circulating levels of matrix metalloproteinase (MMP)-8, MMP-9, and pro-inflammatory markers in patients with metabolic syndrome

Background: Metabolic syndrome (MetS) predisposes to cardiovascular complications. Increased concentrations of pro-inflammatory mediators and imbalanced concentrations of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) may reflect the pathophysiology of MetS. We compared the circulating levels of MMPs, TIMPs, and inflammatory mediators in MetS patients with those found in healthy controls. Methods: We studied 25 healthy subjects and 25 MetS patients. The plasma levels of pro-MMP-2 and pro-MMP-9 were determined by gelatin zymography. The plasma concentrations of MMP-8, MMP-3, TIMP-1, TIMP-2, monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), intercellular adhesion molecule (sICAM-1), and sP-selectin were measured by ELISA kits. Results: We found higher sP-selectin, sICAM-1, MCP-1, and IL-6 (all P<0.05) concentrations in MetS patients compared with healthy controls. No differences in pro-MMP-2, MMP-3, and TIMP-2 levels were found (all P>0.05). However, we found higher pro-MMP-9, MMP-8. and TIMP-1 levels in MetS patients compared with healthy controls (all P<0.05). Conclusions: Patients with MetS have increased circulating concentrations of pro-MMP-9, MMP-8, and TIMP-1 that are associated with increased concentrations of pro-inflammatory mediators and adhesion molecules. These findings suggest that MMPs may have a role in the increased cardiovascular risk of MetS patients. Pharmacological interventions targeting MMPs, especially MMP-9 and MMP-8 deserve further investigation in MetS patients. (C) 2009 Elsevier B.V. All rights reserved.

Human alveolar bone cell proliferation, expression of osteoblastic phenotype, and matrix mineralization on porous titanium produced by powder metallurgy

Objective: This study aimed at investigating the influence of the porous titanium (Ti) structure on the osteogenic cell behaviour. Materials and methods: Porous Ti discs were fabricated by the powder metallurgy process with the pore size typically between 50 and 400 mm and a porosity of 60%. Osteogenic cells obtained from human alveolar bone were cultured until subconfluence and subcultured on dense Ti (control) and porous Ti for periods of up to 17 days. Results: Cultures grown on porous Ti exhibited increased cell proliferation and total protein content, and lower levels of alkaline phosphatase (ALP) activity than on dense Ti. In general, gene expression of osteoblastic markers-runt-related transcription factor 2, collagen type I, alkaline phosphatase, bone morphogenetic protein-7, and osteocalcin was lower at day 7 and higher at day 17 in cultures grown on porous Ti compared with dense Ti, a finding consistent with the enhanced growth rate for such cultures. The amount of mineralized matrix was greater on porous Ti compared with the dense one. Conclusion: These results indicate that the porous Ti is an appropriate substrate for osteogenic cell adhesion, proliferation, and production of a mineralized matrix. Because of the three-dimensional environment it provides, porous Ti should be considered an advantageous substrate for promoting desirable implant surface-bone interactions.

Development of the osteoblastic phenotype in human alveolar bone-derived cells grown on a collagen type I-coated titanium surface

The aim of this study was to evaluate the development of the osteoblastic phenotype in human alveolar bone-derived cells grown on collagen type I-coated titanium (Ti) surface (Col-Ti) obtained by plasma deposition acrylic acid grafting compared with machined Ti (M-Ti). Osteoblastic cells were cultured until subconfluence and subcultured on Col-Ti and M-Ti for periods of up to 21 days. Cultures grown on Col-Ti and M-Ti exhibited similar cell morphology. Cell adhesion, total protein content, and alkaline phosphatase (ALP) activity were not affected by Ti surface modification in all evaluated periods. Growth analyses indicated that there were significantly more cells in cultures grown on Col-Ti at day 3. Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteoprotegerin (OPG) mRNA expression of cells subcultured on Col-Ti was higher, whereas collagen type I (COL) was lower compared with M-Ti. Ti surface modification neither affected the osteocalcin (OC), ALP and receptor activator of NF-kappa B ligand (RANKL) mRNA expression nor the calcium content extracted from mineralized matrix. These results demonstrated that Col-Ti favours cell growth during the proliferative phase (day 3) and osteoblastic differentiation, as demonstrated by changes in mRNA expression profile during the matrix mineralization phase (day 14), suggesting that this Ti surface modification may affect the processes of bone healing and remodelling. To cite this article:Assis AF, Beloti MM, Crippa GE, de Oliveira PT, Morra M, Rosa AL. Development of the osteoblastic phenotype in human alveolar bone-derived cells grown on a collagen type I-coated titanium surface.Clin. Oral Impl. Res. 20, 2009; 240-246.doi: 10.1111/j.1600-0501.2008.01641.x.

Lercanidipine decreases vascular matrix metalloproteinase-2 activity and protects against vascular dysfunction in diabetic rats

Abnormal matrix metalloproteinases (MMPs) activity causes cardiovascular diseases. Because hyperglycemia increase MMPs activities through increased oxidative stress. we hypothesized that antioxidant effects produced by lercanidipine could attenuate the increases in MMP-2 expression/activity in diabetic rats. Control and diabetic (alloxan-induced diabetes) rats received lercanidipine 2.5 mg/kg/day (or tap water) starting three weeks after alloxan (or vehicle) injections. Blood pressure was monitored weekly. After six weeks of treatment, vascular reactivity and structural changes were assessed in aortic rings. MMP-2 levels were determined by gelatin zymography, and MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined by fluorimetry. Lercanidipine produced antihypertensive effects (201 +/- 5 vs. 163 +/- 7 mm Hg in diabetic rats untreated and treated with lercaniclipine, respectively; P < 0.01) and reversed the impairment in endothelium-dependent vasorelaxation in diabetic rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of diabetic rats (both P < 0.001). Lercandipine attenuated the increases in oxidative stress and in MMP-2 (both P < 0.05). While diabetes induced no major structural changes, it caused a 16-fold increase in the ratio of MMP-2/TIMP-2 mRNA expression, which was completely reversed by lercanidipine (both P < 0.001). These results show that antioxidant and beneficial vascular effects produced by lercanidipine in diabetic rats are associated with reversion of the imbalance in vascular MMP-2MMP-2 expression. (C) 2008 Published by Elsevier B.V.