In vitro influence of the extracellular matrix in myoepithelial cells stimulated by malignant conditioned medium

In order to investigate the role of myoepithelial cell and tumor microenvironment in salivary gland neoplasma, we have performed a study towards the effect of different extracellular matrix proteins (basement membrane matrix, type I collagen and fibronectin) on morphology and differentiation of benign myoepithelial cells from pleomorphic adenoma cultured with malignant cell culture medium from squamous cell carcinoma. We have also analyzed the expression of alpha-smooth muscle actin (alpha-SMA) and FGF-2 by immunofluorescence and qPCR. Our immunofluorescence results, supported by qPCR analysis, demonstrated that alpha-SMA and FGF-2 were upregulated in the benign myoepithelial cells from pleomorphic adenoma in all studied conditions on fibronectin substratum. However, the myoepithelial cells on fibronectin substratum did not alter their morphology under malignant conditioned medium stimulation and exhibited a stellate morphology and, occasionally focal adhesions with the substratum. In summary, our data demonstrated that the extracellular matrix exerts an important role in the morphology of the benign myoepithelial cells by the presence of focal adhesions and also inducing increase FGF-2 and alpha-SMA expression by these cells, especially in the fibronectin substratum. (C) 2011 Elsevier Ltd. All rights reserved.

Impact of Protease Inhibitors on Dentin Matrix Degradation by Collagenase

This proof-of-concept study assessed whether the reduction of the degradation of the demineralized organic matrix (DOM) by pre-treatment with protease inhibitors (PI) is effective against dentin matrix loss. Bovine dentin slices were demineralized with 0.87 M citric acid, pH 2.3, for 36 hrs. In sequence, specimens were treated or not (UT, untreated) for 1 min with gels containing epigallocatechin 3-gallate (EGCG, 400 A mu M), chlorhexidine (CHX, 0.012%), FeSO4 (1 mM), NaF (1.23%), or no active compound (P, placebo). Specimens were then stored in artificial saliva (5 days, 37 degrees C) with the addition of collagenase (Clostridium histolyticum, 100 U/mL). We analyzed collagen degradation by assaying hydroxyproline (HYP) in the incubation solutions (n = 5) and evaluated the dentin matrix loss by profilometry (n = 12). Data were analyzed by ANOVA and Tukey's test (p < 0.05). Treatment with gels containing EGCG, CHX, or FeSO4 led to significantly lower HYP concentrations in solution and dentin matrix loss when compared with the other treatments. These results strongly suggest that the preventive effects of the PI tested against dentin erosion are due to their ability to reduce the degradation of the DOM.

Fluoride modulates preosteoblasts viability and matrix metalloproteinases-2 and -9 activities

This study evaluated the influence of fluoride on cell viability and activity of matrix metalloproteinases (MMP) -2 and -9 secreted by preosteoblasts. Preosteoblasts (MC3T3-E1 murine cell line) were cultured in MEM medium supplement with 10% Fetal Bovine Serum (FBS) and nucleosides/ribonucleosides without ascorbic acid. Adherent cells were treated with different concentrations of F (as sodium fluoride-NaF) in medium (5 x 10-6 M, 10-5 M, 10-4 M and 10-3 M) for 24, 48, 72 and 96 h at 37ºC, 5% CO2. Control cells were cultivated in MEM only. After each period, preosteoblast viability was assessed by MTT assay. MMP-2 and -9 activities were performed by gel zymography. Also, alkaline phosphatase (ALP) activity was quantified by colorimetry in all experimental groups. It was shown that cultured cells with the highest dose of F (10-3 M) for 96 h decreased preosteoblast viability while lower doses of F did not alter it, when compared to untreated cells. No differences were observed in ALP activity among groups. Moreover, compared to control, the treatment of cells with F at low dose slightly increased MMP-2 and -9 activities after 24 h. It was concluded that F modulates preosteoblast viability in a dose-dependent manner and also may regulate extracellular matrix remodeling.

Long-term type 1 diabetes impairs decidualization and extracellular matrix remodeling during early embryonic development in mice

Introduction: Endometrial decidualization and associated extracellular matrix (ECM) remodeling are critical events to the establishment of the maternal-fetal interface and successful pregnancy. Here, we investigated the impact of type 1 diabetes on these processes during early embryonic development, in order to contribute to the understanding of the maternal factors associated to diabetic embryopathies. Methods: Alloxan-induced diabetic Swiss female mice were bred after different periods of time to determine the effects of diabetes progression on the development of gestational complications. Furthermore, the analyses focused on decidual development as well as mRNA expression, protein deposition and ultrastructural organization of decidual ECM. Results: Decreased number of implantation sites and decidual dimensions were observed in the group mated 90-110 days after diabetes induction (D), but not in the 50-70D group. Picrosirius staining showed augmentation in the fibrillar collagen network in the 90e110D group and, following immunohistochemical examination, that this was associated with increase in types I and V collagens and decrease in type III collagen and collagen-associated proteoglycans biglycan and lumican. qPCR, however, demonstrated that only type I collagen mRNA levels were increased in the diabetic group. Alterations in the molecular ratio among distinct collagen types and proteoglycans were associated with abnormal collagen fibrillogenesis, analyzed by transmission electron microscopy. Conclusions: Our results support the concept that the development of pregnancy complications is directly related with duration of diabetes (progression of the disease), and that this is a consequence of both systemic factors (i.e. disturbed maternal endocrine-metabolic profile) and uterine factors, including impaired decidualization and ECM remodeling

Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells

Characterization of Human Respiratory Syncytial Virus (HRSV) protein interactions with host cell components is crucial to devise antiviral strategies. Viral nucleoprotein, phosphoprotein and matrix protein genes were optimized for human codon usage and cloned into expression vectors. HEK-293T cells were transfected with these vectors, viral proteins were immunoprecipitated, and co-immunoprecipitated cellular proteins were identified through mass spectrometry. Cell proteins identified with higher confidence scores were probed in the immunoprecipitation using specific antibodies. The results indicate that nucleoprotein interacts with arginine methyl-transferase, methylosome protein and Hsp70. Phosphoprotein interacts with Hsp70 and tropomysin, and matrix with tropomysin and nucleophosmin. Additionally, we performed immunoprecipitation of these cellular proteins in cells infected with HRSV, followed by detection of co-immunoprecipitated viral proteins. The results indicate that these interactions also occur in the context of viral infection, and their potential contribution for a HRSV replication model is discussed.

Electrogeneration of platinum nanoparticles in a matrix of dendrimer-carbon nanotubes

Hybrid materials with enhanced properties can now be obtained by combining nanomaterials such as carbon nanotubes and metallic nanoparticles, where the main challenge is to control fabrication conditions. In this study, we demonstrate that platinum nanoparticles (PtNps) can be electrogenerated within layer-by-layer (LbL) films of polyamidoamine (PAMAM) dendrimers and single-walled carbon nanotubes (SWCNTs), which serve as stabilizing matrices. The advantages of the possible control through electrogeneration were demonstrated with a homogeneous distribution of PtNps over the entire surface of the PAMAM/SWCNT LbL films, whose electroactive sites could be mapped using magnetic force microscopy. The Pt-containing films were used as catalysts for hydrogen peroxide reduction, with a decrease in the reduction potential of 60 mV compared to a Pt film deposited onto bare ITO. By analyzing the mechanisms responsible for hydrogen peroxide reduction, we ascribed the enhanced catalytic activity to synergistic effects between platinum and carbon in the LbL films, which are promising for sensing and fuel cell applications.

A generalization of the Moore-Penrose inverse related to matrix subspaces of C(nxm)

[EN]A natural generalization of the classical Moore-Penrose inverse is presented. The so-called S-Moore-Penrose inverse of a m x n complex matrix A, denoted by As, is defined for any linear subspace S of the matrix vector space Cnxm. The S-Moore-Penrose inverse As is characterized using either the singular value decomposition or (for the nonsingular square case) the orthogonal complements with respect to the Frobenius inner product. These results are applied to the preconditioning of linear systems based on Frobenius norm minimization and to the linearly constrained linear least squares problem.

Aufbau und Inbetriebnahme eines Protonen-Polarimeters an MAMI und Messung der Proton-Polarisation in der Reaktion p (e,e'p) pi 0 in paralleler Kinematik im Bereich der Delta(1232)-Resonanz

Das Detektorsystem des Spektrometers Ader Drei-Spektrometer-Anlage am MainzerElektronenbeschleuniger MAMI wurde im Rahmen dieser Arbeitum ein Fokalebenen-Proton-Polarimeter (FPP) ergänzt.Dazu wurden zwei Horizontale Doppeldriftkammern entworfenund zusammen mit einem Kohlenstoffanalysator hinter derFokalebene des Spektrometers aufgebaut.Zur Berechnung der Spin-Präzession in den inhomogenenMagnetfeldern des Spektrometers wurde das Programm QSPINerstellt und damit eine Spin-Transfer-Matrix (STM) erzeugt,mit der über eine Fitprozedur die Polarisationskomponentender Protonen in der Streuebene an die im FPP gemessenenangepaßt werden können.Mit elastischen Elektron-Proton-Streuexperimenten wurdenfalsche Asymmetrien im FPP untersucht, die berechnete STMbestätigt, die inklusive Proton-Kohlenstoff-Analysierstärkeauf einen größeren Streuwinkelbereich erweitert und dasVerhältnis zwischen dem elektrischen und dem magnetischenSachs-Formfaktor des Protons gemessen.Zur Untersuchung der Coulomb-Quadrupol-Übergangsamplitudebei der Anregung des Nukleons zur Delta(1232)-Resonanzwurden in einem Pion-Elektroproduktions-Experiment am Protonmit dem FPP die Komponenten der Polarisation derRückstoßprotonen in paralleler Kinematik gemessen.

Ein transgenes Mausmodell zur Untersuchung des Einflusses von Matrix Metalloproteinase-2 und -14 auf die Leber

MMP-2 and MMP-14 process extracellular matrix proteins,cytokines, growth factors and adhesion molecules to generatefragments with enhanced or reduced biological activity.In this study, a vectorsystem was developed for theconditional expression of MMP-2 and MMP-14 in the liver oftransgenic mice. For this vectorsystem the murine albuminpromotor was chosen together with the cre/lox system toachieve an inducible MMP-expression in the liver.Only one of the MMP-14 transgenic lines expressed highamounts of active MMP-14 protein after recombination of thelox-P sites. In these mice MMP-14 was able to activate MMP-2and MMP-13 in vivo. However, none of the livers of MMP-14overexpressing mice showed no differences in liverweight,amount of extracellular matrixproteins and rate ofproliferation, apoptosis and tumor-induction when comparedto the liver of wildtype mice.On the other hand overexpression of MMP-2 was embryoniclethal in all MMP-2 transgenic lines. After crossing theMMP-2 transgenic mice with cre deleter mice, a cre mediatedrecombination could be shown at day 6.5 post coitum (pc).Some of the double transgenic embryos of one of thetransgenic lines had severe deformations of the head,especially of the telencephalon and the mesencephalon.It could be shown in this study that disregulation of MMP-2in early embryonic development is lethal but anoverexpression of MMP-14 has no influence on the embryonicdevelopment or the homeostasis of the adult liver.With this conditional vectorsystem it is to possible studythe influnce of MMP-2 and MMP-14 on fibrogenesis,regeneration and tumorgenesis in the liver of mice.

Healing of two and three wall intrabony periodontal defects following treatment with an enamel matrix derivative combined with autogenous bone

BACKGROUND: There are still limited data on the outcomes of regenerative periodontal surgery using a combination of an enamel matrix protein derivative (EMD) and autogenous bone (AB). AIM: To evaluate the healing of deep intrabony defects treated with either a combination EMD+AB or EMD alone. MATERIALS AND METHODS: Forty patients with advanced chronic periodontitis, with one deep intrabony defect, were randomly treated with either EMD+AB (test) or EMD (control). Clinical assessments were performed at baseline and at 1 year after treatment. The primary outcome variable was relative attachment level (RAL). RESULTS: Healing was uneventful in all patients. The test sites showed a reduction in the mean probing pocket depth (PPD) of 5.6 +/- 0.9 mm (p<0.001), a gain in the mean RAL of 4.2 +/- 1.1 mm (p<0.001) and a gain in the mean probing bone level (PBL) of 3.9 +/- 1.0 mm (p<0.001). The control group displayed a mean PPD reduction of 4.6 +/- 0.4 mm (p<0.001), a mean RAL gain of 3.4 +/- 0.8 mm (p<0.001) and a mean PBL gain of 2.8 +/- 0.8 mm (p<0.001). RAL gains of > or =4 mm were measured in 90% of the test defects and in 55% of the controls. PBL gains of > or =4 mm were obtained in 85% of the test defects and in 25% of the control ones. The test treatment resulted in statistically higher PPD reductions, RAL gains and PBL gains compared with the control (p<0.01). CONCLUSIONS: Within their limits, the present results indicate that: (i) at 1 year after surgery, both therapies resulted in statistically significant clinical improvements compared with baseline and (ii) although the combination of EMD+AB resulted in statistically significant higher soft and hard tissue improvements compared with treatment with EMD, the clinical relevance of this finding is unclear.

23Na MR imaging at 7 T after knee matrix-associated autologous chondrocyte transplantation: preliminary results

To evaluate the feasibility of sodium 7-T magnetic resonance (MR) imaging in repaired tissue and native cartilage of patients after matrix-associated autologous chondrocyte transplantation (MACT) and compare results with delayed gadolinium-enhanced MR imaging of cartilage (dGEMRIC) at 3 T.

Bovine pericardium based non-cross linked collagen matrix for successful root coverage, a clinical study in human

Introduction The aim of this study was to clinically assess the capacity of a novel bovine pericardium based, non-cross linked collagen matrix in root coverage. Methods 62 gingival recessions of Miller class I or II were treated. The matrix was adapted underneath a coronal repositioned split thickness flap. Clinical values were assessed at baseline and after six months. Results The mean recession in each patient was 2.2 mm at baseline. 6 Months after surgery 86.7% of the exposed root surfaces were covered. On average 0,3 mm of recession remained. The clinical attachment level changed from 3.5 ± 1.3 mm to 1,8 ( ± 0,7) mm during the observational time period. No statistically significant difference was found in the difference of probing depth. An increase in the width of gingiva was significant. With a baseline value of 1.5 ± 0.9 mm an improvement of 2.4 ± 0.8 mm after six month could be observed. 40 out of 62 recessions were considered a thin biotype at baseline. After 6 months all 62 sites were assessed thick. Conclusions The results demonstrate the capacity of the bovine pericardium based non-cross linked collagen matrix for successful root coverage. This material was able to enhance gingival thickness and the width of keratinized gingiva. The percentage of root coverage achieved thereby is comparable to existing techniques. This method might contribute to an increase of patient's comfort and an enhanced aesthetical outcome.

Four-year results following treatment of intrabony periodontal defects with an enamel matrix derivative alone or combined with a biphasic calcium phosphate

The aim of this study was to evaluate the 4-year clinical outcomes following regenerative surgery in intrabony defects with either EMD + BCP or EMD. Twenty-four patients with advanced chronic periodontitis, displaying one-, two-, or three-walled intrabony defect with a probing depth of at least 6 mm, were randomly treated with either EMD + BCP (test) or EMD alone (control). The following clinical parameters were evaluated at baseline, at 1 year and at 4 years after regenerative surgery: plaque index, gingival index, bleeding on probing, probing depth, gingival recession, and clinical attachment level (CAL). The primary outcome variable was CAL. No differences in any of the investigated parameters were observed at baseline between the two groups. The test group demonstrated a mean CAL change from from 10.8 ± 1.6 mm to 7.4 ± 1.6 mm (p < 0.001) and to 7.6 ± 1.7 mm (p < 0.001) at 1 and 4 years, respectively. In the control group, mean CAL changed from 10.4 ± 1.3 at baseline to 6.9 ± 1.0 mm (p < 0.001) at 1 year and 7.2 ± 1.2 mm (p < 0.001) at 4 years. At 4 years, two defects in the test group and three defects in the control group have lost 1 mm of the CAL gained at 1 year. Compared to baseline, at 4 years, a CAL gain of ≥3 mm was measured in 67% of the defects (i.e., in 8 out of 12) in the test group and in 75% of the defects (i.e., in 9 out of 12) in the control group. There were no statistically significant differences in any of the investigated parameters at 1 and at 4 years between the two groups. Within their limits, the present results indicate that: (a) the clinical improvements obtained with both treatments can be maintained over a period of 4 years, and (b) in two- and three-walled intrabony defects, the addition of BCP did not additionally improve the outcomes obtained with EMD alone. In two- and three-walled intrabony defects, the combination of EMD + BCP did not show any advantage over the use of EMD alone.

Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis

Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-β3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-β/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-β3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-β3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf-β/Bmp activity.