Interface membrane fibroblasts around aseptically loosened endoprostheses express MMP-13

The objective of this article was to assess whether matrix metalloproteinase-13 (MMP-13) is produced by cells of the peri-implant interface tissues and to further characterize these cells. Tissue specimens were collected from the bone-prosthesis interface at the time of revision surgery of clinically loosened hip and knee arthroplasties (n = 27). Synovial tissues from osteoarthritic patients and young patients with mild joint deformity were used as controls (n = 6). Tissue samples were fixed in 4% PFA, decalcified with EDTA, and embedded in paraffin. Sections (4 microm) were stained with hematoxylin/eosin and for the osteoclastic marker enzyme tartrate resistant acid phosphatase. Monocytes/macrophages were characterized with a monoclonal antibody against CD68 and mRNAs encoding MMP-13 and alpha(1) collagen I (COL1A1) were detected by in situ hybridization. Cells expressing transcripts encoding MMP-13 were found in 70% of the interface tissues. These cells colocalized with a cell population expressing COL1A1 mRNA, and were fibroblastic in appearance. MMP-13 expressing cells were found in the close vicinity of osteoclasts and multinuclear giant cells. No signals for transcripts encoding MMP-13 were detected in multinuclear giant cells or in osteoclasts. Control tissues were negative for transcripts encoding MMP-13 mRNA. Fibroblasts of the interface from aseptically loosened endoprostheses selectively express MMP-13. By the expression and the release of MMP-13, these fibroblastic cells may contribute to the local degradation of the extracellular matrix and to bone resorption.

Gelatinase B [matrix metalloproteinase (MMP)-9] and collagenases (MMP-8/-13) are upregulated in cerebrospinal fluid during aseptic and bacterial meningitis in children

We investigated the protein expression of gelatinases [matrix metalloproteinase (MMP)-2 and -9] and collagenases (MMP-8 and -13) in cerebrospinal fluid (CSF) from patients with bacterial (BM, n = 17) and aseptic (AM, n = 14) meningitis. In both, MMP-8 and -9 were increased in 100% of patients, whereas MMP-13 was detectable in 53% and 82% respectively. Three patients with clinical signs of meningitis, without CSF pleocytosis, scored positive for all three MMPs. MMP-8 appeared in two isoforms, granulocyte-type [polymorphonuclear cell (PMN)] and fibroblast/macrophage (F/M) MMP-8. Analysis of kinetic changes from serial lumbar punctures showed that these MMPs are independently regulated, and correlate only partly with CSF cytosis or levels of the endogenous inhibitor, tissue inhibitor of matrix metalloproteinase-1. In vitro, T cells, peripheral blood mononuclear cells (PBMCs) and granulocytes (PMN) release MMP-8 and -9, whereas MMP-13 could be found only in the former two cell types. Using models of exogenous (n-formyl-Met-Leu-Phe, T cell receptor cross-linking) and host-derived stimuli (interleukin-2), the kinetics and the release of the MMP-8, -9 and -13 showed strong variation between these immune cells and suggest release from preformed stocks. In addition, MMP-9 is also synthesized de novo in PBMCs and T cells. In conclusion, invading immune cells contribute only partially to MMPs in CSF during meningitis, and parenchymal cells are an equally relevant source. In this context, in patients with clinical signs of meningitis, but without CSF pleocytosis, MMPs seem to be a highly sensitive marker for intrathecal inflammation. The present data support the concept that broad-spectrum enzyme inhibition targeting gelatinases and collagenases is a potential strategy for adjunctive therapy in infectious meningitis.

Matrix metalloproteinases and angiogenic factors: predictors of survival after radical prostatectomy for clinically organ-confined prostate cancer?

The aim of the present study was to investigate whether biomarkers improve the prediction of recurrence-free, disease-specific, and overall survival in patients with clinically localized prostate cancer. A tissue microarray was constructed from prostate specimens of 278 patients who underwent open radical retropubic prostatectomy for clinically localized prostate cancer. For immunohistochemical studies, antibodies were used against matrix metalloproteinase (MMP)-2, MMP-3, MMP-7, MMP-9, MMP-13, and MMP-19, as well as against vascular endothelial growth factor, hypoxia-induced factor 1 , basic fibroblast growth factor, and cluster of differentiation 31. Univariate and multivariable analyses were performed to evaluate the potential predictors of overall, disease-specific, and recurrence-free survival. In univariate analysis of patients with clinically organ-confined prostate cancer, only higher expression levels of MMP-9 (hazard ratio [0.6], 95% CI 0.45-0.8) had a protective effect in terms of overall survival. This positive effect of high MMP-9 expression was also observed for recurrence-free (HR 0.88, 95% CI 0.78-0.99) and disease-specific survival (HR 0.5, 95% CI 0.36-0.73). In multivariable analysis, none of these potential markers was found to be an independent prognostic factor of survival. Of all MMPs and angiogenic factors tested, MMP-9 expression has the potential as a prognostic marker in patients undergoing radical prostatectomy for clinically organ-confined cases of prostate cancer.

Cannabinoid receptor type I modulates alcohol-induced liver fibrosis

The cannabinoid system (CS) is implicated in the regulation of hepatic fibrosis, steatosis and inflammation, with cannabinoid receptors 1 and 2 (CB1 and CB2) being involved in regulation of pro- and antifibrogenic effects. Daily cannabis smoking is an independent risk factor for the progression of fibrosis in chronic hepatitis C and a mediator of experimental alcoholic steatosis. However, the role and function of CS in alcoholic liver fibrosis (ALF) is unknown so far. Thus, human liver samples from patients with alcoholic liver disease (ALD) were collected for analysis of CB1 expression. In vitro, hepatic stellate cells (HSC) underwent treatment with acetaldehyde, Δ9-tetrahydrocannabinol H(2)O(2), endo- and exocannabinoids (2-arachidonoylglycerol (2-AG) and [THC]), and CB1 antagonist SR141716 (rimonabant). In vivo, CB1 knockout (KO) mice received thioacetamide (TAA)/ethanol (EtOH) to induce fibrosis. As a result, in human ALD, CB1 expression was restricted to areas with advanced fibrosis only. In vitro, acetaldehyde, H(2)O(2), as well as 2-AG and THC, alone or in combination with acetaldehyde, induced CB1 mRNA expression, whereas CB1 blockage with SR141716 dose-dependently inhibited HSC proliferation and downregulated mRNA expression of fibrosis-mediated genes PCα1(I), TIMP-1 and MMP-13. This was paralleled by marked cytotoxicity of SR141716 at high doses (5-10 μmol/L). In vivo, CB1 knockout mice showed marked resistance to alcoholic liver fibrosis. In conclusion, CB1 expression is upregulated in human ALF, which is at least in part triggered by acetaldehyde (AA) and oxidative stress. Inhibition of CB1 by SR141716, or via genetic knock-out protects against alcoholic-induced fibrosis in vitro and in vivo.

Detection of gelatinolytic activity in developing basement membranes of the mouse embryo head by combining sensitive in situ zymography with immunolabeling

Genetic evidence indicates that the major gelatinases MMP-2 and MMP-9 are involved in mammalian craniofacial development. Since these matrix metalloproteinases are secreted as proenzymes that require activation, their tissue distribution does not necessarily reflect the sites of enzymatic activity. Information regarding the spatial and temporal expression of gelatinolytic activity in the head of the mammalian embryo is sparse. Sensitive in situ zymography with dye-quenched gelatin (DQ-gelatin) has been introduced recently; gelatinolytic activity results in a local increase in fluorescence. Using frontal sections of wild-type mouse embryo heads from embryonic day 14.5-15.5, we optimized and validated a simple double-labeling in situ technique for combining DQ-gelatin zymography with immunofluorescence staining. MMP inhibitors were tested to confirm the specificity of the reaction in situ, and results were compared to standard SDS-gel zymography of tissue extracts. Double-labeling was used to show the spatial relationship in situ between gelatinolytic activity and immunostaining for gelatinases MMP-2 and MMP-9, collagenase 3 (MMP-13) and MT1-MMP (MMP-14), a major activator of pro-gelatinases. Strong gelatinolytic activity, which partially overlapped with MMP proteins, was confirmed for Meckel's cartilage and developing mandibular bone. In addition, we combined in situ zymography with immunostaining for extracellular matrix proteins that are potential gelatinase substrates. Interestingly, gelatinolytic activity colocalized precisely with laminin-positive basement membranes at specific sites around growing epithelia in the developing mouse head, such as the ducts of salivary glands or the epithelial fold between tongue and lower jaw region. Thus, this sensitive method allows to associate, with high spatial resolution, gelatinolytic activity with epithelial morphogenesis in the embryo.

Bovine primary chondrocyte culture in synthetic matrix metalloproteinase-sensitive poly(ethylene glycol)-based hydrogels as a scaffold for cartilage repair

A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.

Beta1 integrin deficiency results in multiple abnormalities of the knee joint

The lack of beta1 integrins on chondrocytes leads to severe chondrodysplasia associated with high mortality rate around birth. To assess the impact of beta1 integrin-mediated cell-matrix interactions on the function of adult knee joints, we conditionally deleted the beta1 integrin gene in early limb mesenchyme using the Prx1-cre transgene. Mutant mice developed short limbed dwarfism and had joint defects due to beta1 integrin deficiency in articular regions. The articular cartilage (AC) was structurally disorganized, accompanied by accelerated terminal differentiation, altered shape, and disrupted actin cytoskeleton of the chondrocytes. Defects in chondrocyte proliferation, cytokinesis, and survival resulted in hypocellularity. However, no significant differences in cartilage erosion, in the expression of matrix-degrading proteases, or in the exposure of aggrecan and collagen II cleavage neoepitopes were observed between control and mutant AC. We found no evidence for disturbed activation of MAPKs (ERK1/2, p38, and JNK) in vivo. Furthermore, fibronectin fragment-stimulated ERK activation and MMP-13 expression were indistinguishable in control and mutant femoral head explants. The mutant synovium was hyperplastic and frequently underwent chondrogenic differentiation. beta1-null synoviocytes showed increased proliferation and phospho-focal adhesion kinase expression. Taken together, deletion of beta1 integrins in the limb bud results in multiple abnormalities of the knee joints; however, it does not accelerate AC destruction, perturb cartilage metabolism, or influence intracellular MAPK signaling pathways.

Mechano-Biologie von bovinen Bandscheiben belastet unter Kompression und Torsion

Einleitung: Bandscheiben wirken als Schockabsorbierer in der Wirbelsäule und auf diese wirken meistens komplexe Kräfte, zusammengesetzt aus Kompression, Torsion und Flexion. Die biomechanishe Umgebung einer Bandscheibe ist denn auch geprägt von komplexen Belastungen. Die Forschung über die in vitro Bandscheibenbiologie hat sich bisher um die axiale Kompression konzentriert, wobei die Bedeutung von Torsion und insbesondere dem Zusammenspiel von Kompression und Torsion (="Twisting") praktisch noch nie untersucht wurde an lebenden Organkultur-Explantaten. Wir präsentieren neue mechanobiologische Daten über die Überlebenswahrscheinlichkeit von Bandscheibenzellen kultiviert in einem neuartigen, kompakten Design eines bi-axialen Bioreaktors, um die Bedeutung von Kompression und Torsion zu verstehen. Material/Methode: Bovine Schwanzbandscheiben mit den Endplatten wurden isoliert wie bereits beschrieben [2] und mechanische Belastung wurde angewendet mit einem 2 DoF Bioreaktor für 14 Tage [3]. Die Bandscheiben wurden in verschiedene Belastungsgruppen eingeteilt: 1) Keine Belastung (NL), 2) zyklische Kompression (CC) [8h: axiale Kompression mit 0.6 ± 0.2 MPa, 0.2 Hz], 3) zyklische Torsion (CT) [8h: ± 2° torsion, 0.2 Hz, 0.2 MPa compression], 4) zyklische Kompression und Torsion (CCT) [8h: 0.6 ± 0.2 MPa, 0.2 Hz & ± 2° torsion, 0.2 Hz]. Das Bandscheibengewebe wurde mit LIVE/DEAD gefärbt und miteinem konfokalen Mikroskop visualisiert um die Überlebensrate zu bestimmen. Zell Apoptosis wurde quantifiziert mit einem Caspase 3/7 Kit normalisiert zum totalen Proteingehalt (Bradford). Relative Gen-Expression von wichtigen Genen für die Bandscheibe wurde bestimmt von anabolischen, katabolischen und inflammatorischen Genen mittels real-time RT-PCR. Die Morphologie der Bandscheibenzellen wurde mittels Histologie bestimmt. Ergebnisse: Die Resultate zeigten einen starken Abfall der Zellüberlebenswahrscheinlichkeit im Zentrum der Bandscheiben, dem Nulceus Pulposus (NP), i.e. 10%, in der Gruppe mit CCT. Hingegen die Überlebenswahrscheinlichkeit im Annulus fibrosus (AF) war stabilisiert bei über 60% im NP und im AF in allen anderen Gruppen (Fig 1). Apoptotische Aktivität war statistisch signifikant erhöht in der CC-Gruppe, aber nicht in der CCT-Gruppe, was die Vermutung nahe legt, dass der erhöhte Zellverlust im NP nicht mit Apoptose sondern mit Nekrose erklärt werden kann. Die Gen Expression der anabolischen Gene COL1, COL2 und Biglycan war signifikant erhöht im AF in der CCT Gruppe, ebenfalls waren Remodeling-Gene angeschaltet wie ADAMTS4 und MMP-13 in der CCT Gruppe (Fig. 2). Der Glykosaminoglykan (GAG) Gehalt war generell im AF erhöht in den Gruppen unter mechanischer Belastung, jedoch nicht statistisch signifikant. Schlussfolgerung: Zyklische Torsion kombiniert mit zyklischer Kompression waren in dieser Studie erfolgreich und nach unserem besten Wissen zum ersten Mal an Bandscheibenexplantaten in einer 14- tägigen Organkultur angewendet worden in einem dafür speziell konzipierten Bioreaktor. Die Resultezeigten überraschend einen negativen Effekt bei physiologischen Parametern, was die Belastung (0.6MPa ± 0.2MPa) und die Torsion (± 2°) angeht. Dieser negative Effekt des "Twistings" auf die Überlebenswahrscheinlichkeit der Zellen war jedoch nur regional im NP von Bedeutung, wohingegen im AF keine Effekte zu detektieren waren.

Prognostic value of matrix metalloproteinases in oral squamous cell carcinoma

The aim of this study was to investigate whether there is a correlation between the expressions of four matrix metalloproteinases (MMPs): MMP-2, MMP-7, MMP-9 and MMP-13, and the TNM (tumour-node-metastasis) stages of oral squamous cell carcinoma (OSCC); and to explore the implication of these MMPs in OSCC dissemination. Samples from 61 patients diagnosed with oropharyngeal tumour were studied by immunohistochemistry against MMP-2, MMP-7, MMP-9 and MMP-13. The assessment of immunoreactivity was semi-quantitative. The results showed that MMP-2 and MMP-9 had similar expression patterns in the tumour cells with no changes in the immunoreactivity during tumour progression. MMP-9 always had the highest expression, whereas that of MMP-2 was moderate. MMP-7 showed a significant decrease in expression levels during tumour evolution. MMP-13 had constant expression levels within stage T2 and T3, but showed a remarkable decline in immunoreactivity in stage T4. No significant differences in the MMPs immunoreactivity between tumour cells and stroma were observed. Although strong evidence for the application of MMPs as reliable predictive markers for node metastasis was not acquired, we believe that combining patients' MMPs expression intensity and clinical features may improve the diagnosis and prognosis. Strong evidence for the application of MMPs as reliable predictive markers for node metastasis was not acquired. Application of MMPs as prognostic indicators for the malignancy potential of OSCC might be considered in every case of tumour examination. We believe that combining patients' MMPs expression intensity and clinical features may improve the process of making diagnosis and prognosis.