Collagen Matrix (collagen + matrix)

Distribution by Scientific Domains

Selected Abstracts

Rho plays a central role in regulating local cell-matrix mechanical interactions in 3D culture

CYTOSKELETON, Issue 6 2007
N. Lakshman
Abstract The purpose of this study was to assess quantitatively the role of the small GTPase Rho on cell morphology, f-actin organization, and cell-induced matrix remodeling in 3D culture. Human corneal fibroblasts (HTK) were infected with adenoviruses that express green fluorescent protein (GFP) or GFP-N19Rho (dominant negative Rho). One day later cells were plated inside collagen matrices and allowed to spread for 24 h. Cells were fixed and stained for f-actin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) images were acquired using confocal microscopy. Fourier transform analysis was used to assess local collagen fibril alignment, and changes in cell morphology and collagen density were measured using MetaMorph. The decrease in matrix height was used as an indicator of global matrix contraction. HTK and HTK-GFP cells induced significant global matrix contraction; this was inhibited by N19Rho. HTK and HTK-GFP fibroblasts generally had a bipolar morphology and occasional intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. In contrast, HTK-GFPN19 cells were elongated, and had a more cortical f-actin distribution. Numerous small extensions were also observed along the cell body. In addition, both local collagen fibril density and alignment were significantly reduced. Rho plays a key role in regulating both the morphology and mechanical behavior of corneal fibroblasts in 3D culture. Overall, the data suggest that Rho-kinase dependent cell contractility contributes to global and local matrix remodeling, whereas Rho dependent activation of mDia and/or other downstream effectors regulates the structure and number of cell processes. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]

CD44 variant isoform v10 is expressed on tumor-infiltrating lymphocytes and mediates hyaluronan-independent heterotypic cell,cell adhesion to melanoma cells

T. K. Weimann
Abstract: CD44 is a family of cell-surface receptors on human lymphocytes that act as co-stimulatory molecules leading to the induction of effector functions in T cells. We have analyzed primary cutaneous malignant melanomas with clinical and histologic signs of tumor regression using immunohistochemistry and observed the predominant expression of the CD44 variant isoform v10 on CD3 CD4/CD8 co-expressing tumor-infiltrating lymphocytes (TIL). We further analyzed the role of CD44v10 in adhesion of lymphocytes to human melanoma cells. In contrast to CD44, lymphatic cells, CD44v10+ lymphatic cells strongly bound to cultured human melanoma cells and to frozen tissue samples of melanomas. Antibody blocking studies revealed a hyaluronan-, integrin-, and selectin-independent pathway of adhesion. Furthermore, CD44v10+ lymphatic cells exhibited significantly higher invasiveness in three-dimensional collagen matrices as compared with CD44H+ and CD44-negative lymphocytes. These results indicate that expression of CD44v10 on TIL may mediate adhesion to melanoma cells and result in gain of novel invasive properties. [source]

Lack of desquamation , the Achilles heel of the reconstructed epidermis

M. Ponec
Synopsis The use of human skin equivalents for screening tests aiming to assess repetitive application of various test agents is hampered by the lack of desquamation in vitro. The present study was undertaken to examine whether the desquamation can be induced by various treatments including mechanical stress, application of various agents that should decrease the surface pH and calcium level, activate the enzymes involved in desquamation process or UV irradiation. In addition, the effect of ,-hydroxyacids, known to enhance desquamation and to improve the stratum corneum barrier function in vivo, was examined as well. Human epidermis reconstructed on de-epidermized dermis or on fibroblast-populated collagen matrices during a 2-week culture at the air,liquid interface underwent various treatments during an additional 3-week period. The effects of treatments were evaluated on the basis of tissue morphology and lipid composition. The results of the present study revealed that cell shedding could only be induced by a mild repetitive mechanical treatment. The lack of desquamation, under most in vitro conditions, has a practical consequence, since it may hamper the use of reconstructed epidermis for various screening studies aiming to examine the repetitive exposure to topical agents or UV irradiation. The gradual thickening of the stratum corneum will lead to its higher resistance to the environmental stimuli and in this way affect the outcome of the tests. Furthermore, from the results obtained in the present study, it became evident that one should be careful in selecting endpoints when, for example, the effects of agents known to modulate melanogenesis are examined. Résumé L'utilization d'équivalents cutanés humains dans les procédures de criblage, afin d'estimer l'action répétée de divers agents, est entravée par l'absence de desquamation in vitro. La présente étude a été entreprise afin de déterminer dans quelle mesure la desquamation peut être induite par différents traitements tels que stress mécanique, application d'agents divers qui conduiraient à une chute du pH de surface et du taux de Calcium, activeraient les enzymes impliquées dans le processus de desquamation, ou l'irradiation UV. De plus, l'effet des , hydroxy-acides, connus pour favouriser la desquamation et d'améliorer la fonction barrière du Stratum-Corneum in vivo, a étéétudié. L'épiderme humain reconstruit sur un derme dé-épidermisé ou sur des matrices de collagène colonisées par des fibroblastes pendant 2 semaines de culture, en interface air × liquide, a subi divers traitements pendant une période additionnelle de 3 semaines. Les effets de ces traitements étaient évalués sur des critères morphologiques du tissu ainsi que la composition en lipides. Les résultats de cette étude montrent que l'élimination cellulaire ne peut être induite que par un léger traitement mécanique répété. L'absence de desquamation dans la plupart des conditions in vitro a une conséquence pratique puisqu'elle peut entraver l'utilization de l'épiderme reconstruit à des fins diverses de criblage en vue d'appréhender les expositions répétées à des agents topiques, ou l'irradiation UV. L'épaississement progressif du Stratum-Corneum lui confèrera une résistance accrue aux stimuli environnementaux qui, en retour, modifiera les résultats des tests. De plus, les résultats de cette présente étude impliquent à l'évidence une précaution dans la sélection des cinétiques de mesures lorsque, par exemple, les effets des agents connus pour moduler la mélanogénèse sont étudiés. [source]

In vitro study comparing two collageneous membranes in view of their clinical application for rotator cuff tendon regeneration

Milena Fini
Abstract Tenocytes were isolated from the rotator cuff tendons of healthy (HT) and glucocorticoid (GC)-treated rats (GCT) and were cultured on polystyrene wells (TCP) as control, and on 2 de-cellularized collagen matrices: porcine small intestinal submucosa (SIS), and human dermal matrix (Graftjacket®, GJ). At 3 and 7 days cell proliferation and synthesis were evaluated. Proliferation of HT tenocytes increased between experimental times for both tested membranes, but already at 3 days, HT tenocytes cultured on GJ showed the highest WST-1 value. The collagen-I (CICP) synthesis on GJ membrane did not change between experimental times and was significantly higher than TCP and SIS at 7 days. Proteoglycans (PG), and fibronectin (FBN) synthesis increased when HT were cultured on GJ, between experimental times, and both PG and FBN synthesis on GJ membrane were higher than TCP and SIS at 7 days. GC determined decreases in cell proliferation, CICP and PG syntheses at 3 days of culture on TCP when compared to HT tenocytes while a decrease in WST-1 was maintained at 7 days. CICP, PG and FBN (only at 3 days) syntheses were significantly higher in GCT tenocytes cultured on GJ. The negative effects on GC on GCT tenocytes cultured on membrane were particularly evident on SIS for CICP (,18%) and FBN (,67%) synthesis. The obtained results support the conclusion that GJ is more suitable than SIS as a scaffold for in situ tissue engineering and for the in vitro bioengineering of tendons to heal massive tears of the rotator cuff tendon. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:98,107, 2007 [source]

Close dependence of fibroblast proliferation on collagen scaffold matrix stiffness

E. Hadjipanayi
Abstract Human dermal fibroblasts (HDFs) in free-floating collagen matrices show minimal proliferation, although this may increase when the matrix is ,under tension'. We have investigated the detailed mechanics underlying one of the possible controls of this important cell behaviour, in particular the hypothesis that this is a response to substrate stiffness. Hyperhydrated collagen gels were plastic-compressed (PC) to give a predetermined collagen density and stiffness. Mechanical properties were tested using a dynamic mechanical analyser; cell number by Alamar blue assay. In the stiffest PC matrices, cell proliferation was rapid and seeding density-dependent, with a population doubling time of 2 days. In contrast, compliant attached matrices showed a 4 day lag period and a doubling time of 6 days. HDF growth was directly related to matrix stiffness, such that increasing stiffness using a range of compression levels (0,75% fluid removal) supported increasing proliferation rate, doubling times and matrix elastic modulus. HDF quiescence in compliant matrices was reversible, such that increasing stiffness in situ by compression at 1 and 5 days initiated proliferation. We conclude that collagen matrix stiffness regulates proliferation of fibroblasts (a duro-response), with important implications for understanding fibroblast,matrix feedback controls during wound healing and the design and regulation of engineered connective tissues based on collagen and other hydrogel-based scaffolds. Copyright © 2008 John Wiley & Sons, Ltd. [source]

Biomechanical findings in rats undergoing fascial reconstruction with graft materials suggested as an alternative to polypropylene ,,

M.L. Konstantinovic
Abstract Aims Graft materials used for pelvic floor reinforcement should still be considered as investigational and, therefore, evaluated experimentally and within clinical trials. The present report describes our biomechanical findings in rats implanted with selected novel implant materials, which in recent years have been suggested as alternatives to plain polypropylene (PP) meshes. Methods Full thickness abdominal wall defects were primarily repaired by the implant of interest. Experiments involved eight different implant materials: two partly degradable synthetic implants, that is, a hybrid of polyglactin 910 with PP (Vypro II) and collagen coated PP (Pelvitex); two non-cross linked (Surgisis, InteX,n LP) and two cross-linked materials (Pelvicol, Pelvisoft) and two porous modifications of InteX,n LP and Pelvicol implants. At different time points (7, 14, 30, and 90 days), the implants and surrounding host tissue (explant) were harvested and tensiometry was performed. Tensile strength and location of breakage were recorded. Results In general resorbable non-cross linked collagen matrices and porous materials were weaker after 90 days; similar behavior was seen for implant materials alone and their construction with the surrounding native tissue. Both non-porous and porous modification of InteX,n LP appeared at 90 days as a very thin layer of collagen that was two-thirds, respectively one-third of the initial thickness. Conclusions In experimental conditions, sufficient strength was obtained only after 3 months, and PP containing constructs appeared as the strongest though reconstruction with Pelvicol showed comparable outcomes. Lower values for strength of non-cross linked and porous collagen materials are questioning their efficacy for pelvic floor reconstruction. Neurourol. Urodynam. 29:488,493, 2010. © 2009 Wiley-Liss, Inc. [source]

Ultrastructural study of the jaw structures in two species of Ampharetidae (Annelida: Polychaeta)

ACTA ZOOLOGICA, Issue 3 2004
Alexander B. Tzetlin
Abstract Two species of jaw bearing Ampharetidae (Adercodon pleijeli (Mackie 1994) and Ampharete sp. B) were investigated in order to describe the microanatomy of the mouth parts and especially jaws of these enigmatic polychaetes. The animals of both studied species have 14,18 mouth tentacles that are about 30 µm in diameter each. In both species, the ventral pharyngeal organ is well developed and situated on the ventral side of the buccal cavity. It is composed of a ventral muscle bulb and investing muscles. The bulb consists of posterior and anterior parts separated by a deep median transversal groove. In both species, the triangular teeth or denticles are arranged in a single transversal row on the surface of the posterior part of the ventral bulb just in front of its posterior edge. There are 36 denticles in Adercodon pleijeli and 50 in Ampharete sp. B. The height of the denticles (6,12 µm) is similar in both species. Each tooth is composed of two main layers. The outer one (dental) is the electron-dense sclerotized layer that covers the tooth. The inner one consists of long microvilli with a collagen matrix between them. The thickness of the dental layer ranges from 0.95 to 0.6 µm. The jaws of the studied worms may play a certain role in scraping off microfouling. The fine structure of the jaws in Ampharetidae is very similar to that of the mandibles of Dorvilleidae, the mandibles and the maxillae of Lumbrineridae, Eunicidae and Onuphidae, and the jaws of other Aciculata. This type of jaw is characterized by unlimited growth and the absence of replacement. The occurrence of jaws in a few smaller Ampharetidae is considered as an apomorphic state. [source]

Biomimetic Formation of Hydroxyapatite/collagen Matrix Composite

Y. Wang
A composite of collagen (COL) and hydroxyapatite(HA) was prepared using a biomimetic approach, which performs a direct nucleation of HA on self- assembled collagen matrix. This research may be helpful to understand the possible mechanisms for collagen-mediated mineralization in general and the COL/HCA composite can be considered a new particularly attractive material for human bone tissue implantation. [source]

Dynamic Hydrogels: Switching of 3D Microenvironments Using Two-Component Naturally Derived Extracellular Matrices

Brian M. Gillette
This article describes fabrication of a two-component extracellular matrix (ECM) in which one component acts as a stable structural element and another component gels or dissolves reversibly (a modulatory component). Using a composite collagen-alginate ECM, reversible crosslinking of the alginate (the modulatory component) via application of calcium or citrate modulates cell mobility in a 3D collagen matrix (the structural component). [source]

Preparation and characterization of complex gel of type I collagen and aluminosilicate containing imogolite nanofibers

Asuka Nakano
Abstract Complex gel materials of Type I collagen and aluminosilicate containing imogolite nanofibers were prepared as opaque gel by mixing an acidic fine dispersion of aluminosilicate with an acidic solution of collagen. The product was stained blue by Coomassie Brilliant Blue (CBB), indicating that the gel contained collagen. A white sponge was obtained after lyophilization of the complex gel. Elemental analysis revealed that the complex contains C, H, N, Al, and Si atoms; and the compositional ratio of aluminosilicate/collagen (w/w) was calculated as 0.75 for the complex gel when aluminosilicate was mixed with an equal quantity of collagen. Transmission electron microscope (TEM) observation showed that aluminosilicate nanofibers were homogeneously distributed in the collagen matrix. The thermogravimetric analysis (TGA) curve of the complex was not a simple summation of each components, and especially, the weight loss step corresponding to detachment of the adsorbed water observed in aluminosilicate became difficult to distinguish, suggesting that the adsorbed water was removed in the complexation. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010 [source]

Growth of osteoblast-like cells on biomimetic apatite-coated chitosan scaffolds

I. Manjubala
Abstract Porous scaffold materials that can provide a framework for the cells to adhere, proliferate, and create extracellular matrix are considered to be suitable materials for bone regeneration. Interconnected porous chitosan scaffolds were prepared by freeze-drying method, and were mineralized by calcium and phosphate solution by double-diffusion method to form nanoapatite in chitosan matrix. The mineralized chitosan scaffold contains hydroxyapatite nanocrystals on the surface and also within the pore channels of the scaffold. To assess the effect of apatite and porosity of the scaffolds on cells, human osteoblast (SaOS-2) cells were cultured on unmineralized and mineralized chitosan scaffolds. The cell growth on the mineralized scaffolds and on the pure chitosan scaffold shows a similar growth trend. The total protein content and alkaline phosphatase enzyme activity of the cells grown on scaffolds were quantified, and were found to increase over time in mineralized scaffold after 1 and 3 weeks of culture. The electron microscopy of the cell-seeded scaffolds showed that most of the outer macropores became sealed off by a continuous layer of cells. The cells spanned around the pore wall and formed extra cellular matrix, consisting mainly of collagen in mineralized scaffolds. The hydroxyproline content also confirmed the formation of the collagen matrix by cells in mineralized scaffolds. This study demonstrated that the presence of apatite nanocrystals in chitosan scaffolds does not significantly influence the growth of cells, but does induce the formation of extracellular matrix and therefore has the potential to serve for bone tissue engineering. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2008 [source]

siRNA mediated inhibition of MMP-1 reduces invasive potential of a human chondrosarcoma cell line

Xiaoling Jiang
Matrix metalloproteinases (MMPs) play a crucial role in tumor cell invasion and metastasis. Expression of MMP-1 has been reported as a prognostic predictor of recurrence in human chondrosarcoma, and studies using human chondrosarcoma cell lines indicate that MMP-1 expression levels correlate with in vitro invasiveness. These observations suggest that MMP-1 activity has a central role in cell egress from the primary tumor at an early step in the metastatic cascade. In this study, siRNA was used to investigate whether knock down of the MMP-1 gene could be used to inhibit invasiveness in a human chondrosarcoma cell line. The inhibitory effect of siRNA on endogenous MMP-1 gene expression and protein synthesis was demonstrated via RT-PCR, Northern blotting, Western blotting, collagenase activity assay, and an in vitro cell migration assay. The siRNA inhibited MMP-1 expression specifically, since it did not affect the expression of endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) nor other collagenases. Most importantly, the siRNA mediated reduction in MMP-1 expression correlated with a decreased ability of chondrosarcoma cells to invade a Type I collagen matrix. The reduction of invasive behavior demonstrated by human chondrosarcoma cells transfected with MMP-1 siRNA and the specificity of this inhibition supports the hypothesis that this metalloproteinase molecule is involved in initiation of chondrosarcoma metastasis. © 2004 Wiley-Liss, Inc. [source]

Ex vivo bone morphogenetic protein-2 gene delivery using gingival fibroblasts promotes bone regeneration in rats

Joong-Ho Shin
Shin J-H, Kim K-H, Kim S-H, Koo K-T, Kim T-I, Seol Y-J, Ku Y, Rhyu I-C, Chung C-P, Lee Y-M. Ex vivo bone morphogenetic protein-2 gene delivery using gingival fibroblasts promotes bone regeneration in rats. J Clin Periodontol 2009; 37: 305,311. doi: 10.1111/j.1600-051X.2009.01522.x. Abstract Aim: The aim of the present study was to investigate bone regeneration following ex vivo bone morphogenetic protein-2 (BMP-2) gene delivery using human gingival fibroblasts (HGFs) in rat calvarial defects. Materials and Methods: An 8 mm craniotomy defect was created in Sprague,Dawley rats. The animals were divided into four groups: (1) non-grafted group, the defect was left empty; (2) collagen matrix group, the defect was filled with collagen matrix only; (3) HGF group, the defect was filled with non-transduced HGFs on collagen matrix; (4) BMP-2/HGF group, the defect was filled with BMP-2 gene-transduced HGFs on collagen matrix. Animals were sacrificed at 2 and 4 weeks after surgery, and micro-computed tomographic and histologic observations were performed. Results: The BMP-2/HGF group showed promoted osseous healing of calvarial defects, as compared with the other groups. At both 2 and 4 weeks, regenerated bone area was significantly greater in the BMP-2/HGF group than the other three groups. Quite a few number of transplanted HGFs were observed within the regenerated bone tissues. Conclusions: The results of this study suggest that ex vivo BMP-2 gene delivery induces prominent bone regeneration in vivo and HGFs may be useful as target cells for ex vivo gene therapy. [source]

A New Biological Matrix for Septal Occlusion

The ideal septal occluder scaffold should promote the healthiest and most complete healing response possible while eventually facilitating the full resorption of the material, leaving "native" tissue behind. An excellent biocompatibility of the scaffold tissue is a prerequisite for quick, complete, and firm ingrowth of the device, optimizing outcomes and minimizing the potential for complications. Intestinal collagen layer (ICL) is a highly purified (acellular) bioengineered type-1 collagen derived from porcine submucosa. It is gradually resorbed by the host organism and subsequently replaced by the host tissue. CardioSEAL® occluders were modified by substituting the conventional polyester fabric for an intestinal collagen layer (ICL). Percutaneous transcatheter closure of interventionally created atrial septal defects was performed in lambs using these modified occluders. A complete pathomorphological investigation including histology was carried out after 2, 4, and 12 weeks follow-up. Standard CardioSEAL implants served as a control group. After 2 weeks in vivo the devices were already covered completely by neo-endothelium. Compared with the conventional synthetic scaffold, ICL devices showed a quicker endothelialization, decreased thrombogenicity, and superior biocompatibility with no significant cellular infiltration observed in the histology of explants with ICL fabrics. After 3 months in vivo the collagen layer remained mechanically intact, but began to show the first histological signs of mild disintegration, gradual resorption, and remodeling. In conclusion, short-term results from preliminary in vivo experiments using a bioengineered collagen matrix as the occluder tissue scaffold showed excellent biocompatibility. This resulted in superior overall results: quicker endothelialization, a decreased thrombogenicity, and decreased immunological host response. (J Interven Cardiol 2003;16:149,152) [source]

Quantification of the graphical details of collagen fibrils in transmission electron micrographs

Y. Xia
A novel 2D image analysis technique is demonstrated. Using the digitized images of articular cartilage from transmission electron microscopy (TEM), this technique performs a localized ,vector' analysis at each region that is large enough to include several or tens of collagen fibrils but small enough to provide a fine resolution for the whole tissue. For each small and localized region, the morphology of the collagen fibrils can be characterized by three quantities essential to the nature of the tissue: the concentration of the fibrils, the overall orientation of the fibrils, and the anisotropy of the fibrils. This technique is capable of providing new insight to the existing technology by assigning quantitative attributes to the qualitative graphics. The assigned quantities are sensitive to the fine structure of the collagen matrix and meaningful in the architectural nature of the collagen matrix. These quantities could provide a critical linkage between the ultrastructure of the tissue and the macroscopic behaviours of the material. In addition, coarse-graining the microscopic resolution of EM without compromising the essential features of the tissue's structure provides a direct view of the tissue's morphology and permits direct correlations and comparisons among interdisciplinary techniques. [source]

In-vitro nasal drug delivery studies: comparison of derivatised, fibrillar and polymerised collagen matrix-based human nasal primary culture systems for nasal drug delivery studies

Remigius Uchenna Agu
The aim of this study was to establish a collagen matrix-based nasal primary culture system for drug delivery studies. Nasal epithelial cells were cultured on derivatised (Cellagen membrane CD-24), polymerised (Vitrogen gel) and fibrillar (Vitrogen film) collagen substrata. Cell morphology was assessed by microscopy. The cells were further characterised by measurement of ciliary beat frequency (CBF), transepithelial resistance (TER), permeation of sodium fluorescein, mitochondrial dehydrogenase (MDH) activity and lactate dehydrogenase (LDH) release upon cell exposure to sodium tauro-24, 25 dihydrofusidate (STDHF). Among the three collagen substrata investigated, the best epithelial differentiated phenotype (monolayer with columnar/cuboidal morphology) occurred in cells grown on Cellagen membrane CD-24 between day 4 and day 11. Cell culture reproducibility was better with Cellagen membrane CD-24 (90%) in comparison with Vitrogen gel (70%) and Vitrogen film (< 10%). TER was higher in cells grown on Vitrogen gel than on Cellagen membrane CD-24 and Vitrogen film. The apparent permeability coefficient (Papp × 10,7 cm s,1) of sodium fluorescein in these conditions was 0.45 ± 0.08 (Vitrogen gel) and 1.91 ± 0.00 (Cellagen membrane CD-24). Except for LDH release, CBF and cell viability were comparable for all the substrata. Based on MDH activity, LDH release, CBF, TER and permeation studies, Cellagen membrane CD-24- and Vitrogen gel-based cells were concluded to be functionally suitable for in-vitro nasal drug studies. Vitrogen film-based cultures may be limited to metabolism and cilio-toxicity studies. [source]

Molecular structural analysis of noncarious cervical sclerotic dentin using Raman spectroscopy

Changqi Xu
Abstract Molecular structure of the sclerotic dentin in noncarious cervical lesions (NCCLs) including both the inorganic phase and organic phase was investigated using Raman spectroscopy. It was found that NCCL sclerotic dentin was hypermineralized with the mineral/matrix ratios 2,3 times higher than those of normal dentin, which was caused by both the increase of mineral content and decrease of organic matrix (collagen) content in the sclerotic dentin. For the inorganic phase, the phosphate band (PO43,, ,1, symmetric stretching vibrational mode) in NCCL sclerotic dentin was shifted from 960 to 963 cm,1, and the width of this band was decreased from 16.4 to 10.4 cm,1, indicating that the degree of mineral crystallinity in NCCL sclerotic dentin was higher than that of normal dentin. In addition, the carbonate content in the mineral of NCCL sclerotic dentin was less than that of normal dentin. As compared to the inorganic phase, the changes within the organic phase were not dramatic. However, the changes in collagen cross-link density along with other spectral changes were still detectable. There was a noteworthy reduction in the ratio of nonreducible to reducible cross-links in the NCCL sclerotic dentin, indicating that cross-link breaks occurred in the collagen matrix of the lesions. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Interaction between myostatin and extracellular matrix components

Takayuki MIURA
ABSTRACT Myostatin, a member of the TGF-, superfamily, is a negative regulator of skeletal muscle mass. We have recently demonstrated that decorin binds to myostatin in vitro, and that immobilized decorin within the collagen matrix prevents myostatin-mediated inhibition of myoblast proliferation. However, little is known about other ECM molecules that bind to myostatin and modulate its activity. Thus, in the present study, we investigated the interaction of several other ECM molecules with myostatin. We here show that fibromodulin, fibronectin and laminin bind to myostatin in the presence of Zn2+ with a dissociation constant (KD) of 10,10,10,8 mol/L. Fibromodulin shows the highest affinity for myostatin among them. These results suggest that these ECM molecules may modulate myostatin activity like decorin does. [source]

ADAM15 exerts an antiapoptotic effect on osteoarthritic chondrocytes via up-regulation of the X-linked inhibitor of apoptosis

Beate Böhm
Objective To investigate the capacity of ADAM15, a disintegrin metalloproteinase that is up-regulated in osteoarthritic (OA) cartilage, to protect chondrocytes against apoptosis induced by growth factor deprivation and genotoxic stress. Methods Caspase 3/7 activity was determined in primary OA and ADAM15-transfected T/C28a4 chondrocytes upon exposure to the DNA-damaging agent camptothecin or serum withdrawal. Camptothecin-induced cytotoxicity was determined by measuring cellular ATP content. (Anti-)apoptotic proteins were analyzed by immunoblotting, and levels of messenger RNA (mRNA) for X-linked inhibitor of apoptosis (XIAP) were determined using real-time polymerase chain reaction. RNA interference was applied for down-regulation of ADAM15 and XIAP expression. Immunohistochemistry analysis of normal and OA cartilage samples was performed using XIAP- and ADAM15-specific antibodies. Results ADAM15-transfected chondrocytes cultured on a collagen matrix displayed significantly reduced caspase 3/7 activity upon serum or intermittent matrix withdrawal, compared with vector-transfected control cells. Apoptosis induction by camptothecin exposure also led to significantly elevated caspase 3/7 activity and reduced cell viability of the vector-transfected compared with ADAM15-transfected chondrocytes. Increased levels of activated caspase 3 and cleaved poly(ADP-ribose) polymerase were detected in the vector controls. XIAP, an inhibitor of activated caspase 3, was significantly up-regulated (,3-fold) at the protein and mRNA levels in ADAM15-transfected chondrocytes upon camptothecin treatment. Specific down-regulation of either ADAM15 or XIAP in OA chondrocytes led to significant sensitization to camptothecin-induced caspase 3/7 activity. Immunohistochemical analysis revealed low to moderate XIAP expression in normal specimens and markedly increased XIAP staining, colocalizing with ADAM15, in OA cartilage. Conclusion ADAM15 conveys antiapoptotic properties to OA chondrocytes that might sustain their potential to better resist the influence of death-inducing stimuli under pathophysiologic conditions. [source]

Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritis

Mary-Clare Miller
Objective A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage, and this process requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen-degrading matrix metalloproteinases (MMPs), membrane type 1 MMP (MT1-MMP), in synovial pannus invasiveness. Methods The expression and localization of MT1-MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemical analysis of RA joint specimens. The functional role of MT1-MMP was analyzed by 3-dimensional (3-D) collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, or GM6001. The effect of adenoviral expression of a dominant-negative MT1-MMP construct lacking a catalytic domain was also examined. Results MT1-MMP was highly expressed at the pannus,cartilage junction in RA joints. Freshly isolated rheumatoid synovial tissue and isolated RA synovial fibroblasts invaded into a 3-D collagen matrix in an MT1-MMP,dependent manner. Invasion was blocked by TIMP-2 and GM6001 but not by TIMP-1. Invasion was also inhibited by the overexpression of a dominant-negative MT1-MMP, which inhibits collagenolytic activity and proMMP-2 activation by MT1-MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1-MMP,dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix. Conclusion MT1-MMP serves as an essential collagen-degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1-MMP,dependent invasion may represent a novel therapeutic strategy for RA. [source]

Encapsulation of adult human mesenchymal stem cells within collagen-agarose microenvironments,

Anna Batorsky
Abstract Reliable control over the process of cell differentiation is a major challenge in moving stem cell-based therapies forward. The composition of the extracellular matrix (ECM) is known to play an important role in modulating differentiation. We have developed a system to encapsulate adult human mesenchymal stem cells (hMSC) within spherical three-dimensional (3D) microenvironments consisting of a defined mixture of collagen Type I and agarose polymers. These protein-based beads were produced by emulsification of liquid hMSC-matrix suspensions in a silicone fluid phase and subsequent gelation to form hydrogel beads, which were collected by centrifugation and placed in culture. Bead size and size distribution could be varied by changing the encapsulation parameters (impeller speed and blade separation), and beads in the range of 30,150 microns in diameter were reliably produced. Collagen concentrations up to 40% (wt/wt) could be incorporated into the bead matrix. Visible light and fluorescence microscopy confirmed that the collagen matrix was uniformly distributed throughout the beads. Cell viability post-encapsulation was in the range of 75,90% for all bead formulations (similar to control slab gels) and remained at this level for 8 days in culture. Fluorescent staining of the actin cytoskeleton revealed that hMSC spreading increased with increasing collagen concentration. This system of producing 3D microenvironments of defined matrix composition therefore offers a way to control cell-matrix interactions and thereby guide hMSC differentiation. The bead format allows the use of small amounts of matrix proteins, and such beads can potentially be used as a cell delivery vehicle in tissue repair applications. © 2005 Wiley Periodicals, Inc. [source]

The salmon vertebral body develops through mineralization of two preformed tissues that are encompassed by two layers of bone

Kari Nordvik
Abstract The teleost backbone consists of amphicoelous vertebrae and intervertebral ligaments, both of which include notochord-derived structures. On the basis of a sequential developmental study of the vertebral column of Atlantic salmon (Salmo salar L.) from the egg stage up to early fry stage (300,2500 day-degrees) we show that the vertebral body consists of four layers or compartments, two of which are formed through mineralization of preformed collagenous tissue (the notochordal sheath and the intervertebral ligament) and two of which are formed through ossification. The three inner layers have ordered lamellar collagen matrixes, which alternate perpendicularly from layer to layer, whereas the outer layer consists of cancellous bone with a woven matrix. The bone layers also differ in osteocyte content. In this study we describe the structural details of the layers, and their modes of formation. The results are compared with previous descriptions, and possible phylogenetic implications are discussed. [source]

Recombinant human platelet-derived growth factor BB (rhPDGF-BB) and beta-tricalcium phosphate/collagen matrix enhance fracture healing in a diabetic rat model

Loay Al-Zube
Abstract Diabetes mellitus is a common systemic disease that has been associated with poor fracture healing outcomes. The mechanism through which diabetes impairs bone regeneration is unknown. One possible mechanism may be related to either decreased or uncoordinated release of local growth factors at the fracture site. Indeed, previous studies have found reduced platelet-derived growth factor (PDGF) levels in the fracture callus of diabetic rats, suggesting that local application of PDGF may overcome the negative effects of diabetes and promote fracture healing. To test this hypothesis, low (22 µg) and high (75 ug) doses of recombinant human PDGF-BB (rhPDGF-BB) were applied directly to femur fracture sites in BB Wistar diabetic rats that were then compared to untreated or vehicle-treated animals. rhPDGF-BB treatment significantly increased early callus cell proliferation compared to that in control specimens. Low dose rhPDGF-BB treatment significantly increased callus peak torque values (p,<,0.05) at 8 weeks after fracture as compared to controls. High dose rhPDGF-BB treatment increased callus bone area at 12 weeks postfracture. These data indicate that rhPDGF-BB treatment ameliorates the effects of diabetes on fracture healing by promoting early cellular proliferation that ultimately leads to more bone formation. Local application of rhPDGF-BB may be a new therapeutic approach to treat diabetes-impaired fracture healing. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1074,1081, 2009 [source]