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Matrix Synthesis (matrix + synthesis)
Kinds of Matrix Synthesis Selected AbstractsThree-dimensional Culture of Human Nucleus Pulposus Cells in Fibrin Clot: Comparisons on Cellular Proliferation and Matrix Synthesis With Cells in AlginateARTIFICIAL ORGANS, Issue 1 2008Shu-Hua Yang Abstract:, Regeneration of nucleus pulposus (NP) tissue may stop or reverse early intervertebral disk (IVD) degeneration. Cellular proliferation and matrix synthesis can be promoted by incorporation of cells and bioscaffolds. However, insertion of preshaped solid bioscaffolds may damage remaining IVD integrity. Fibrin clots can be introduced in a minimally invasive manner with polymerization in desired three-dimensional shape and retention of cells. In this study, we investigated the cellular proliferation and matrix synthesis of human NP cells in the fibrin clots in vitro. Monolayer-expanded cells were embedded in fibrin clot or alginate and were cultivated in vitro for 2 weeks. Increased DNA content and decreased expression of apoptosis stimulating fragment (Fas)-associated death-domain protein in fibrin scaffolds suggested higher cellular proliferation and reduced apoptosis. Superior proteoglycan synthesis was found in fibrin scaffolds. As expression of collagens I and X increased and SOX9 expression decreased, fibrin scaffolds tended to promote fibrotic transformation and inhibit chondrogenesis. Adjustments of fibrin preparations are needed to make it more suitable for IVD regeneration. [source] Differential modulation of rat hepatic stellate phenotype by natural and synthetic retinoidsHEPATOLOGY, Issue 1 2004Karine Hellemans Activation of hepatic stellate cells (HSC) is a central event in the pathogenesis of liver fibrosis during chronic liver injury. We examined the expression of retinoic acid (RAR) and retinoid X receptors (RXR) during HSC activation and evaluated the influence of natural and synthetic retinoic acids (RA) on the phenotype of culture-activated HSC. The expression of the major RAR/RXR subtypes and isoforms was analyzed by Northern hybridization. Presence of functional receptor proteins was established by gel shift analysis. Retinoic acids, RAR, and RXR selective agonists and an RAR antagonist were used to evaluate the effects of retinoid signalling on matrix synthesis by Northern blotting and immunoprecipitation, and on cell proliferation by BrdU incorporation. The 9- cisRA and synthetic RXR agonists reduced HSC proliferation and synthesis of collagen I and fibronectin. All- trans RA and RAR agonists both reduced the synthesis of collagen I, collagen III, and fibronectin, but showed a different effect on cell proliferation. Synthetic RAR agonists did not affect HSC proliferation, indicating that ATRA inhibits cell growth independent of its interaction with RARs. In contrast, RAR specific antagonists enhance HSC proliferation and demonstrate that RARs control proliferation in a negative way. In conclusion, natural RAs and synthetic RAR or RXR specific ligands exert differential effects on activated HSC. Our observations may explain prior divergent results obtained following retinoid administration to cultured stellate cells or to animals subjected to fibrogenic stimuli. (HEPATOLOGY 2004;39:97,108.) [source] Exhaustive approach to the coupling matrix synthesis problem and application to the design of high degree asymmetric filtersINTERNATIONAL JOURNAL OF RF AND MICROWAVE COMPUTER-AIDED ENGINEERING, Issue 1 2007Richard J. Cameron Abstract In this paper a new approach to the synthesis of coupling matrices for microwave filters is presented. The new approach represents an advance on existing direct and optimization methods for coupling matrix synthesis, in that it will exhaustively discover all possible coupling matrix solutions for a network if more than one exists. This enables a selection to be made of the set of coupling values, resonator frequency offsets, parasitic coupling tolerance, etc. that will be best suited to the technology it is intended to realize the microwave filter with. To demonstrate the use of the method, the case of the recently introduced "extended box" coupling matrix configuration is taken. The extended box is a new class of filter configuration adapted to the synthesis of asymmetric filtering characteristics of any degree. For this configuration the number of solutions to the coupling matrix synthesis problem appears to be high and offers therefore some flexibility that can be used during the design phase. We illustrate this by carrying out the synthesis process of two asymmetric filters of 8th and 10th degree. In the first example a ranking criterion is defined in anticipation of a dual mode realization and allows the selection of a "best" coupling matrix out of 16 possible ones. For the 10th degree filter a new technique of approximate synthesis is presented, yielding some simplifications of the practical realization of the filter as well as of its computer aided tuning phase. © 2006 Wiley Periodicals, Inc. Int J RF and Microwave CAE, 2007. [source] Disc structure function and its potential for repairINTERNATIONAL JOURNAL OF RHEUMATIC DISEASES, Issue 1 2002J. Melrose The intervertebral disc (IVD) is the largest predominantly avascular, aneural, alymphatic structure of the human body. It provides articulation between adjoining vertebral bodies and also acts as a weight-bearing cushion dissipating axially applied spinal loads. The IVD is composed of an outer collagen-rich annulus fibrosus (AF) and a central proteoglycan (PG)-rich nucleus pulposus (NP). Superior and inferior cartilaginous endplates (CEPs), thin layers of hyaline-like cartilage, cover the ends of the vertebral bodies. The AF is composed of concentric layers (lamellae) which contain variable proportions of type I and II collagen, this tissue has high tensile strength. The NP in contrast is a gelatinous PG-rich tissue which provides weight-bearing properties to the composite disc structure. With the onset of age, cells in the NP progressively die as this tissue becomes depleted of PGs, less hydrated and more fibrous as the disc undergoes an age-dependent fibrocartilaginous transformation. Such age-dependent cellular and matrix changes can decrease the discs' biomechanical competence and trauma can further lead to failure of structural components of the disc. Annular defects are fairly common and include vertebral rim-lesions, concentric (circumferential) annular tears (separation of adjacent annular lamellae) and radial annular tears (clefts which initiate within the NP). While vascular in-growth around annular tears has been noted, evidence from human post-mortem studies indicate they have a limited ability to undergo repair. Several experimental approaches are currently under evaluation for their ability to promote the repair of such annular lesions. These include growth of AF fibrochondrocytes on a resorbable polycaprolactone (PCL) bio-membrane.1 Sheets of fibrochondrocytes lay down type-I collagen and actin stress fibres on PCL. These matrix components are important for the spatial assembly of the collagenous lamella during annular development and correct phenotypic expression of cells in biomatrices.1 An alternative approach employs preparation of tissue engineered IVDs where AF and NP cells are separately cultured in polyglycolic acid and sodium alginate biomatrices, either separately or within a manifold designed to reproduce the required IVD dimensions for its use as a prospective implant device.2 AF and NP cells have also been grown on tissue culture inserts after their recovery from alginate bead culture to form plugs of tissue engineered cartilage.3 A key component in this latter strategy was the stimulation of the high density disc cell cultures with osteogenic protein-1 (OP-1) 200 ng/mL.3 This resulted in the production of tissue engineered AF and NP plugs with compositions, histochemical characteristics and biomechanical properties approaching those of the native disc tissues.2,3 Such materials hold reat promise in future applications as disc or annular implants. The introduction of appropriate genes into disc cells by gene transduction methodology using adenoviral vectors or ,gene-gun' delivery systems also holds considerable promise for the promotion of disc repair processes.4 Such an approach with the OP-1 gene is particularly appealing.5 The anchoring of discal implants to vertebral bodies has also been evaluated by several approaches. A 3D fabric based polyethylene biocomposite holds much promise as one such anchorage device6 while biological glues used to seal fibrocartilaginous structures such as the AF and meniscus8 following surgical intervention, also hold promise in this area. Several very promising new experimental approaches and strategies are therefore currently under evaluation for the improvement of discal repair. The aforementioned IVD defects are a common cause of disc failure and sites of increased nerve in-growth in symptomatic IVDs in man and are thus often sources of sciatic-type pain. Annular defects such as those described above have formerly been considered incapable of undergoing spontaneous repair thus a clear need exists for interventions which might improve on their repair. Based on the rapid rate of progress and the examples outlined above one may optimistically suggest that a successful remedy to this troublesome clinical entity will be developed in the not so distant future. References 1JohnsonWEBet al. (2001) Directed cytoskeletal orientation and intervertebral disc cell growth: towards the development of annular repair techniques. Trans Orthop Res Soc26, 894. 2MizunoHet al. (2001) Tissue engineering of a composite intervertebral disc. Trans Orthop Res Soc26, 78. 3MatsumotoTet al. (2001) Formation of transplantable disc shaped tissues by nucleus pulposus and annulus fibrosus cells: biochemical and biomechanical properties. Trans Orthop Res Soc26, 897. 4NishidaKet al. (2000) Potential applications of gene therapy to the treatment of intervertebral disc disorders. Clin Orthop Rel Res379 (Suppl), S234,S241. 5MatsumotoTet al. (2001) Transfer of osteogenic protein-1 gene by gene gun system promotes matrix synthesis in bovine intervertebral disc and articular cartilage cells. Trans Orthop Res Soc26, 30. 6ShikinamiY , Kawarada (1998) Potential application of a triaxial three-dimensional fabric (3-DF) as an implant. Biomaterials19, 617,35. [source] TNF-, Mediates p38 MAP Kinase Activation and Negatively Regulates Bone Formation at the Injured Growth Plate in Rats,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2006Fiona H Zhou Abstract TNF-, is known to inhibit osteoblast differentiation in vitro and yet it is essential for bone fracture repair. Roles of TNF-, in the bony repair of injured growth plate were examined in young rats treated with a TNF-, antagonist. The results show that TNF-, mediates p38 activation, which influences the recruitment, proliferation, and osteoblast differentiation of mesenchymal cells and negatively regulates bone formation at the injured growth plate. Introduction: TNF-, inhibits expression of osteoblast differentiation factor cbfa1 and osteoblast differentiation in vitro and yet TNF-, signaling is essential for bone fracture healing. Roles of TNF-, in the bony repair of injured growth plate cartilage are unknown. Materials and Methods: Roles of TNF-, in the activation of p38 mitogen activated protein (MAP) kinase and the subsequent bony repair of the injured growth plate were examined in young rats receiving the TNF-, inhibitor ENBREL or saline control. Activation of p38 was determined by Western blot analysis and immunohistochemistry. Inflammatory cell counts on day 1, measurements of repair tissue proportions, and counting of proliferative mesenchymal cells on day 8 at growth plate injury site were carried out (n = 6). Expression of inflammatory cytokines TNF-, and IL-1,, fibrogenic growth factor (FGF)-2, cbfa1, and bone protein osteocalcin at the injured growth plate was assessed by quantitative RT-PCR. Effects of TNF-, signaling on proliferation, migration, and apoptosis of rat bone marrow mesenchymal cells (rBMMCs) and the regulatory roles of p38 in these processes were examined using recombinant rat TNF-,, ENBREL, and the p38 inhibitor SB239063 in cultured primary rBMMCs. Results: p38 activation was induced in the injured growth plate during the initial inflammatory response, and activated p38 was immunolocalized in inflammatory cells at the injury site and in the adjacent growth plate. In addition, activation of p38 was blocked in rats treated with TNF-, antagonist, suggesting a role of TNF-, in p38 activation. Whereas TNF-, inhibition did not alter inflammatory infiltrate and expression of TNF-, and IL-1, at the injured growth plate on day 1, it reduced mesenchymal infiltrate and cell proliferation and FGF-2 expression on day 8. Consistently, TNF-, increased proliferation and migration of rBMMCs in vitro, whereas p38 inhibition reduced rBMMC proliferation and migration. At the injured growth plate on day 8, TNF-, inhibition increased expression of cbfa1 and osteocalcin and increased trabecular bone formation at the injury site. There was a significant inverse correlation between TNF-, and cbfa1 expression levels, suggesting a negative relationship between TNF-, and cbfa1 in this in vivo model. Conclusions: These observations suggest that TNF-, activates p38 MAP kinase during the inflammatory response at the injured growth plate, and TNF-,-p38 signaling seems to be required for marrow mesenchymal cell proliferation and migration at the growth plate injury site and in cell culture. Furthermore, TNF signaling has an inhibitory effect on bone formation at the injured growth plate by suppressing bone cell differentiation and bone matrix synthesis at the injury site. [source] Strong Static Magnetic Field Stimulates Bone Formation to a Definite Orientation In Vitro and In Vivo,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2002Hiroko Kotani Ph.D. Abstract The induction of bone formation to an intentional orientation is a potentially viable clinical treatment for bone disorders. Among the many chemical and physical factors, a static magnetic field (SMF) of tesla order can regulate the shapes of blood cells and matrix fibers. This study investigated the effects of a strong SMF (8 T) on bone formation in both in vivo and in vitro systems. After 60 h of exposure to the SMF, cultured mouse osteoblastic MC3T3-E1 cells were transformed to rodlike shapes and were orientated in the direction parallel to the magnetic field. Although this strong SMF exposure did not affect cell proliferation, it up-regulated cell differentiation and matrix synthesis as determined by ALP and alizarin red stainings, respectively. The SMF also stimulated ectopic bone formation in and around subcutaneously implanted bone morphogenetic protein (BMP) 2-containing pellets in mice, in which the orientation of bone formation was parallel to the magnetic field. It is concluded that a strong SMF has the potency not only to stimulate bone formation, but also to regulate its orientation in both in vitro and in vivo models. This is the first study to show the regulation of the orientation of adherent cells by a magnetic field. We propose that the combination of a strong SMF and a potent osteogenic agent such as BMP possibly may lead to an effective treatment of bone fractures and defects. [source] Collagen Metabolism Is Markedly Altered in the Hypertrophic Cartilage of Growth Plates from Rats with Growth Impairment Secondary to Chronic Renal FailureJOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2001Jesús Álvarez Abstract Skeletal growth depends on growth plate cartilage activity, in which matrix synthesis by chondrocytes is one of the major processes contributing to the final length of a bone. On this basis, the present work was undertaken to ascertain if growth impairment secondary to chronic renal insufficiency is associated with disturbances of the extracellular matrix (ECM) of the growth plate. By combining stereological and in situ hybridization techniques, we examined the expression patterns of types II and X collagens and collagenase-3 in tibial growth plates of rats made uremic by subtotal nephrectomy (NX) in comparison with those of sham-operated rats fed ad libitum (SAL) and sham-operated rats pair-fed with NX (SPF). NX rats were severely uremic, as shown by markedly elevated serum concentrations of urea nitrogen, and growth retarded, as shown by significantly decreased longitudinal bone growth rates. NX rats showed disturbances in the normal pattern of chondrocyte differentiation and in the rates and degree of substitution of hypertrophic cartilage with bone, which resulted in accumulation of cartilage at the hypertrophic zone. These changes were associated with an overall decrease in the expression of types II and X collagens, which was especially marked in the abnormally extended zone of the hypertrophic cartilage. Unlike collagen, the expression of collagenase-3 was not disturbed severely. Electron microscopic analysis proved that changes in gene expression were coupled to alterations in the mineralization as well as in the collagen fibril architecture at the hypertrophic cartilage. Because the composition and structure of the ECM have a critical role in regulating the behavior of the growth plate chondrocytes, results obtained are consistent with the hypothesis that alteration of collagen metabolism in these cells could be a key process underlying growth retardation in uremia. [source] Intracellular dynamics of Smad-mediated TGF, signalingJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2003Robert M. Greene The transforming growth factor-, (TGF,) family represents a class of signaling molecules that plays a central role in morphogenesis, growth, and cell differentiation during normal embryonic development. Members of this growth factor family are particularly vital to development of the mammalian secondary palate where they regulate palate mesenchymal cell proliferation and extracellular matrix synthesis. Such regulation is particularly critical since perturbation of either cellular process results in a cleft of the palate. While the cellular and phenotypic effects of TGF, on embryonic craniofacial tissue have been extensively catalogued, the specific genes that function as downstream mediators of TGF, action in the embryo during palatal ontogenesis are poorly defined. Embryonic palatal tissue in vivo and murine embryonic palate mesenchymal (MEPM) cells in vitro secrete and respond to TGF,. In the current study, elements of the Smad component of the TGF, intracellular signaling system were identified and characterized in cells of the embryonic palate and functional activation of the Smad pathway by TGF,1, TGF,2, and TGF,3 was demonstrated. TGF,-initiated Smad signaling in cells of the embryonic palate was found to result in: (1) phosphorylation of Smad 2; (2) nuclear translocation of the Smads 2, 3, and 4 protein complex; (3) binding of Smads 3 and 4 to a consensus Smad binding element (SBE) oligonucleotide; (4) transactivation of transfected reporter constructs, containing TGF,-inducible Smad response elements; and (4) increased expression of gelatinases A and B (endogenous genes containing Smad response elements) whose expression is critical to matrix remodeling during palatal ontogenesis. Collectively, these data point to the presence of a functional Smad-mediated TGF, signaling system in cells of the developing murine palate. J. Cell. Physiol. 197: 261,271, 2003. © 2003 Wiley-Liss, Inc. [source] Autocrine growth factors in human periodontal ligament cells cultured on enamel matrix derivativeJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 2 2001Staale P. Lyngstadaas Abstract Objective: Enamel extracellular matrix proteins in the form of the enamel matrix derivative EMDOGAIN® (EMD) have been successfully employed to mimic natural cementogenesis to restore fully functional periodontal ligament, cementum and alveolar bone in patients with severe periodontitis. When applied to denuded root surfaces EMD forms a matrix that locally facilitates regenerative responses in the adjacent periodontal tissues. The cellular mechanism(s), e.g. autocrine growth factors, extracellular matrix synthesis and cell growth, underlying PDL regeneration with EMD is however poorly investigated. Material and Methods: Human periodontal ligament (PDL) cells were cultured on EMD and monitored for cellular attachment rate, proliferation, DNA replication and metabolism. Furthermore, intracellular cyclic-AMP levels and autocrine production of selected growth factors were monitored by immunological assays. Controls included PDL and epithelial cells in parallel cultures. Results: PDL cell attachment rate, growth and metabolism were all significantly increased when EMD was present in cultures. Also, cells exposed to EMD showed increased intracellular cAMP signalling and autocrine production of TGF-,1, IL-6 and PDGF AB when compared to controls. Epithelial cells increased cAMP and PDGF AB secretion when EMD was present, but proliferation and growth were inhibited. Conclusion: Cultured PDL cells exposed to EMD increase attachment rate, growth rate and metabolism, and subsequently release several growth factors into the medium. The cellular interaction with EMD generates an intracellular cAMP signal, after which cells secrete TGF-,1, IL-6 and PDGF AB. Epithelial cell growth however, is inhibited by the same signal. This suggest that EMD favours mesenchymal cell growth over epithelium, and that autocrine growth factors released by PDL cells exposed to EMD contribute to periodontal healing and regeneration in a process mimicking natural root development. [source] Sustained hypoxia enhances chondrocyte matrix synthesisJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2009Christian H. Coyle Abstract Articular cartilage is an avascular tissue with chondrocytes in the deeper zones existing under conditions of sustained hypoxia. Using a hypoxic chamber to provide controlled hypoxia, this study was performed to determine whether sustained hypoxia enhances the production of cartilage matrix proteins. Freshly isolated primary bovine articular chondrocytes were encapsulated in three-dimensional alginate beads and maintained at 2% oxygen with media changes using media pre-equilibrated to 2% oxygen. Immunolocalization of HIF-1, was performed to verify hypoxic conditions. Sustained hypoxia resulted in an increase in proteoglycan synthesis after only 1 day, as measured by 35S-sulfate incorporation. This increase was maintained for the duration of the 17 day study. After 17 days of hypoxic culture, increases in total type II collagen and COL2A1 gene expression were probed by indirect immunofluorescence, type II collagen ELISA, and real-time qPCR; in addition, increased glycosaminoglycan deposition was observed as determined by chemical analysis. These studies show that sustained hypoxia enhances articular chondrocyte matrix synthesis and viability in three-dimensional alginate culture. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 793,799, 2009 [source] Acceleration of cartilage repair by genetically modified chondrocytes over expressing bone morphogenetic protein-7JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 4 2003Chisa Hidaka Background: Cartilage has a limited capacity to heal. Although chondrocyte transplantation is a useful therapeutic strategy, the repair process can be lengthy. Previously we have shown that over expression of bone morphogenetic protein-7 (BMP-7) in chondrocytes by adenovirus-mediated gene transfer leads to increased matrix synthesis and cartilage-like tissue formation in vitro. In this context we hypothesized that implantation of genetically modified chondrocytes expressing BMP-7 would accelerate the formation of hyaline-like repair tissue in an equine model of cartilage defect repair. Methods: Chondrocytes treated with adenovirus vector encoding BMP-7 (AdBMP-7) or as control, an adenovirus vector encoding an irrelevant gene (Escherichia coli cytosine deaminase, AdCD) were implanted into extensive (15 mm diameter) articular cartilage defects in the patellofemoral joints of 10 horses. Biopsies were performed to evaluate early healing at 4 weeks. At the terminal time point of 8 months, repairs were assessed for morphology, MRI appearance, compressive strength, biochemical composition and persistence of implanted cells. Results: Four weeks after surgery AdBMP-7-treated repairs showed an increased level of BMP-7 expression and accelerated healing, with markedly more hyaline-like morphology than control. Quantitative real-time polymerase chain reaction (PCR) analysis of the repair tissue 8 months after surgery showed that few implanted cells persisted. By this time, the controls had healed similarly to the AdBMP-7-treated defects, and no difference was detected in the morphologic, biochemical or biomechanical properties of the repair tissues from the two treatment groups. Conclusions: Implantation of genetically modified chondrocytes expressing BMP-7 accelerates the appearance of hyaline-like repair tissue in experimental cartilage defects. Clinical relevance: Rehabilitation after cell-based cartilage repair can be prolonged, leading to decreased patient productivity and quality of life. This study shows the feasibility of using genetically modified chondrocytes to accelerate cartilage healing. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Molecular fingerprinting of TGFß-treated embryonic maxillary mesenchymal cellsORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 4 2003M.M. Pisano Abstract The transforming growth factor-ß (TGFß) family represents a class of signaling molecules that plays a central role in normal embryonic development, specifically in development of the craniofacial region. Members of this family are vital to development of the secondary palate where they regulate maxillary and palate mesenchymal cell proliferation and extracellular matrix synthesis. The function of this growth factor family is particularly critical in that perturbation of either process results in a cleft of the palate. While the cellular and phenotypic effects of TGFß on embryonic craniofacial tissue have been extensively cataloged, the specific genes that function as downstream mediators of TGFß in maxillary/palatal development are poorly defined. Gene expression arrays offer the ability to conduct a rapid, simultaneous assessment of hundreds to thousands of differentially expressed genes in a single study. Inasmuch as the downstream sequelae of TGFß action are only partially defined, a complementary DNA (cDNA) expression array technology (Clontech's AtlasTM Mouse cDNA Expression Arrays), was utilized to delineate a profile of differentially expressed genes from TGFß-treated primary cultures of murine embryonic maxillary mesenchymal cells. Hybridization of a membrane-based cDNA array (1178 genes) was performed with 32P-labeled cDNA probes synthesized from RNA isolated from either TGFß-treated or vehicle-treated embryonic maxillary mesenchymal cells. Resultant phosphorimages were subject to AtlasImageTM analysis in order to determine differences in gene expression between control and TGFß-treated maxillary mesenchymal cells. Of the 1178 arrayed genes, 552 (47%) demonstrated detectable levels of expression. Steady state levels of 22 genes were up-regulated, while those of 8 other genes were down-regulated, by a factor of twofold or greater in response to TGFß. Affected genes could be grouped into three general functional categories: transcription factors and general DNA-binding proteins; growth factors/signaling molecules; and extracellular matrix and related proteins. The extent of hybridization of each gene was evaluated by comparison with the abundant, constitutively expressed mRNAs: ubiquitin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ornithine decarboxylase (ODC), cytoplasmic beta-actin and 40S ribosomal protein. No detectable changes were observed in the expression levels of these genes in response to TGFß treatment. Gene expression profiling results were verified by Real-Time quantitative polymerase chain reaction. Utilization of cDNA microarray technology has enabled us to delineate a preliminary transcriptional map of TGFß responsiveness in embryonic maxillary mesenchymal cells. The profile of differentially expressed genes offers revealing insights into potential molecular regulatory mechanisms employed by TGFß in orchestrating craniofacial ontogeny. [source] Orthodontically stressed periodontium of transgenic mouse as a model for studying mechanical response in bone: The effect on the number of osteoblastsORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 2 2000Dubravko Pavlin A better understanding of cellular and molecular mechanisms involved in response to mechanical stress is a prerequisite for future improvements in orthodontic treatment. To expand the application of molecular biology techniques in this area of research, we developed and characterized a mouse tooth movement model. The aim of this study was to biomechanically characterize this model and to evaluate the effect of orthodontic stress on the proliferation of periodontal osteoblasts. We used an orthodontic coil spring appliance with a low force/deflection rate, which produced an average force of 10,12 g. This design provided a predictable tipping movement of the molar with the center of rotation at the level of root apices. Histological observations of paradental tissues revealed a response favoring a fast onset of tooth movement and deposition of new osteoid starting after 3 days of treatment. The effect of treatment on the histomorpometric parameter of the number of osteoclasts per unit bone perimeter was determined after 1, 2, 3, 4, 6, and 12 days of treatment. Starting with day 2, the osteoblast number showed a modest but consistent increase in treated periodontal sites at all time-points, ranging from 14 to 39% and becoming significant only at day 6. Only a moderate increase in the number of osteoblasts in the areas of otherwise intense bone matrix synthesis suggests that, during bone formation, proliferation of cells has a smaller role compared to a marked increase in differentiation of individual cells. The mouse model, which allows for a controlled, reproducible, orthodontic mechanical loading, can be applied to both wild-type and transgenic animals and should enhance the research of the transduction of mechanical orthodontic signal into a biological response. [source] Pulmonary fibrosis: Cellular and molecular eventsPATHOLOGY INTERNATIONAL, Issue 3 2003Mohammed S. Razzaque Connective tissue remodeling of the interstitium is an important feature of chronic lung diseases encompassing interstitial inflammatory changes and subsequent pulmonary fibrosis. The early inflammatory phase is usually associated with the release of several cytokines and chemokines by activated resident cells and infiltrating cells which, in turn, help further recruit inflammatory mononuclear cells. Cytokines and growth factors secreted by inflammatory cells and by interstitial cells (fibroblasts and myofibroblasts) play an important role in the fibrogenic phase of pulmonary fibrosis by inducing matrix synthesis. In addition, matrix-degrading enzymes and their inhibitors also contribute to extracellular matrix (ECM) remodeling in pulmonary fibrosis. This review addresses the pathophysiology of wound healing and different phases of pulmonary fibrosis. [source] Involvement of the Klotho Protein in Dentin Formation and MineralizationTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 2 2008Hironobu Suzuki Abstract Klotho -deficient mice exhibit multiple pathological conditions resembling human aging. Our previous study showed alterations in the distribution of osteocytes and in the bone matrix synthesis in klotho -deficient mice. Although the bone and tooth share morphological features such as mineralization processes and components of the extracellular matrix, little information is available on how klotho deletion influences tooth formation. The present study aimed to elucidate the altered histology of incisors of klotho -deficient mice,comparing the findings with those from their wild-type littermates, by using immunohistochemistry for alkaline phosphatase (ALP), osteopontin, and dentin matrix protein-1 (DMP-1), terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) detection for apoptosis, and electron probe microanalyzer (EPMA) analysis on calcium (Ca), phosphate (P), and magnesium (Mg). Klotho -deficient incisors exhibited disturbed layers of odontoblasts, predentin, and dentin, resulting in an obscure dentin-predentinal border at the labial region. Several odontoblast-like cells without ALP activity were embedded in the labial dentin matrix, and immunopositivity for DMP-1 and osteopontin was discernible in the matrix surrounding these embedded odontoblast-like cells. TUNEL detection demonstrated an apoptotic reaction in the embedded odontoblast-like cells and pulpal cells in the klotho -deficient mice. EPMA revealed lower concentrations of Ca, P, and Mg in the klotho -deficient dentin, except for the dentin around abnormal odontoblast-like cells. These findings suggest the involvement of the klotho gene in dentinogenesis and its mineralization. Anat Rec, 2007. © 2008 Wiley-Liss, Inc. [source] Gene therapy for cartilage defectsTHE JOURNAL OF GENE MEDICINE, Issue 12 2005Magali Cucchiarini Abstract Focal defects of articular cartilage are an unsolved problem in clinical orthopaedics. These lesions do not heal spontaneously and no treatment leads to complete and durable cartilage regeneration. Although the concept of gene therapy for cartilage damage appears elegant and straightforward, current research indicates that an adaptation of gene transfer techniques to the problem of a circumscribed cartilage defect is required in order to successfully implement this approach. In particular, the localised delivery into the defect of therapeutic gene constructs is desirable. Current strategies aim at inducing chondrogenic pathways in the repair tissue that fills such defects. These include the stimulation of chondrocyte proliferation, maturation, and matrix synthesis via direct or cell transplantation-mediated approaches. Among the most studied candidates, polypeptide growth factors have shown promise to enhance the structural quality of the repair tissue. A better understanding of the basic scientific aspects of cartilage defect repair, together with the identification of additional molecular targets and the development of improved gene-delivery techniques, may allow a clinical translation of gene therapy for cartilage defects. The first experimental steps provide reason for cautious optimism. Copyright © 2005 John Wiley & Sons, Ltd. [source] Transcriptional profiling and biochemical analysis of mechanically induced cartilaginous tissues in a rat modelARTHRITIS & RHEUMATISM, Issue 4 2010Kristy T. Salisbury Palomares Objective To characterize patterns of molecular expression that lead to cartilage formation in vivo in a postnatal setting, by profiling messenger RNA expression across the time course of mechanically induced chondrogenesis. Methods Retired breeder Sprague-Dawley rats underwent a noncritical-sized transverse femoral osteotomy. Experimental animals (n = 45) were subjected to bending stimulation (60° cyclic motion in the sagittal plane for 15 minutes/day) of the osteotomy gap beginning on day 10 after the operation. Control animals (n = 32) experienced continuous rigid fixation. Messenger RNA isolated on days 10, 17, 24, and 38 after surgery was analyzed using a microarray containing 608 genes involved in skeletal development, tissue differentiation, fracture healing, and mechanotransduction. The glycosaminoglycan (GAG) content in the stimulated tissues was compared with that in native articular cartilage as a means of assessing the progression of chondrogenic development of the tissues. Results The majority of the 100 genes that were differentially expressed were up-regulated in response to mechanical stimulation. Many of these genes are associated with articular cartilage development and maintenance, diarthrodial joint development, cell adhesion, extracellular matrix synthesis, signal transduction, and skeletal development. Quantitative real-time polymerase chain reaction results were consistent with the microarray findings. The GAG content of the stimulated tissues increased over time and was no different from that of articular cartilage on day 38 after surgery. Conclusion Our findings indicate that mechanical stimulation causes up-regulation of genes that are principally involved in joint cavity morphogenesis and critical to articular cartilage function. Further study of this type of stimulation may identify key signaling events required for postnatal hyaline cartilage formation. [source] Associations of 25 structural, degradative, and inflammatory candidate genes with lumbar disc desiccation, bulging, and height narrowingARTHRITIS & RHEUMATISM, Issue 2 2009Tapio Videman Objective To examine the allelic diversity of structural, inflammatory, and matrix-modifying gene candidates and their association with disc degeneration. Methods Subjects were 588 men ages 35,70 years. We investigated associations of single-nucleotide polymorphisms in AGC1 and in 12 collagen, 8 interleukin, and 4 matrix metalloproteinase genes with quantitative magnetic resonance imaging measurements of disc desiccation and disc bulging and height narrowing scores, after controlling for age and suspected risk factors. Analyses were performed using QTDT software. P values were derived from 1,000 permutations, and empirical P values for global significance also were applied. Results Twelve of the 99 variants in 25 selected candidate genes provided evidence of association (P < 0.05) with disc signal intensity in the upper and/or lower lumbar regions. Allelic variants of AGC1 (rs1042631; P = 0.001), COL1A1 (rs2075555; P = 0.005), COL9A1 (rs696990; P = 0.00008), and COL11A2 (rs2076311; P = 0.018) genes provided the most significant evidence of association with disc signal intensity. The same variants of AGC1 (P = 0.010) and COL9A1 (P = 0.014), as well as variants in the COL11A1 gene (rs1463035 [P = 0.004]; rs1337185 [P = 0.015]) were also associated with disc bulging, as was AGC1 with disc height narrowing (rs1516797; P = 0.005). In addition, 4 allelic variants in the immunologic candidate genes (rs2071375 in IL1A [P = 0.027]; rs1420100 in IL18RAP [P = 0.005]) were associated with disc signal intensity. Conclusion Genetic variants account for interindividual differences in disc matrix synthesis and degradation. The accuracy of the quantitative disc signal intensity measurements we used likely enhanced our ability to observe these associations. Our findings shed light on possible mechanisms of degeneration and support the view that disc degeneration is a polygenetic condition. [source] Bone Morphogenetic Protein-6-loaded Chitosan Scaffolds Enhance the Osteoblastic Characteristics of MC3T3-E1 CellsARTIFICIAL ORGANS, Issue 1 2010Abdullah C. Akman Abstract The purpose of this study is to investigate the convenience of bone morphogenetic protein-6 (BMP-6)-loaded chitosan scaffolds with preosteoblastic cells for bone tissue engineering. MC3T3-E1 cells were seeded into three different groups: chitosan scaffolds, BMP-6-loaded chitosan scaffolds, and chitosan scaffolds with free BMP-6 in culture medium. Tissue-engineered constructs were characterized by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay, scanning electron microscopy (SEM), mineralization assay (von Kossa), alkaline phosphatase (ALP) activity, and osteocalcin (OCN) assays. BMP-6-loaded chitosan scaffolds supported proliferation of the MC3T3-E1 mouse osteogenic cells in a similar pattern as the unloaded chitosan scaffolds group and as the chitosan scaffolds with free BMP-6 group. SEM images of the cell-seeded scaffolds revealed significant acceleration of extracellular matrix synthesis in BMP-6-loaded chitosan scaffolds. Both levels of ALP and OCN were higher in BMP-6-loaded chitosan scaffold group compared with the other two groups. In addition, BMP-6-loaded scaffolds showed strong staining in mineralization assays. These findings suggest that BMP-6-loaded chitosan scaffold supports cellular functions of the osteoblastic cells; therefore, this scaffold is considered as a new promising vehicle for bone tissue engineering applications. [source] Three-dimensional Culture of Human Nucleus Pulposus Cells in Fibrin Clot: Comparisons on Cellular Proliferation and Matrix Synthesis With Cells in AlginateARTIFICIAL ORGANS, Issue 1 2008Shu-Hua Yang Abstract:, Regeneration of nucleus pulposus (NP) tissue may stop or reverse early intervertebral disk (IVD) degeneration. Cellular proliferation and matrix synthesis can be promoted by incorporation of cells and bioscaffolds. However, insertion of preshaped solid bioscaffolds may damage remaining IVD integrity. Fibrin clots can be introduced in a minimally invasive manner with polymerization in desired three-dimensional shape and retention of cells. In this study, we investigated the cellular proliferation and matrix synthesis of human NP cells in the fibrin clots in vitro. Monolayer-expanded cells were embedded in fibrin clot or alginate and were cultivated in vitro for 2 weeks. Increased DNA content and decreased expression of apoptosis stimulating fragment (Fas)-associated death-domain protein in fibrin scaffolds suggested higher cellular proliferation and reduced apoptosis. Superior proteoglycan synthesis was found in fibrin scaffolds. As expression of collagens I and X increased and SOX9 expression decreased, fibrin scaffolds tended to promote fibrotic transformation and inhibit chondrogenesis. Adjustments of fibrin preparations are needed to make it more suitable for IVD regeneration. [source] Developmental gene expression profiling of mammalian, fetal orofacial tissueBIRTH DEFECTS RESEARCH, Issue 12 2004Partha Mukhopadhyay Abstract BACKGROUND The embryonic orofacial region is an excellent developmental paradigm that has revealed the centrality of numerous genes encoding proteins with diverse and important biological functions in embryonic growth and morphogenesis. DNA microarray technology presents an efficient means of acquiring novel and valuable information regarding the expression, regulation, and function of a panoply of genes involved in mammalian orofacial development. METHODS To identify differentially expressed genes during mammalian orofacial ontogenesis, the transcript profiles of GD-12, GD-13, and GD-14 murine orofacial tissue were compared utilizing GeneChip arrays from Affymetrix. Changes in gene expression were verified by TaqMan quantitative real-time PCR. Cluster analysis of the microarray data was done with the GeneCluster 2.0 Data Mining Tool and the GeneSpring software. RESULTS Expression of >50% of the ,12,000 genes and expressed sequence tags examined in this study was detected in GD-12, GD-13, and GD-14 murine orofacial tissues and the expression of several hundred genes was up- and downregulated in the developing orofacial tissue from GD-12 to GD-13, as well as from GD-13 to GD-14. Such differential gene expression represents changes in the expression of genes encoding growth factors and signaling molecules; transcription factors; and proteins involved in epithelial-mesenchymal interactions, extracellular matrix synthesis, cell adhesion, proliferation, differentiation, and apoptosis. Following cluster analysis of the microarray data, eight distinct patterns of gene expression during murine orofacial ontogenesis were selected for graphic presentation of gene expression patterns. CONCLUSIONS This gene expression profiling study identifies a number of potentially unique developmental participants and serves as a valuable aid in deciphering the complex molecular mechanisms crucial for mammalian orofacial development. Supplementary material for this article can be found at http://www.mrw.interscience.wiley.com/suppmat/1542-0752/suppmat/2004/70/v70.mukhopadhyay.html Birth Defects Research (Part A), 2004. © 2004 Wiley-Liss, Inc. [source] | |||||||||||||