Influence of tissue- and cell-scale extracellular matrix distribution on the mechanical properties of tissue engineered cartilage

NARCIS (Netherlands)

Khoshgoftar, M.; Wilson, W.; Ito, K.; Donkelaar, van C.C.

2013-01-01

The insufficient load-bearing capacity of today’s tissue- engineered (TE) cartilage limits its clinical application. Generally, cartilage TE studies aim to increase the extracellular matrix (ECM) content, as this is thought to determine the load-bearing properties of the cartilage. However, there

Evaluation of viability and proliferative activity of human urothelial cells cultured onto xenogenic tissue-engineered extracellular matrices.

LENUS (Irish Health Repository)

Davis, Niall F

2011-04-01

To evaluate the viability and proliferative activity of human urothelial cells (HUCs) cultured on tissue-engineered extracellular matrix scaffolds and to assess the potential of extracellular matrixes to support the growth of HUCs in their expected in vivo urine environment.

Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells.

Science.gov (United States)

Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

2015-10-15

Collagen is a widely used naturally occurring biomaterial for scaffolding, whereas mesenchymal stem cells (MSCs) represent a promising cell source in tissue engineering and regenerative medicine. It is generally known that cells are able to remodel their environment by simultaneous degradation of the scaffolds and deposition of newly synthesized extracellular matrix. Nevertheless, the interactions between MSCs and collagen biomaterials are poorly known, and the strategies enhancing the extracellular matrix deposition are yet to be defined. In this study, we aim to investigate the fate of collagen when it is in contact with MSCs and hypothesize that protease inhibition will enhance their extracellular deposition of collagen fibrils. Specifically, human MSCs (hMSCs) were exposed to fluorescence-labeled collagen with and without intracellular or extracellular protease inhibitors (or both) before tracing the collagen at both intracellular and extracellular spaces. Collagen were internalized by hMSCs and degraded intracellularly in lysosomes. In the presence of protease inhibitors, both intracellular collagen fibril growth and extracellular deposition of collagen fibrils were enhanced. Moreover, protease inhibitors work synergistically with ascorbic acid, a well-known matrix deposition-enhancing reagent, in further enhancing collagen fibril deposition at the extracellular space. These findings provide a better understanding of the interactions between hMSCs and collagen biomaterials and suggest a method to manipulate matrix remodeling and deposition of hMSCs, contributing to better scaffolding for tissue engineering and regenerative medicine.

Simple and high yielding method for preparing tissue specific extracellular matrix coatings for cell culture.

Science.gov (United States)

DeQuach, Jessica A; Mezzano, Valeria; Miglani, Amar; Lange, Stephan; Keller, Gordon M; Sheikh, Farah; Christman, Karen L

2010-09-27

The native extracellular matrix (ECM) consists of a highly complex, tissue-specific network of proteins and polysaccharides, which help regulate many cellular functions. Despite the complex nature of the ECM, in vitro cell-based studies traditionally assess cell behavior on single ECM component substrates, which do not adequately mimic the in vivo extracellular milieu. We present a simple approach for developing naturally derived ECM coatings for cell culture that provide important tissue-specific cues unlike traditional cell culture coatings, thereby enabling the maturation of committed C2C12 skeletal myoblast progenitors and human embryonic stem cells differentiated into cardiomyocytes. Here we show that natural muscle-specific coatings can (i) be derived from decellularized, solubilized adult porcine muscle, (ii) contain a complex mixture of ECM components including polysaccharides, (iii) adsorb onto tissue culture plastic and (iv) promote cell maturation of committed muscle progenitor and stem cells. This versatile method can create tissue-specific ECM coatings, which offer a promising platform for cell culture to more closely mimic the mature in vivo ECM microenvironment.

Tissue-Derived Extracellular Matrix Bioscaffolds: Emerging Applications in Cartilage and Meniscus Repair.

Science.gov (United States)

Monibi, Farrah A; Cook, James L

2017-08-01

Musculoskeletal injuries are a common problem in orthopedic practice. Given the long-term consequences of unaddressed cartilage and meniscal pathology, a number of treatments have been attempted to stimulate repair or to replace the injured tissue. Despite advances in orthopedic surgery, effective treatments for cartilage and meniscus injuries remain a significant clinical challenge. Tissue engineering is a developing field that aims to regenerate injured tissues with a combination of cells, scaffolds, and signals. Many natural and synthetic scaffold materials have been developed and tested for the repair and restoration of a number of musculoskeletal tissues. Among these, biological scaffolds derived from cell and tissue-derived extracellular matrix (ECM) have shown great promise in tissue engineering given the critical role of the ECM for maintaining the biological and biomechanical properties, structure, and function of native tissues. This review article presents emerging applications for tissue-derived ECM scaffolds in cartilage and meniscus repair. We examine normal ECM composition and the current and future methods for potential treatment of articular cartilage and meniscal defects with decellularized scaffolds.

Mechanical phenotyping of cells and extracellular matrix as grade and stage markers of lung tumor tissues.

Science.gov (United States)

Panzetta, Valeria; Musella, Ida; Rapa, Ida; Volante, Marco; Netti, Paolo A; Fusco, Sabato

2017-07-15

The mechanical cross-talk between cells and the extra-cellular matrix (ECM) regulates the properties, functions and healthiness of the tissues. When this is disturbed it changes the mechanical state of the tissue components, singularly or together, and cancer, along with other diseases, may start and progress. However, the bi-univocal mechanical interplay between cells and the ECM is still not properly understood. In this study we show how a microrheology technique gives us the opportunity to evaluate the mechanics of cells and the ECM at the same time. The mechanical phenotyping was performed on the surgically removed tissues of 10 patients affected by adenocarcinoma of the lung. A correlation between the mechanics and the grade and stage of the tumor was reported and compared to the mechanical characteristics of the healthy tissue. Our findings suggest a sort of asymmetric modification of the mechanical properties of the cells and the extra-cellular matrix in the tumor, being the more compliant cell even though it resides in a stiffer matrix. Overall, the simultaneous mechanical characterization of the tissues constituents (cells and ECM) provided new support for diagnosis and offered alternative points of analysis for cancer mechanobiology. When the integrity of the mechanical cross-talk between cells and the extra-cellular matrix is disturbed cancer, along with other diseases, may initiate and progress. Here, we show how a new technique gives the opportunity to evaluate the mechanics of cells and the ECM at the same time. It was applied on surgically removed tissues of 10 patients affected by adenocarcinoma of the lung and a correlation between the mechanics and the grade and stage of the tumor was reported and compared to the mechanical characteristics of the healthy tissue. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Basic components of connective tissues and extracellular matrix: elastin, fibrillin, fibulins, fibrinogen, fibronectin, laminin, tenascins and thrombospondins.

Science.gov (United States)

Halper, Jaroslava; Kjaer, Michael

2014-01-01

Collagens are the most abundant components of the extracellular matrix and many types of soft tissues. Elastin is another major component of certain soft tissues, such as arterial walls and ligaments. Many other molecules, though lower in quantity, function as essential components of the extracellular matrix in soft tissues. Some of these are reviewed in this chapter. Besides their basic structure, biochemistry and physiology, their roles in disorders of soft tissues are discussed only briefly as most chapters in this volume deal with relevant individual compounds. Fibronectin with its muldomain structure plays a role of "master organizer" in matrix assembly as it forms a bridge between cell surface receptors, e.g., integrins, and compounds such collagen, proteoglycans and other focal adhesion molecules. It also plays an essential role in the assembly of fibrillin-1 into a structured network. Laminins contribute to the structure of the extracellular matrix (ECM) and modulate cellular functions such as adhesion, differentiation, migration, stability of phenotype, and resistance towards apoptosis. Though the primary role of fibrinogen is in clot formation, after conversion to fibrin by thrombin, it also binds to a variety of compounds, particularly to various growth factors, and as such fibrinogen is a player in cardiovascular and extracellular matrix physiology. Elastin, an insoluble polymer of the monomeric soluble precursor tropoelastin, is the main component of elastic fibers in matrix tissue where it provides elastic recoil and resilience to a variety of connective tissues, e.g., aorta and ligaments. Elastic fibers regulate activity of TGFβs through their association with fibrillin microfibrils. Elastin also plays a role in cell adhesion, cell migration, and has the ability to participate in cell signaling. Mutations in the elastin gene lead to cutis laxa. Fibrillins represent the predominant core of the microfibrils in elastic as well as non

Extracellular histones in tissue injury and inflammation.

Science.gov (United States)

Allam, Ramanjaneyulu; Kumar, Santhosh V R; Darisipudi, Murthy N; Anders, Hans-Joachim

2014-05-01

Neutrophil NETosis is an important element of host defense as it catapults chromatin out of the cell to trap bacteria, which then are killed, e.g., by the chromatin's histone component. Also, during sterile inflammation TNF-alpha and other mediators trigger NETosis, which elicits cytotoxic effects on host cells. The same mechanism should apply to other forms of regulated necrosis including pyroptosis, necroptosis, ferroptosis, and cyclophilin D-mediated regulated necrosis. Beyond these toxic effects, extracellular histones also trigger thrombus formation and innate immunity by activating Toll-like receptors and the NLRP3 inflammasome. Thereby, extracellular histones contribute to the microvascular complications of sepsis, major trauma, small vessel vasculitis as well as acute liver, kidney, brain, and lung injury. Finally, histones prevent the degradation of extracellular DNA, which promotes autoimmunization, anti-nuclear antibody formation, and autoimmunity in susceptible individuals. Here, we review the current evidence on the pathogenic role of extracellular histones in disease and discuss how to target extracellular histones to improve disease outcomes.

hMSCs Cultured on Plant-Derived Tissue Engineering Extracellular Matrix in a Microgravity Environment

Data.gov (United States)

National Aeronautics and Space Administration — The objective of this proposal is to fabricate an all plant-derived renewable, biodegradable complete mimic of the bone extracellular matrix (ECM). For the first...

Extracellular matrix of dental pulp stem cells: Applications in pulp tissue engineering using somatic MSCs

Directory of Open Access Journals (Sweden)

Sriram eRavindran

2014-01-01

Full Text Available Dental Caries affects approximately 90% of the world’s population. At present, the clinical treatment for dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality. Tissue engineering can potentially solve this problem by enabling regeneration of a functional pulp tissue. Dental pulp stem cells (DPSCs have been shown to be an excellent source for pulp regeneration. However, limited availability of these cells hinders its potential for clinical translation. We have investigated the possibility of using somatic mesenchymal stem cells from other sources for dental pulp tissue regeneration using a biomimetic dental pulp extracellular matrix (ECM incorporated scaffold. Human periodontal ligament stem cells (PDLSCs and human bone marrow stromal cells (HMSCs were investigated for their ability to differentiate towards an odontogenic lineage. In vitro real-time PCR results coupled with histological and immunohistochemical examination of the explanted tissues confirmed the ability of PDLSCs and HMSCs to form a vascularized pulp-like tissue. These findings indicate that the dental pulp stem derived ECM scaffold stimulated odontogenic differentiation of PDLSCs and HMSCs without the need for exogenous addition of growth and differentiation factors. This study represents a translational perspective toward possible therapeutic application of using a combination of somatic stem cells and extracellular matrix for pulp regeneration.

Proteoglycan changes in the extracellular matrix of lung tissue from patients with pulmonary emphysema

NARCIS (Netherlands)

van Straaten, JFM; Coers, W; Noordhoek, JA; Flipsen, JTM; Kauffman, HF; Timens, W; Postma, DS

To characterize the changes in the extracellular matrix in smoking-related pulmonary emphysema, we undertook immunohistochemical studies in lung tissues from controls (n = 7), from patients with mild (n = 11) and severe (n = 8) emphysema, and from patients with lung fibrosis (n = 6). We studied

Abundant extracellular myelin in the meninges of patients with multiple sclerosis.

Science.gov (United States)

Kooi, E-J; van Horssen, J; Witte, M E; Amor, S; Bø, L; Dijkstra, C D; van der Valk, P; Geurts, J J G

2009-06-01

In multiple sclerosis (MS) myelin debris has been observed within MS lesions, in cerebrospinal fluid and cervical lymph nodes, but the route of myelin transport out of the brain is unknown. Drainage of interstitial fluid from the brain parenchyma involves the perivascular spaces and leptomeninges, but the presence of myelin debris in these compartments has not been described. To determine whether myelin products are present in the meninges and perivascular spaces of MS patients. Formalin-fixed brain tissue containing meninges from 29 MS patients, 9 non-neurological controls, 6 Alzheimer's disease, 5 stroke, 5 meningitis and 7 leucodystrophy patients was investigated, and immunohistochemically stained for several myelin proteins [proteolipid protein (PLP), myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)]. On brain material from MS patients and (non)neurological controls, PLP immunostaining was used to systematically investigate the presence of myelin debris in the meninges, using a semiquantitative scale. Extensive extracellular presence of myelin particles, positive for PLP, MBP, MOG and CNPase in the leptomeninges of MS patients, was observed. Myelin particles were also observed in perivascular spaces of MS patients. Immunohistochemical double-labelling for macrophage and dendritic cell markers and PLP confirmed that the vast majority of myelin particles were located extracellularly. Extracellular myelin particles were virtually absent in meningeal tissue of non-neurological controls, Alzheimer's disease, stroke, meningitis and leucodystrophy cases. In MS leptomeninges and perivascular spaces, abundant extracellular myelin can be found, whereas this is not the case for controls and other neurological disease. This may be relevant for understanding sustained immunogenicity or, alternatively, tolerogenicity in MS.

Single-nanotube tracking reveals the nanoscale organization of the extracellular space in the live brain

Science.gov (United States)

Godin, Antoine G.; Varela, Juan A.; Gao, Zhenghong; Danné, Noémie; Dupuis, Julien P.; Lounis, Brahim; Groc, Laurent; Cognet, Laurent

2017-03-01

The brain is a dynamic structure with the extracellular space (ECS) taking up almost a quarter of its volume. Signalling molecules, neurotransmitters and nutrients transit via the ECS, which constitutes a key microenvironment for cellular communication and the clearance of toxic metabolites. The spatial organization of the ECS varies during sleep, development and aging and is probably altered in neuropsychiatric and degenerative diseases, as inferred from electron microscopy and macroscopic biophysical investigations. Here we show an approach to directly observe the local ECS structures and rheology in brain tissue using super-resolution imaging. We inject single-walled carbon nanotubes into rat cerebroventricles and follow the near-infrared emission of individual nanotubes as they diffuse inside the ECS for tens of minutes in acute slices. Because of the interplay between the nanotube geometry and the ECS local environment, we can extract information about the dimensions and local viscosity of the ECS. We find a striking diversity of ECS dimensions down to 40 nm, and as well as of local viscosity values. Moreover, by chemically altering the extracellular matrix of the brains of live animals before nanotube injection, we reveal that the rheological properties of the ECS are affected, but these alterations are local and inhomogeneous at the nanoscale.

Neutrophil Extracellular Traps in Ulcerative Colitis

DEFF Research Database (Denmark)

Bjerg Bennike, Tue; Carlsen, Thomas Gelsing; Ellingsen, Torkell

2015-01-01

microscopy and confocal microscopy. RESULTS: We identified and quantified 5711 different proteins with proteomics. The abundance of the proteins calprotectin and lactotransferrin in the tissue correlated with the degree of tissue inflammation as determined by histology. However, fecal calprotectin did...... not correlate. Forty-six proteins were measured with a statistically significant differences in abundances between the UC colon tissue and controls. Eleven of the proteins with increased abundances in the UC biopsies were associated with neutrophils and neutrophil extracellular traps. The findings were...... validated by microscopy, where an increased abundance of neutrophils and the presence of neutrophil extracellular traps by extracellular DNA present in the UC colon tissue were confirmed. CONCLUSIONS: Neutrophils, induced neutrophil extracellular traps, and several proteins that play a part in innate...

Volume transmission in health and disease: communication via the brain extracellular space

Czech Academy of Sciences Publication Activity Database

Syková, Eva

2003-01-01

Roč. 85, č. 2 (2003), s. 2 ISSN 0022-3042. [Meeting of the European Society for Neurochemistry /14./. Varšava, 01.06.2003-04.06.2003] Institutional research plan: CEZ:AV0Z5039906 Keywords : volume transmission * extracellular space Subject RIV: FH - Neurology Impact factor: 4.825, year: 2003

Extracellular vesicles, tissue factor, cancer and thrombosis – discussion themes of the ISEV 2014 Educational Day

Directory of Open Access Journals (Sweden)

Chris Gardiner

2015-03-01

Full Text Available Although the association between cancer and venous thromboembolism (VTE has long been known, the mechanisms are poorly understood. Circulating tissue factor–bearing extracellular vesicles have been proposed as a possible explanation for the increased risk of VTE observed in some types of cancer. The International Society for Extracellular Vesicles (ISEV and International Society on Thrombosis and Haemostasis (ISTH held a joint Educational Day in April 2014 to discuss the latest developments in this field. This review discusses the themes of that event and the ISEV 2014 meeting that followed.

Collagenous Extracellular Matrix Biomaterials for Tissue Engineering: Lessons from the Common Sea Urchin Tissue

Science.gov (United States)

Goh, Kheng Lim; Holmes, David F.

2017-01-01

Scaffolds for tissue engineering application may be made from a collagenous extracellular matrix (ECM) of connective tissues because the ECM can mimic the functions of the target tissue. The primary sources of collagenous ECM material are calf skin and bone. However, these sources are associated with the risk of having bovine spongiform encephalopathy or transmissible spongiform encephalopathy. Alternative sources for collagenous ECM materials may be derived from livestock, e.g., pigs, and from marine animals, e.g., sea urchins. Collagenous ECM of the sea urchin possesses structural features and mechanical properties that are similar to those of mammalian ones. However, even more intriguing is that some tissues such as the ligamentous catch apparatus can exhibit mutability, namely rapid reversible changes in the tissue mechanical properties. These tissues are known as mutable collagenous tissues (MCTs). The mutability of these tissues has been the subject of on-going investigations, covering the biochemistry, structural biology and mechanical properties of the collagenous components. Recent studies point to a nerve-control system for regulating the ECM macromolecules that are involved in the sliding action of collagen fibrils in the MCT. This review discusses the key attributes of the structure and function of the ECM of the sea urchin ligaments that are related to the fibril-fibril sliding action—the focus is on the respective components within the hierarchical architecture of the tissue. In this context, structure refers to size, shape and separation distance of the ECM components while function is associated with mechanical properties e.g., strength and stiffness. For simplicity, the components that address the different length scale from the largest to the smallest are as follows: collagen fibres, collagen fibrils, interfibrillar matrix and collagen molecules. Application of recent theories of stress transfer and fracture mechanisms in fibre reinforced

Collagenous Extracellular Matrix Biomaterials for Tissue Engineering: Lessons from the Common Sea Urchin Tissue.

Science.gov (United States)

Goh, Kheng Lim; Holmes, David F

2017-04-25

Scaffolds for tissue engineering application may be made from a collagenous extracellular matrix (ECM) of connective tissues because the ECM can mimic the functions of the target tissue. The primary sources of collagenous ECM material are calf skin and bone. However, these sources are associated with the risk of having bovine spongiform encephalopathy or transmissible spongiform encephalopathy. Alternative sources for collagenous ECM materials may be derived from livestock, e.g., pigs, and from marine animals, e.g., sea urchins. Collagenous ECM of the sea urchin possesses structural features and mechanical properties that are similar to those of mammalian ones. However, even more intriguing is that some tissues such as the ligamentous catch apparatus can exhibit mutability, namely rapid reversible changes in the tissue mechanical properties. These tissues are known as mutable collagenous tissues (MCTs). The mutability of these tissues has been the subject of on-going investigations, covering the biochemistry, structural biology and mechanical properties of the collagenous components. Recent studies point to a nerve-control system for regulating the ECM macromolecules that are involved in the sliding action of collagen fibrils in the MCT. This review discusses the key attributes of the structure and function of the ECM of the sea urchin ligaments that are related to the fibril-fibril sliding action-the focus is on the respective components within the hierarchical architecture of the tissue. In this context, structure refers to size, shape and separation distance of the ECM components while function is associated with mechanical properties e.g., strength and stiffness. For simplicity, the components that address the different length scale from the largest to the smallest are as follows: collagen fibres, collagen fibrils, interfibrillar matrix and collagen molecules. Application of recent theories of stress transfer and fracture mechanisms in fibre reinforced

Collagenous Extracellular Matrix Biomaterials for Tissue Engineering: Lessons from the Common Sea Urchin Tissue

Directory of Open Access Journals (Sweden)

Kheng Lim Goh

2017-04-01

Full Text Available Scaffolds for tissue engineering application may be made from a collagenous extracellular matrix (ECM of connective tissues because the ECM can mimic the functions of the target tissue. The primary sources of collagenous ECM material are calf skin and bone. However, these sources are associated with the risk of having bovine spongiform encephalopathy or transmissible spongiform encephalopathy. Alternative sources for collagenous ECM materials may be derived from livestock, e.g., pigs, and from marine animals, e.g., sea urchins. Collagenous ECM of the sea urchin possesses structural features and mechanical properties that are similar to those of mammalian ones. However, even more intriguing is that some tissues such as the ligamentous catch apparatus can exhibit mutability, namely rapid reversible changes in the tissue mechanical properties. These tissues are known as mutable collagenous tissues (MCTs. The mutability of these tissues has been the subject of on-going investigations, covering the biochemistry, structural biology and mechanical properties of the collagenous components. Recent studies point to a nerve-control system for regulating the ECM macromolecules that are involved in the sliding action of collagen fibrils in the MCT. This review discusses the key attributes of the structure and function of the ECM of the sea urchin ligaments that are related to the fibril-fibril sliding action—the focus is on the respective components within the hierarchical architecture of the tissue. In this context, structure refers to size, shape and separation distance of the ECM components while function is associated with mechanical properties e.g., strength and stiffness. For simplicity, the components that address the different length scale from the largest to the smallest are as follows: collagen fibres, collagen fibrils, interfibrillar matrix and collagen molecules. Application of recent theories of stress transfer and fracture mechanisms in fibre

Influence of the temporal deposition of extracellular matrix on the mechanical properties of tissue-engineered cartilage

NARCIS (Netherlands)

Khoshgoftar, M.; Wilson, W.; Ito, K.; Donkelaar, van C.C.

2014-01-01

Enhancement of the load-bearing capacity of tissue engineered (TE) cartilage is expected to improve the clinical outcome of implantations. Generally, cartilage TE studies aim to increase the total extracellular matrix (ECM) content to improve implant mechanical properties. Besides the ECM content,

Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering.

Science.gov (United States)

El-Amin, S F; Lu, H H; Khan, Y; Burems, J; Mitchell, J; Tuan, R S; Laurencin, C T

2003-03-01

The nature of the extracellular matrix (ECM) is crucial in regulating cell functions via cell-matrix interactions, cytoskeletal organization, and integrin-mediated signaling. In bone, the ECM is composed of proteins such as collagen (CO), fibronectin (FN), laminin (LM), vitronectin (VN), osteopontin (OP) and osteonectin (ON). For bone tissue engineering, the ECM should also be considered in terms of its function in mediating cell adhesion to biomaterials. This study examined ECM production, cytoskeletal organization, and adhesion of primary human osteoblastic cells on biodegradable matrices applicable for tissue engineering, namely polylactic-co-glycolic acid 50:50 (PLAGA) and polylactic acid (PLA). We hypothesized that the osteocompatible, biodegradable polymer surfaces promote the production of bone-specific ECM proteins in a manner dependent on polymer composition. We first examined whether the PLAGA and PLA matrices could support human osteoblastic cell growth by measuring cell adhesion at 3, 6 and 12h post-plating. Adhesion on PLAGA was consistently higher than on PLA throughout the duration of the experiment, and comparable to tissue culture polystyrene (TCPS). ECM components, including CO, FN, LM, ON, OP and VN, produced on the surface of the polymers were quantified by ELISA and localized by immunofluorescence staining. All of these proteins were present at significantly higher levels on PLAGA compared to PLA or TCPS surfaces. On PLAGA, OP and ON were the most abundant ECM components, followed by CO, FN, VN and LN. Immunofluorescence revealed an extracellular distribution for CO and FN, whereas OP and ON were found both intracellularly as well as extracellularly on the polymer. In addition, the actin cytoskeletal network was more extensive in osteoblasts cultured on PLAGA than on PLA or TCPS. In summary, we found that osteoblasts plated on PLAGA adhered better to the substrate, produced higher levels of ECM molecules, and showed greater cytoskeletal

Protein Dynamics in the Plant Extracellular Space

Directory of Open Access Journals (Sweden)

Leonor Guerra-Guimarães

2016-07-01

Full Text Available The extracellular space (ECS or apoplast is the plant cell compartment external to the plasma membrane, which includes the cell walls, the intercellular space and the apoplastic fluid (APF. The present review is focused on APF proteomics papers and intends to draw information on the metabolic processes occurring in the ECS under abiotic and biotic stresses, as well as under non-challenged conditions. The large majority of the proteins detected are involved in “cell wall organization and biogenesis”, “response to stimulus” and “protein metabolism”. It becomes apparent that some proteins are always detected, irrespective of the experimental conditions, although with different relative contribution. This fact suggests that non-challenged plants have intrinsic constitutive metabolic processes of stress/defense in the ECS. In addition to the multiple functions ascribed to the ECS proteins, should be considered the interactions established between themselves and with the plasma membrane and its components. These interactions are crucial in connecting exterior and interior of the cell, and even simple protein actions in the ECS can have profound effects on plant performance. The proteins of the ECS are permanently contributing to the high dynamic nature of this plant compartment, which seems fundamental to plant development and adaptation to the environmental conditions.

A Model of Effective Diffusion and Tortuosity in the Extracellular Space of the Brain

Czech Academy of Sciences Publication Activity Database

Hrabě, J.; Hrabětová, S.; Segeth, Karel

2004-01-01

Roč. 87, - (2004), s. 1606-1617 ISSN 0006-3495 R&D Projects: GA ČR GA201/04/1503 Institutional research plan: CEZ:AV0Z1019905 Keywords : extracellular space * tortuosity * diffusion Subject RIV: BA - General Mathematics Impact factor: 4.585, year: 2004

Basic Components of Vascular Connective Tissue and Extracellular Matrix.

Science.gov (United States)

Halper, Jaroslava

2018-01-01

Though the composition of the three layers constituting the blood vessel wall varies among the different types of blood vessels, and some layers may even be missing in capillaries, certain basic components, and properties are shared by all blood vessels, though each histologically distinct layer contains a unique complement of extracellular components, growth factors and cytokines, and cell types as well. The structure and composition of vessel layers informs and is informed by the function of the particular blood vessel. The adaptation of the composition and the resulting function of the extracellular matrix (ECM) to changes in circulation/blood flow and a variety of other extravascular stimuli can be characterized as remodeling spearheaded by vascular cells. There is a surprising amount of cell traffic among the three layers. It starts with endothelial cell mediated transmigration of inflammatory cells from the bloodstream into the subendothelium, and then into tissue adjoining the blood vessel. Smooth muscle cells and a variety of adventitial cells reside in tunica media and tunica externa, respectively. The latter cells are a mixture of progenitor/stem cells, fibroblasts, myofibroblasts, pericytes, macrophages, and dendritic cells and respond to endothelial injury by transdifferentiation as they travel into the two inner layers, intima and media for corrective mission in the ECM composition. This chapter addresses the role of various vascular cell types and ECM components synthesized by them in maintenance of normal structure and in their contribution to major pathological processes, such as atherosclerosis, organ fibrosis, and diabetic retinopathy. © 2018 Elsevier Inc. All rights reserved.

Functional characterization of detergent-decellularized equine tendon extracellular matrix for tissue engineering applications.

Directory of Open Access Journals (Sweden)

Daniel W Youngstrom

Full Text Available Natural extracellular matrix provides a number of distinct advantages for engineering replacement orthopedic tissue due to its intrinsic functional properties. The goal of this study was to optimize a biologically derived scaffold for tendon tissue engineering using equine flexor digitorum superficialis tendons. We investigated changes in scaffold composition and ultrastructure in response to several mechanical, detergent and enzymatic decellularization protocols using microscopic techniques and a panel of biochemical assays to evaluate total protein, collagen, glycosaminoglycan, and deoxyribonucleic acid content. Biocompatibility was also assessed with static mesenchymal stem cell (MSC culture. Implementation of a combination of freeze/thaw cycles, incubation in 2% sodium dodecyl sulfate (SDS, trypsinization, treatment with DNase-I, and ethanol sterilization produced a non-cytotoxic biomaterial free of appreciable residual cellular debris with no significant modification of biomechanical properties. These decellularized tendon scaffolds (DTS are suitable for complex tissue engineering applications, as they provide a clean slate for cell culture while maintaining native three-dimensional architecture.

Intra-operative on-line discrimination of kidney cancer from normal tissue by IR ATR spectroscopy of extracellular fluid

Science.gov (United States)

Urboniene, V.; Velicka, M.; Ceponkus, J.; Pucetaite, M.; Jankevicius, F.; Sablinskas, V.; Steiner, G.

2016-03-01

Determination of cancerous and normal kidney tissues during partial, simple or radical nephrectomy surgery was performed by using differences in the IR absorption spectra of extracellular fluid taken from the corresponding tissue areas. The samples were prepared by stamping of the kidney tissue on ATR diamond crystal. The spectral measurements were performed directly in the OR during surgery for 58 patients. It was found that intensities of characteristic spectral bands of glycogen (880-1200 cm-1) in extracellular fluid are sensitive to the type of the tissue and can be used as spectral markers of tumours. Characteristic spectral band of lactic acid (1730 cm-1) - product of the anaerobic glycolysis, taking place in the cancer cells is not suitable for use as a spectral marker of cancerous tissue, since it overlaps with the band of carbonyl stretch in phospholipids and fatty acids. Results of hierarchical cluster analysis of the spectra show that the spectra of healthy and tumour tissue films can be reliably separated into two groups. On the other hand, possibility to differentiate between tumours of different types and grades remains in question. While the fluid from highly malignant G3 tumour tissue contains highly pronounced glycogen spectral bands and can be well separated from benign and G1 tumours by principal component analysis, the variations between spectra from sample to sample prevent from obtaining conclusive results about the grouping between different tumour types and grades. The proposed method is instant and can be used in situ and even in vivo.

Supercritical carbon dioxide extracted extracellular matrix material from adipose tissue

Energy Technology Data Exchange (ETDEWEB)

Wang, Jun Kit; Luo, Baiwen; Guneta, Vipra [School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); Li, Liang; Foo, Selin Ee Min [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Dai, Yun; Tan, Timothy Thatt Yang [School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459 (Singapore); Tan, Nguan Soon [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, 61 Biopolis Drive, Proteos, Singapore 138673 (Singapore); KK Research Centre, KK Women' s and Children' s Hospital, 100 Bukit Timah Road, Singapore 229899 (Singapore); Choong, Cleo, E-mail: cleochoong@ntu.edu.sg [School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); KK Research Centre, KK Women' s and Children' s Hospital, 100 Bukit Timah Road, Singapore 229899 (Singapore); Wong, Marcus Thien Chong [Plastic, Reconstructive & Aesthetic Surgery, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore 308433 (Singapore)

2017-06-01

Adipose tissue is a rich source of extracellular matrix (ECM) material that can be isolated by delipidating and decellularizing the tissue. However, the current delipidation and decellularization methods either involve tedious and lengthy processes or require toxic chemicals, which may result in the elimination of vital proteins and growth factors found in the ECM. Hence, an alternative delipidation and decellularization method for adipose tissue was developed using supercritical carbon dioxide (SC-CO{sub 2}) that eliminates the need of any harsh chemicals and also reduces the amount of processing time required. The resultant SC-CO{sub 2}-treated ECM material showed an absence of nuclear content but the preservation of key proteins such as collagen Type I, collagen Type III, collagen Type IV, elastin, fibronectin and laminin. In addition, other biological factors such as glycosaminoglycans (GAGs) and growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were also retained. Subsequently, the resulting SC-CO{sub 2}-treated ECM material was used as a bioactive coating on tissue culture plastic (TCP). Four different cell types including adipose tissue-derived mesenchymal stem cells (ASCs), human umbilical vein endothelial cells (HUVECs), immortalized human keratinocyte (HaCaT) cells and human monocytic leukemia cells (THP-1) were used in this study to show that the SC-CO{sub 2}-treated ECM coating can be potentially used for various biomedical applications. The SC-CO{sub 2}-treated ECM material showed improved cell-material interactions for all cell types tested. In addition, in vitro scratch wound assay using HaCaT cells showed that the presence of SC-CO{sub 2}-treated ECM material enhanced keratinocyte migration whilst the in vitro cellular studies using THP-1-derived macrophages showed that the SC-CO{sub 2}-treated ECM material did not evoke pro-inflammatory responses from the THP-1-derived macrophages. Overall

Supercritical carbon dioxide extracted extracellular matrix material from adipose tissue.

Science.gov (United States)

Wang, Jun Kit; Luo, Baiwen; Guneta, Vipra; Li, Liang; Foo, Selin Ee Min; Dai, Yun; Tan, Timothy Thatt Yang; Tan, Nguan Soon; Choong, Cleo; Wong, Marcus Thien Chong

2017-06-01

Adipose tissue is a rich source of extracellular matrix (ECM) material that can be isolated by delipidating and decellularizing the tissue. However, the current delipidation and decellularization methods either involve tedious and lengthy processes or require toxic chemicals, which may result in the elimination of vital proteins and growth factors found in the ECM. Hence, an alternative delipidation and decellularization method for adipose tissue was developed using supercritical carbon dioxide (SC-CO 2 ) that eliminates the need of any harsh chemicals and also reduces the amount of processing time required. The resultant SC-CO 2 -treated ECM material showed an absence of nuclear content but the preservation of key proteins such as collagen Type I, collagen Type III, collagen Type IV, elastin, fibronectin and laminin. In addition, other biological factors such as glycosaminoglycans (GAGs) and growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were also retained. Subsequently, the resulting SC-CO 2 -treated ECM material was used as a bioactive coating on tissue culture plastic (TCP). Four different cell types including adipose tissue-derived mesenchymal stem cells (ASCs), human umbilical vein endothelial cells (HUVECs), immortalized human keratinocyte (HaCaT) cells and human monocytic leukemia cells (THP-1) were used in this study to show that the SC-CO 2 -treated ECM coating can be potentially used for various biomedical applications. The SC-CO 2 -treated ECM material showed improved cell-material interactions for all cell types tested. In addition, in vitro scratch wound assay using HaCaT cells showed that the presence of SC-CO 2 -treated ECM material enhanced keratinocyte migration whilst the in vitro cellular studies using THP-1-derived macrophages showed that the SC-CO 2 -treated ECM material did not evoke pro-inflammatory responses from the THP-1-derived macrophages. Overall, this study shows the efficacy

Supercritical carbon dioxide extracted extracellular matrix material from adipose tissue

International Nuclear Information System (INIS)

Wang, Jun Kit; Luo, Baiwen; Guneta, Vipra; Li, Liang; Foo, Selin Ee Min; Dai, Yun; Tan, Timothy Thatt Yang; Tan, Nguan Soon; Choong, Cleo; Wong, Marcus Thien Chong

2017-01-01

Adipose tissue is a rich source of extracellular matrix (ECM) material that can be isolated by delipidating and decellularizing the tissue. However, the current delipidation and decellularization methods either involve tedious and lengthy processes or require toxic chemicals, which may result in the elimination of vital proteins and growth factors found in the ECM. Hence, an alternative delipidation and decellularization method for adipose tissue was developed using supercritical carbon dioxide (SC-CO 2 ) that eliminates the need of any harsh chemicals and also reduces the amount of processing time required. The resultant SC-CO 2 -treated ECM material showed an absence of nuclear content but the preservation of key proteins such as collagen Type I, collagen Type III, collagen Type IV, elastin, fibronectin and laminin. In addition, other biological factors such as glycosaminoglycans (GAGs) and growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were also retained. Subsequently, the resulting SC-CO 2 -treated ECM material was used as a bioactive coating on tissue culture plastic (TCP). Four different cell types including adipose tissue-derived mesenchymal stem cells (ASCs), human umbilical vein endothelial cells (HUVECs), immortalized human keratinocyte (HaCaT) cells and human monocytic leukemia cells (THP-1) were used in this study to show that the SC-CO 2 -treated ECM coating can be potentially used for various biomedical applications. The SC-CO 2 -treated ECM material showed improved cell-material interactions for all cell types tested. In addition, in vitro scratch wound assay using HaCaT cells showed that the presence of SC-CO 2 -treated ECM material enhanced keratinocyte migration whilst the in vitro cellular studies using THP-1-derived macrophages showed that the SC-CO 2 -treated ECM material did not evoke pro-inflammatory responses from the THP-1-derived macrophages. Overall, this study shows the efficacy

Human Adipose Tissue Derived Extracellular Matrix and Methylcellulose Hydrogels Augments and Regenerates the Paralyzed Vocal Fold.

Science.gov (United States)

Kim, Dong Wook; Kim, Eun Ji; Kim, Eun Na; Sung, Myung Whun; Kwon, Tack-Kyun; Cho, Yong Woo; Kwon, Seong Keun

2016-01-01

Vocal fold paralysis results from various etiologies and can induce voice changes, swallowing complications, and issues with aspiration. Vocal fold paralysis is typically managed using injection laryngoplasty with fat or synthetic polymers. Injection with autologous fat has shown excellent biocompatibility. However, it has several disadvantages such as unpredictable resorption rate, morbidities associated with liposuction procedure which has to be done in operating room under general anesthesia. Human adipose-derived extracellular matrix (ECM) grafts have been reported to form new adipose tissue and have greater biostability than autologous fat graft. Here, we present an injectable hydrogel that is constructed from adipose tissue derived soluble extracellular matrix (sECM) and methylcellulose (MC) for use in vocal fold augmentation. Human sECM derived from adipose tissue was extracted using two major steps-ECM was isolated from human adipose tissue and was subsequently solubilized. Injectable sECM/MC hydrogels were prepared by blending of sECM and MC. Sustained vocal fold augmentation and symmetric vocal fold vibration were accomplished by the sECM/MC hydrogel in paralyzed vocal fold which were confirmed by laryngoscope, histology and a high-speed imaging system. There were increased number of collagen fibers and fatty granules at the injection site without significant inflammation or fibrosis. Overall, these results indicate that the sECM/MC hydrogel can enhance vocal function in paralyzed vocal folds without early resorption and has potential as a promising material for injection laryngoplasty for stable vocal fold augmentation which can overcome the shortcomings of autologous fat such as unpredictable duration and morbidity associated with the fat harvest.

Silk fibroin based biomimetic artificial extracellular matrix for hepatic tissue engineering applications

International Nuclear Information System (INIS)

Kasoju, Naresh; Bora, Utpal

2012-01-01

Hepatic tissue engineering, which aims to construct artificial liver tissues, requires a suitable extracellular matrix (ECM) for growth and proliferation of metabolically active hepatocytes. The current paper describes the development of a biomimetic artificial ECM, for hepatic tissue engineering applications, by mimicking the architectural features and biochemical composition of native ECM. Electrospinning was chosen as the fabrication technique of choice, while regenerated silk fibroin (RSF) and galactosylated chitosan (GalCS) were chosen as materials of choice. Poly(ethylene oxide) was used as a processing aid. Methodical optimization studies were performed to obtain smooth and continuous nanofibers with homogenous size distribution. Extensive characterization studies were performed to determine its morphological, physical, chemical/structural, thermal and cytotoxicity properties. Subsequently, detailed in vitro hepatocyte compatibility studies were performed using HepG2 cell line. Remarkably, the studies revealed that the growth, viability, metabolic activity and proliferation of hepatocytes were relatively superior on RSF–GalCS scaffold than on pure RSF and pure GalCS. In summary, the electrospun nanofibrous RSF–GalCS scaffold tries to mimic both architectural and biochemical features of native ECM, and hence could be an appropriate scaffold for in vitro engineering of hepatic tissue. However, additional experiments are needed to confirm the superiority in characteristic functionality of hepatocytes growing on RSF–GalCS scaffold in relation to RSF and GalCS scaffolds, and to test its behavior in vivo. (paper)

Extracellular matrix as a driver for lung regeneration.

Science.gov (United States)

Balestrini, Jenna L; Niklason, Laura E

2015-03-01

Extracellular matrix has manifold roles in tissue mechanics, guidance of cellular behavior, developmental biology, and regenerative medicine. Over the past several decades, various pre-clinical and clinical studies have shown that many connective tissues may be replaced and/or regenerated using suitable extracellular matrix scaffolds. More recently, decellularization of lung tissue has shown that gentle removal of cells can leave behind a "footprint" within the matrix that may guide cellular adhesion, differentiation and homing following cellular repopulation. Fundamental issues like understanding matrix composition and micro-mechanics remain difficult to tackle, largely because of a lack of available assays and tools for systematically characterizing intact matrix from tissues and organs. This review will critically examine the role of engineered and native extracellular matrix in tissue and lung regeneration, and provide insights into directions for future research and translation.

Update on controls for isolation and quantification methodology of extracellular vesicles derived from adipose tissue mesenchymal stem cells

NARCIS (Netherlands)

M. Franquesa (Marcella); M.J. Hoogduijn (Martin); E. Ripoll (Elia); F. Luk (Franka); M. Salih (Mahdi); M.G.H. Betjes (Michiel); J. Torras; C.C. Baan (Carla); J. Grinyo (Josep); A. Merino (Ana)

2014-01-01

textabstractThe research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of

Integrins and extracellular matrix in mechanotransduction

Directory of Open Access Journals (Sweden)

Ramage L

2011-12-01

Full Text Available Lindsay RamageQueen’s Medical Research Institute, University of Edinburgh, Edinburgh, UKAbstract: Integrins are a family of cell surface receptors which mediate cell–matrix and cell–cell adhesions. Among other functions they provide an important mechanical link between the cells external and intracellular environments while the adhesions that they form also have critical roles in cellular signal-transduction. Cell–matrix contacts occur at zones in the cell surface where adhesion receptors cluster and when activated the receptors bind to ligands in the extracellular matrix. The extracellular matrix surrounds the cells of tissues and forms the structural support of tissue which is particularly important in connective tissues. Cells attach to the extracellular matrix through specific cell-surface receptors and molecules including integrins and transmembrane proteoglycans. Integrins work alongside other proteins such as cadherins, immunoglobulin superfamily cell adhesion molecules, selectins, and syndecans to mediate cell–cell and cell–matrix interactions and communication. Activation of adhesion receptors triggers the formation of matrix contacts in which bound matrix components, adhesion receptors, and associated intracellular cytoskeletal and signaling molecules form large functional, localized multiprotein complexes. Cell–matrix contacts are important in a variety of different cell and tissue properties including embryonic development, inflammatory responses, wound healing, and adult tissue homeostasis. This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.Keywords: ligand binding, α subunit, ß subunit, focal adhesion, cell differentiation, mechanical loading, cell–matrix interaction

Human Adipose Tissue Derived Extracellular Matrix and Methylcellulose Hydrogels Augments and Regenerates the Paralyzed Vocal Fold.

Directory of Open Access Journals (Sweden)

Dong Wook Kim

Full Text Available Vocal fold paralysis results from various etiologies and can induce voice changes, swallowing complications, and issues with aspiration. Vocal fold paralysis is typically managed using injection laryngoplasty with fat or synthetic polymers. Injection with autologous fat has shown excellent biocompatibility. However, it has several disadvantages such as unpredictable resorption rate, morbidities associated with liposuction procedure which has to be done in operating room under general anesthesia. Human adipose-derived extracellular matrix (ECM grafts have been reported to form new adipose tissue and have greater biostability than autologous fat graft. Here, we present an injectable hydrogel that is constructed from adipose tissue derived soluble extracellular matrix (sECM and methylcellulose (MC for use in vocal fold augmentation. Human sECM derived from adipose tissue was extracted using two major steps-ECM was isolated from human adipose tissue and was subsequently solubilized. Injectable sECM/MC hydrogels were prepared by blending of sECM and MC. Sustained vocal fold augmentation and symmetric vocal fold vibration were accomplished by the sECM/MC hydrogel in paralyzed vocal fold which were confirmed by laryngoscope, histology and a high-speed imaging system. There were increased number of collagen fibers and fatty granules at the injection site without significant inflammation or fibrosis. Overall, these results indicate that the sECM/MC hydrogel can enhance vocal function in paralyzed vocal folds without early resorption and has potential as a promising material for injection laryngoplasty for stable vocal fold augmentation which can overcome the shortcomings of autologous fat such as unpredictable duration and morbidity associated with the fat harvest.

Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

Energy Technology Data Exchange (ETDEWEB)

Matsuzaki, Shinichi [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Ishizuka, Tamotsu, E-mail: tamotsui@showa.gunma-u.ac.jp [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Yamada, Hidenori; Kamide, Yosuke; Hisada, Takeshi; Ichimonji, Isao; Aoki, Haruka; Yatomi, Masakiyo [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Komachi, Mayumi [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Tsurumaki, Hiroaki; Ono, Akihiro; Koga, Yasuhiko [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Dobashi, Kunio [Gunma University Graduate School of Health Sciences, Maebashi 371-8511 (Japan); Mogi, Chihiro; Sato, Koichi; Tomura, Hideaki [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Mori, Masatomo [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan)

2011-10-07

Highlights: {yields} The involvement of extracellular acidification in airway remodeling was investigated. {yields} Extracellular acidification alone induced CTGF production in human ASMCs. {yields} Extracellular acidification enhanced TGF-{beta}-induced CTGF production in human ASMCs. {yields} Proton-sensing receptor OGR1 was involved in acidic pH-stimulated CTGF production. {yields} OGR1 may play an important role in airway remodeling in asthma. -- Abstract: Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-{beta}-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G{sub q/11} protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP{sub 3}) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G{sub q/11} protein and inositol-1,4,5-trisphosphate-induced Ca{sup 2+} mobilization in human ASMCs.

Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

International Nuclear Information System (INIS)

Matsuzaki, Shinichi; Ishizuka, Tamotsu; Yamada, Hidenori; Kamide, Yosuke; Hisada, Takeshi; Ichimonji, Isao; Aoki, Haruka; Yatomi, Masakiyo; Komachi, Mayumi; Tsurumaki, Hiroaki; Ono, Akihiro; Koga, Yasuhiko; Dobashi, Kunio; Mogi, Chihiro; Sato, Koichi; Tomura, Hideaki; Mori, Masatomo; Okajima, Fumikazu

2011-01-01

Highlights: → The involvement of extracellular acidification in airway remodeling was investigated. → Extracellular acidification alone induced CTGF production in human ASMCs. → Extracellular acidification enhanced TGF-β-induced CTGF production in human ASMCs. → Proton-sensing receptor OGR1 was involved in acidic pH-stimulated CTGF production. → OGR1 may play an important role in airway remodeling in asthma. -- Abstract: Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-β-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G q/11 protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP 3 ) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G q/11 protein and inositol-1,4,5-trisphosphate-induced Ca 2+ mobilization in human ASMCs.

Cartilaginous extracellular matrix-modified chitosan hydrogels for cartilage tissue engineering.

Science.gov (United States)

Choi, Bogyu; Kim, Soyon; Lin, Brian; Wu, Benjamin M; Lee, Min

2014-11-26

Cartilaginous extracellular matrix (ECM) components such as type-II collagen (Col II) and chondroitin sulfate (CS) play a crucial role in chondrogenesis. However, direct clinical use of natural Col II or CS as scaffolds for cartilage tissue engineering is limited by their instability and rapid enzymatic degradation. Here, we investigate the incorporation of Col II and CS into injectable chitosan hydrogels designed to gel upon initiation by exposure to visible blue light (VBL) in the presence of riboflavin. Unmodified chitosan hydrogel supported proliferation and deposition of cartilaginous ECM by encapsulated chondrocytes and mesenchymal stem cells. The incorporation of native Col II or CS into chitosan hydrogels further increased chondrogenesis. The incorporation of Col II, in particular, was found to be responsible for the enhanced cellular condensation and chondrogenesis observed in modified hydrogels. This was mediated by integrin α10 binding to Col II, increasing cell-matrix adhesion. These findings demonstrate the potential of cartilage ECM-modified chitosan hydrogels as biomaterials to promote cartilage regeneration.

The pharmacokinetics of the interstitial space in humans

OpenAIRE

Levitt, David G

2003-01-01

Background The pharmacokinetics of extracellular solutes is determined by the blood-tissue exchange kinetics and the volume of distribution in the interstitial space in the different organs. This information can be used to develop a general physiologically based pharmacokinetic (PBPK) model applicable to most extracellular solutes. Methods The human pharmacokinetic literature was surveyed to tabulate the steady state and equilibrium volume of distribution of the solutes mannitol, EDTA, morphi...

Use of UV-irradiated bacteriophage T6 to kill extracellular bacteria in tissue culture infectivity assays

International Nuclear Information System (INIS)

Shaw, D.R.; Maurelli, A.T.; Goguen, J.D.; Straley, S.C.; Curtiss, R. III

1983-01-01

The authors have utilized 'lysis from without' mediated by UV-inactivated bacteriophage T6 to eliminate extracellular bacteria in experiments measuring the internalization, intracellular survival and replication of Yersinia pestis within mouse peritoneal macrophages and of Shigella flexneri within a human intestinal epithelial cell line. The technique described has the following characteristics: (a) bacterial killing is complete within 15 min at 37 0 C, with a >10 3 -fold reduction in colony-forming units (CFU); (b) bacteria within cultured mammalian cells are protected from killing by UV-inactivated T6; (c) the mammalian cells are not observably affected by exposure to UV-inactivated T6. This technique has several advantages over the use of antibiotics to eliminate extracellular bacteria and is potentially widely applicable in studies of the interactions between pathogenic bacteria and host phagocytic cells as well as other target tissues. (Auth.)

Tissue-specific extracellular matrix coatings for the promotion of cell proliferation and maintenance of cell phenotype.

Science.gov (United States)

Zhang, Yuanyuan; He, Yujiang; Bharadwaj, Shantaram; Hammam, Nevin; Carnagey, Kristen; Myers, Regina; Atala, Anthony; Van Dyke, Mark

2009-08-01

Recent studies have shown that extracellular matrix (ECM) substitutes can have a dramatic impact on cell growth, differentiation and function. However, these ECMs are often applied generically and have yet to be developed for specific cell types. In this study, we developed tissue-specific ECM-based coating substrates for skin, skeletal muscle and liver cell cultures. Cellular components were removed from adult skin, skeletal muscle, and liver tissues, and the resulting acellular matrices were homogenized and dissolved. The ECM solutions were used to coat culture dishes. Tissue matched and non-tissue matched cell types were grown on these coatings to assess adhesion, proliferation, maintenance of phenotype and cell function at several time points. Each cell type showed better proliferation and differentiation in cultures containing ECM from their tissue of origin. Although subtle compositional differences in the three ECM types were not investigated in this study, these results suggest that tissue-specific ECMs provide a culture microenvironment that is similar to the in vivo environment when used as coating substrates, and this new culture technique has the potential for use in drug development and the development of cell-based therapies.

ISDoT: in situ decellularization of tissues for high-resolution imaging and proteomic analysis of native extracellular matrix

DEFF Research Database (Denmark)

Mayorca-Guiliani, Alejandro E.; Madsen, Chris D.; Cox, Thomas R.

2017-01-01

The extracellular matrix (ECM) is a master regulator of cellular phenotype and behavior. It has a crucial role in both normal tissue homeostasis and disease pathology. Here we present a fast and efficient approach to enhance the study of ECM composition and structure. Termed in situ...... decellularization of tissues (ISDoT), it allows whole organs to be decellularized, leaving native ECM architecture intact. These three-dimensional decellularized tissues can be studied using high-resolution fluorescence and second harmonic imaging, and can be used for quantitative proteomic interrogation of the ECM....... Our method is superior to other methods tested in its ability to preserve the structural integrity of the ECM, facilitate high-resolution imaging and quantitatively detect ECM proteins. In particular, we performed high-resolution sub-micron imaging of matrix topography in normal tissue and over...

Proteomic Analysis of Human Tendon and Ligament: Solubilization and Analysis of Insoluble Extracellular Matrix in Connective Tissues.

Science.gov (United States)

Sato, Nori; Taniguchi, Takako; Goda, Yuichiro; Kosaka, Hirofumi; Higashino, Kosaku; Sakai, Toshinori; Katoh, Shinsuke; Yasui, Natsuo; Sairyo, Koichi; Taniguchi, Hisaaki

2016-12-02

Connective tissues such as tendon, ligament and cartilage are mostly composed of extracellular matrix (ECM). These tissues are insoluble, mainly due to the highly cross-linked ECM proteins such as collagens. Difficulties obtaining suitable samples for mass spectrometric analysis render the application of modern proteomic technologies difficult. Complete solubilization of them would not only elucidate protein composition of normal tissues but also reveal pathophysiology of pathological tissues. Here we report complete solubilization of human Achilles tendon and yellow ligament, which is achieved by chemical digestion combined with successive protease treatment including elastase. The digestion mixture was subjected to liquid chromatography-mass spectrometry. The low specificity of elastase was overcome by accurate mass analysis achieved using FT-ICR-MS. In addition to the detailed proteome of both tissues, we also quantitatively determine the major protein composition of samples, by measuring peak area of some characteristic peptides detected in tissue samples and in purified proteins. As a result, differences between human Achilles tendon and yellow ligament were elucidated at molecular level.

Effect of Ionic Diffusion on Extracellular Potentials in Neural Tissue.

Directory of Open Access Journals (Sweden)

Geir Halnes

2016-11-01

Full Text Available Recorded potentials in the extracellular space (ECS of the brain is a standard measure of population activity in neural tissue. Computational models that simulate the relationship between the ECS potential and its underlying neurophysiological processes are commonly used in the interpretation of such measurements. Standard methods, such as volume-conductor theory and current-source density theory, assume that diffusion has a negligible effect on the ECS potential, at least in the range of frequencies picked up by most recording systems. This assumption remains to be verified. We here present a hybrid simulation framework that accounts for diffusive effects on the ECS potential. The framework uses (1 the NEURON simulator to compute the activity and ionic output currents from multicompartmental neuron models, and (2 the electrodiffusive Kirchhoff-Nernst-Planck framework to simulate the resulting dynamics of the potential and ion concentrations in the ECS, accounting for the effect of electrical migration as well as diffusion. Using this framework, we explore the effect that ECS diffusion has on the electrical potential surrounding a small population of 10 pyramidal neurons. The neural model was tuned so that simulations over ∼100 seconds of biological time led to shifts in ECS concentrations by a few millimolars, similar to what has been seen in experiments. By comparing simulations where ECS diffusion was absent with simulations where ECS diffusion was included, we made the following key findings: (i ECS diffusion shifted the local potential by up to ∼0.2 mV. (ii The power spectral density (PSD of the diffusion-evoked potential shifts followed a 1/f2 power law. (iii Diffusion effects dominated the PSD of the ECS potential for frequencies up to several hertz. In scenarios with large, but physiologically realistic ECS concentration gradients, diffusion was thus found to affect the ECS potential well within the frequency range picked up in

Delivery of viral vectors to tumor cells: extracellular transport, systemic distribution, and strategies for improvement.

Science.gov (United States)

Wang, Yong; Yuan, Fan

2006-01-01

It is a challenge to deliver therapeutic genes to tumor cells using viral vectors because (i) the size of these vectors are close to or larger than the space between fibers in extracellular matrix and (ii) viral proteins are potentially toxic in normal tissues. In general, gene delivery is hindered by various physiological barriers to virus transport from the site of injection to the nucleus of tumor cells and is limited by normal tissue tolerance of toxicity determined by local concentrations of transgene products and viral proteins. To illustrate the obstacles encountered in the delivery and yet limit the scope of discussion, this review focuses only on extracellular transport in solid tumors and distribution of viral vectors in normal organs after they are injected intravenously or intratumorally. This review also discusses current strategies for improving intratumoral transport and specificity of viral vectors.

Integrated transcriptomic and proteomic analysis of the molecular cargo of extracellular vesicles derived from porcine adipose tissue-derived mesenchymal stem cells

OpenAIRE

Eirin, Alfonso; Zhu, Xiang-Yang; Puranik, Amrutesh S.; Woollard, John R.; Tang, Hui; Dasari, Surendra; Lerman, Amir; van Wijnen, Andre J.; Lerman, Lilach O.

2017-01-01

Background Mesenchymal stromal/stem cell (MSC) transplantation is a promising therapy for tissue regeneration. Extracellular vesicles (EVs) released by MSCs act as their paracrine effectors by delivering proteins and genetic material to recipient cells. To assess how their cargo mediates biological processes that drive their therapeutic effects, we integrated miRNA, mRNA, and protein expression data of EVs from porcine adipose tissue-derived MSCs. Methods Simultaneous expression profiles of m...

The source of net ultrafiltration during hemodialysis is mostly the extracellular space regardless of hydration status.

Science.gov (United States)

Jeong, Hyeonju; Lim, Chae-Wan; Choi, Hye-Min; Oh, Dong-Jin

2016-01-01

Fluid shifts are common in patients undergoing chronic hemodialysis (HD) during the intradialytic periods, as several liters of fluid are removed during ultrafiltration (UF). Some patients have experienced frequent intradialytic hypotension (IDH). However, the characteristics of fluid shifts and which fluid space is affected remain controversial. Therefore, we designed this study to evaluate the fluid spaces most affected by UF and to determine whether hydration status influences the fluid shifts during HD. This was a prospective cohort study of 40 patients undergoing HD. We measured the patient's fluid spaces using a whole-body bioimpedance apparatus to evaluate the changes in the fluid spaces before HD and 1-4 hours of HD and 30 minutes after HD. UF achieved during HD by the 40 patients (age, 60.0 ± 5.2 years; 50% men; 50% of patients with diabetes; body weight, 61.3 ± 10.5 kg) was 2.18 ± 0.78 L (measured fluid overload, 2.15 ± 1.24 L). 1) Mean relative reduction of total body water and extracellular water was reduced from the start to the end of HD. 2) However, mean relative reduction of intracellular water was not reduced from the start to the end of HD. 3) No significant differences in fluid shifts were observed according to hydration status. The source of net UF during HD is mostly the extracellular space regardless of hydration status. Thus, IDH may be related to differences in the interstitial fluid shift to the vascular space. © 2015 International Society for Hemodialysis.

Extracellular space diffusion and extrasynaptic transmission

Czech Academy of Sciences Publication Activity Database

Vargová, Lýdia; Syková, Eva

2008-01-01

Roč. 57, Suppl.3 (2008), S89-S99 ISSN 0862-8408 R&D Projects: GA MŠk 1M0538; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50390512 Keywords : Diffusion * Extracellular volume * Tortuosity Subject RIV: FH - Neurology Impact factor: 1.653, year: 2008

Laminated electrospun nHA/PHB-composite scaffolds mimicking bone extracellular matrix for bone tissue engineering.

Science.gov (United States)

Chen, Zhuoyue; Song, Yue; Zhang, Jing; Liu, Wei; Cui, Jihong; Li, Hongmin; Chen, Fulin

2017-03-01

Electrospinning is an effective means to generate nano- to micro-scale polymer fibers resembling native extracellular matrix for tissue engineering. However, a major problem of electrospun materials is that limited pore size and porosity may prevent adequate cellular infiltration and tissue ingrowth. In this study, we first prepared thin layers of hydroxyapatite nanoparticle (nHA)/poly-hydroxybutyrate (PHB) via electrospinning. We then laminated the nHA/PHB thin layers to obtain a scaffold for cell seeding and bone tissue engineering. The results demonstrated that the laminated scaffold possessed optimized cell-loading capacity. Bone marrow mesenchymal stem cells (MSCs) exhibited better adherence, proliferation and osteogenic phenotypes on nHA/PHB scaffolds than on PHB scaffolds. Thereafter, we seeded MSCs onto nHA/PHB scaffolds to fabricate bone grafts. Histological observation showed osteoid tissue formation throughout the scaffold, with most of the scaffold absorbed in the specimens 2months after implantation, and blood vessels ingrowth into the graft could be observed in the graft. We concluded that electrospun and laminated nanoscaled biocomposite scaffolds hold great therapeutic potential for bone regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

Extracellular Histones Increase Tissue Factor Activity and Enhance Thrombin Generation by Human Blood Monocytes.

Science.gov (United States)

Gould, Travis J; Lysov, Zakhar; Swystun, Laura L; Dwivedi, Dhruva J; Zarychanski, Ryan; Fox-Robichaud, Alison E; Liaw, Patricia C

2016-12-01

Sepsis is characterized by systemic activation of inflammatory and coagulation pathways in response to infection. Recently, it was demonstrated that histones released into the circulation by dying/activated cells may contribute to sepsis pathology. Although the ability of extracellular histones to modulate the procoagulant activities of several cell types has been investigated, the influence of histones on the hemostatic functions of circulating monocytes is unknown. To address this, we investigated the ability of histones to modulate the procoagulant potential of THP-1 cells and peripheral blood monocytes, and examined the effects of plasmas obtained from septic patients to induce a procoagulant phenotype on monocytic cells. Tissue factor (TF) activity assays were performed on histone-treated THP-1 cells and blood monocytes. Exposure of monocytic cells to histones resulted in increases in TF activity, TF antigen, and phosphatidylserine exposure. Histones modulate the procoagulant activity via engagement of Toll-like receptors 2 and 4, and this effect was abrogated with inhibitory antibodies. Increased TF activity of histone-treated cells corresponded to enhanced thrombin generation in plasma determined by calibrated automated thrombography. Finally, TF activity was increased on monocytes exposed to plasma from septic patients, an effect that was attenuated in plasma from patients receiving unfractionated heparin (UFH). Our studies suggest that increased levels of extracellular histones found in sepsis contribute to dysregulated coagulation by increasing TF activity of monocytes. These procoagulant effects can be partially ameliorated in sepsis patients receiving UFH, thereby identifying extracellular histones as a potential therapeutic target for sepsis treatment.

Mechanical influence of tissue culture plates and extracellular matrix on mesenchymal stem cell behavior: A topical review.

Science.gov (United States)

Tatullo, Marco; Marrelli, Massimo; Falisi, Giovanni; Rastelli, Claudio; Palmieri, Francesca; Gargari, Marco; Zavan, Barbara; Paduano, Francesco; Benagiano, Vincenzo

2016-03-01

Tissue engineering applications need a continuous development of new biomaterials able to generate an ideal cell-extracellular matrix interaction. The stem cell fate is regulated by several factors, such as growth factors or transcription factors. The most recent literature has reported several publications able to demonstrate that environmental factors also contribute to the regulation of stem cell behavior, leading to the opinion that the environment plays the major role in the cell differentiation.The interaction between mesenchymal stem cells (MSCs) and extracellular environment has been widely described, and it has a crucial role in regulating the cell phenotype. In our laboratory (Tecnologica Research Institute, Crotone, Italy), we have recently studied how several physical factors influence the distribution and the morphology of MSCs isolated from dental pulp, and how they are able to regulate stem cell differentiation. Mechanical and geometrical factors are only a small part of the environmental factors able to influence stem cell behavior, however, this influence should be properly known: in fact, this assumption must be clearly considered during those studies involving MSCs; furthermore, these interactions should be considered as an important bias that involves an high number of studies on the MSCs, since in worldwide laboratories the scientists mostly use tissue culture plates for their experiments. © The Author(s) 2015.

Mesenchymal stem cells from different murine tissues have differential capacity to metabolize extracellular nucleotides.

Science.gov (United States)

Iser, Isabele C; Bracco, Paula A; Gonçalves, Carlos E I; Zanin, Rafael F; Nardi, Nance B; Lenz, Guido; Battastini, Ana Maria O; Wink, Márcia R

2014-10-01

Mesenchymal stem cells (MSCs) have shown a great potential for cell-based therapy and many different therapeutic purposes. Despite the recent advances in the knowledge of MSCs biology, their biochemical and molecular properties are still poorly defined. Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-5'-nucleotidase (eNT/CD73) are widely expressed enzymes that hydrolyze extracellular nucleotides, generating an important cellular signaling cascade. Currently, studies have evidenced the relationship between the purinergic system and the development, maintenance, and differentiation of stem cells. The objective of this study is to identify the NTPDases and eNT/CD73 and compare the levels of nucleotide hydrolysis on MSCs isolated from different murine tissues (bone marrow, lung, vena cava, kidney, pancreas, spleen, skin, and adipose tissue). MSCs from all tissues investigated expressed the ectoenzymes at different levels. In MSCs from pancreas and adipose tissue, the hydrolysis of triphosphonucleosides was significantly higher when compared to the other cells. The diphosphonucleosides were hydrolyzed at a higher rate by MSC from pancreas when compared to MSC from other tissues. The differential nucleotide hydrolysis activity and enzyme expression in these cells suggests that MSCs play different roles in regulating the purinergic system in these tissues. Overall MSCs are an attractive adult-derived cell population for therapies, however, the fact that ecto-nucleotide metabolism can affect the microenvironment, modulating important events, such as immune response, makes the assessment of this metabolism an important part of the characterization of MSCs to be applied therapeutically. © 2014 Wiley Periodicals, Inc.

Extracellular fluid volume expansion and third space sequestration at the site of small bowel anastomoses.

Science.gov (United States)

Chan, S T; Kapadia, C R; Johnson, A W; Radcliffe, A G; Dudley, H A

1983-01-01

Intestinal surgery is usually associated with the parenteral administration of sodium and water, sometimes in amounts considerably in excess of excretory capacity. We have studied the effect of this situation on the water content of the gut at and 5 cm from a single-layer end-to-end anastomosis in the rabbit. Water content was measured by desiccation. One group of animals (group 1) did not receive intravenous therapy. The second group (group 2) received 5 ml kg-1 h-1 of Hartmann's solution during the operative period and thereafter to a total volume of 200 ml by 48 h. In group 1 there was a 5-10 per cent increase in tissue weight both at the anastomotic site and at 5 cm (P less than 0.01, Mann-Whitney U test) on the first 3 days. Thereafter, water content at the anastomosis persisted, but resolved in normal gut. In group 2 a further 5 per cent increase in weight over group 1 occurred (P less than 0.01), persistent at the anastomotic site over 5 days, though resolving elsewhere after 2 days. Extracellular fluid volume expansion exaggerates an anatomical third space present in the region of an anastomosis. At the suture line, oedema so induced is persistent and could be deleterious.

Tissue Engineering Organs for Space Biology Research

Science.gov (United States)

Vandenburgh, H. H.; Shansky, J.; DelTatto, M.; Lee, P.; Meir, J.

1999-01-01

Long-term manned space flight requires a better understanding of skeletal muscle atrophy resulting from microgravity. Atrophy most likely results from changes at both the systemic level (e.g. decreased circulating growth hormone, increased circulating glucocorticoids) and locally (e.g. decreased myofiber resting tension). Differentiated skeletal myofibers in tissue culture have provided a model system over the last decade for gaining a better understanding of the interactions of exogenous growth factors, endogenous growth factors, and muscle fiber tension in regulating protein turnover rates and muscle cell growth. Tissue engineering these cells into three dimensional bioartificial muscle (BAM) constructs has allowed us to extend their use to Space flight studies for the potential future development of countermeasures.

Laminated electrospun nHA/PHB-composite scaffolds mimicking bone extracellular matrix for bone tissue engineering

Energy Technology Data Exchange (ETDEWEB)

Chen, Zhuoyue [Lab of Tissue Engineering, Faculty of Life Science, Northwest University, 229 TaiBai North Road, Xi' an, Shaanxi Province 710069 (China); Provincial Key Laboratory of Biotechnology of Shaanxi, Northwest University, 229 TaiBai North Road, Xi' an, Shaanxi Province 710069 (China); Song, Yue [Lab of Tissue Engineering, Faculty of Life Science, Northwest University, 229 TaiBai North Road, Xi' an, Shaanxi Province 710069 (China); Zhang, Jing [Lab of Tissue Engineering, Faculty of Life Science, Northwest University, 229 TaiBai North Road, Xi' an, Shaanxi Province 710069 (China); Provincial Key Laboratory of Biotechnology of Shaanxi, Northwest University, 229 TaiBai North Road, Xi' an, Shaanxi Province 710069 (China); Key Laboratory of Resource Biology and Modern Biotechnology in Western China, Ministry of Education, Northwest University, 229 TaiBai North Road, Xi' an, Shaanxi Province, 710069 (China); Liu, Wei [Lab of Tissue Engineering, Faculty of Life Science, Northwest University, 229 TaiBai North Road, Xi' an, Shaanxi Province 710069 (China); Cui, Jihong, E-mail: cjh@nwu.edu.cn [Lab of Tissue Engineering, Faculty of Life Science, Northwest University, 229 TaiBai North Road, Xi' an, Shaanxi Province 710069 (China); Provincial Key Laboratory of Biotechnology of Shaanxi, Northwest University, 229 TaiBai North Road, Xi' an, Shaanxi Province 710069 (China); Key Laboratory of Resource Biology and Modern Biotechnology in Western China, Ministry of Education, Northwest University, 229 TaiBai North Road, Xi' an, Shaanxi Province, 710069 (China); and others

2017-03-01

Electrospinning is an effective means to generate nano- to micro-scale polymer fibers resembling native extracellular matrix for tissue engineering. However, a major problem of electrospun materials is that limited pore size and porosity may prevent adequate cellular infiltration and tissue ingrowth. In this study, we first prepared thin layers of hydroxyapatite nanoparticle (nHA)/poly-hydroxybutyrate (PHB) via electrospinning. We then laminated the nHA/PHB thin layers to obtain a scaffold for cell seeding and bone tissue engineering. The results demonstrated that the laminated scaffold possessed optimized cell-loading capacity. Bone marrow mesenchymal stem cells (MSCs) exhibited better adherence, proliferation and osteogenic phenotypes on nHA/PHB scaffolds than on PHB scaffolds. Thereafter, we seeded MSCs onto nHA/PHB scaffolds to fabricate bone grafts. Histological observation showed osteoid tissue formation throughout the scaffold, with most of the scaffold absorbed in the specimens 2 months after implantation, and blood vessels ingrowth into the graft could be observed in the graft. We concluded that electrospun and laminated nanoscaled biocomposite scaffolds hold great therapeutic potential for bone regeneration. - Highlights: • We laminated the nHA/PHB layers to obtain a scaffold for bone tissue engineering. • The laminated scaffold performed optimized cell-loading capacity. • MSCs exhibited osteogenic phenotypes on the laminated scaffold. • Osteoid tissue formed throughout the laminated scaffold after 2 months in vivo. The laminated bio-composite scaffolds can be applied to bone regeneration.

Laminated electrospun nHA/PHB-composite scaffolds mimicking bone extracellular matrix for bone tissue engineering

International Nuclear Information System (INIS)

Chen, Zhuoyue; Song, Yue; Zhang, Jing; Liu, Wei; Cui, Jihong

2017-01-01

Electrospinning is an effective means to generate nano- to micro-scale polymer fibers resembling native extracellular matrix for tissue engineering. However, a major problem of electrospun materials is that limited pore size and porosity may prevent adequate cellular infiltration and tissue ingrowth. In this study, we first prepared thin layers of hydroxyapatite nanoparticle (nHA)/poly-hydroxybutyrate (PHB) via electrospinning. We then laminated the nHA/PHB thin layers to obtain a scaffold for cell seeding and bone tissue engineering. The results demonstrated that the laminated scaffold possessed optimized cell-loading capacity. Bone marrow mesenchymal stem cells (MSCs) exhibited better adherence, proliferation and osteogenic phenotypes on nHA/PHB scaffolds than on PHB scaffolds. Thereafter, we seeded MSCs onto nHA/PHB scaffolds to fabricate bone grafts. Histological observation showed osteoid tissue formation throughout the scaffold, with most of the scaffold absorbed in the specimens 2 months after implantation, and blood vessels ingrowth into the graft could be observed in the graft. We concluded that electrospun and laminated nanoscaled biocomposite scaffolds hold great therapeutic potential for bone regeneration. - Highlights: • We laminated the nHA/PHB layers to obtain a scaffold for bone tissue engineering. • The laminated scaffold performed optimized cell-loading capacity. • MSCs exhibited osteogenic phenotypes on the laminated scaffold. • Osteoid tissue formed throughout the laminated scaffold after 2 months in vivo. The laminated bio-composite scaffolds can be applied to bone regeneration.

Ectopic mineralization disorders of the extracellular matrix of connective tissue: molecular genetics and pathomechanisms of aberrant calcification.

Science.gov (United States)

Li, Qiaoli; Jiang, Qiujie; Uitto, Jouni

2014-01-01

Ectopic mineralization of connective tissues is a complex process leading to deposition of calcium phosphate complexes in the extracellular matrix, particularly affecting the skin and the arterial blood vessels and common in age-associated disorders. A number of initiating and contributing metabolic and environmental factors are linked to aberrant mineralization in these diseases, making the identification of precise pathomechanistic pathways exceedingly difficult. However, there has been significant recent progress in understanding the ectopic mineralization processes through study of heritable single-gene disorders, which have allowed identification of discrete pathways and contributing factors leading to aberrant connective tissue mineralization. These studies have provided support for the concept of an intricate mineralization/anti-mineralization network present in peripheral connective tissues, providing a perspective to development of pharmacologic approaches to limit the phenotypic consequences of ectopic mineralization. This overview summarizes the current knowledge of ectopic heritable mineralization disorders, with accompanying animal models, focusing on pseudoxanthoma elasticum and generalized arterial calcification of infancy, two autosomal recessive diseases manifesting with extensive connective tissue mineralization in the skin and the cardiovascular system. © 2013.

Extracellular Matrix Hydrogel Promotes Tissue Remodeling, Arteriogenesis, and Perfusion in a Rat Hindlimb Ischemia Model

Directory of Open Access Journals (Sweden)

Jessica L. Ungerleider, BS

2016-01-01

Full Text Available Although surgical and endovascular revascularization can be performed in peripheral arterial disease (PAD, 40% of patients with critical limb ischemia do not have a revascularization option. This study examines the efficacy and mechanisms of action of acellular extracellular matrix-based hydrogels as a potential novel therapy for treating PAD. We tested the efficacy of using a tissue-specific injectable hydrogel derived from decellularized porcine skeletal muscle (SKM and compared this to a new human umbilical cord-derived matrix (hUC hydrogel, which could have greater potential for tissue regeneration because of the younger age of the tissue source. In a rodent hindlimb ischemia model, both hydrogels were injected 1-week post-surgery and perfusion was regularly monitored with laser speckle contrast analysis to 35 days post-injection. There were significant improvements in hindlimb tissue perfusion and perfusion kinetics with both biomaterials. Histologic analysis indicated that the injected hydrogels were biocompatible, and resulted in arteriogenesis, rather than angiogenesis, as well as improved recruitment of skeletal muscle progenitors. Skeletal muscle fiber morphology analysis indicated that the muscle treated with the tissue-specific SKM hydrogel more closely matched healthy tissue morphology. Whole transcriptome analysis indicated that the SKM hydrogel caused a shift in the inflammatory response, decreased cell death, and increased blood vessel and muscle development. These results show the efficacy of an injectable ECM hydrogel alone as a potential therapy for treating patients with PAD. Our results indicate that the SKM hydrogel improved functional outcomes through stimulation of arteriogenesis and muscle progenitor cell recruitment.

Space research on organs and tissues

Science.gov (United States)

Tischler, Marc E.; Morey-Holton, Emily

1993-01-01

Studies in space on various physiological systems have and will continue to provide valuable information on how they adapt to reduced gravitational conditions, and how living in a 1 g (gravity) environment has guided their development. Muscle and bone are the most notable tissues that respond to unweighting caused by lack of gravity. The function of specific muscles and bones relates directly to mechanical loading, so that removal of 'normal forces' in space, or in bedridden patients, causes dramatic loss of tissue mass. The cardiovascular system is also markedly affected by reduced gravity. Adaptation includes decreased blood flow to the lower extremities, thus decreasing the heart output requirement. Return to 1 g is associated with a period of reconditioning due to the deconditioning that occurs in space. Changes in the cardiovascular system are also related to responses of the kidney and certain endocrine (hormone-producing) organs. Changes in respiratory function may also occur, suggesting an effect on the lungs, though this adaptation is poorly understood. The neurovestibular system, including the brain and organs of the inner ear, must adapt to the disorientation caused by lack of gravity. Preliminary findings have been reported for liver. Additionally, endocrine organs responsible for release of hormones such as insulin, growth hormone, glucocorticoids, and thyroid hormone may respond to spaceflight.

Matrilin-2, an extracellular adaptor protein, is needed for the regeneration of muscle, nerve and other tissues

Directory of Open Access Journals (Sweden)

Éva Korpos

2015-01-01

Full Text Available The extracellular matrix (ECM performs essential functions in the differentiation, maintenance and remodeling of tissues during development and regeneration, and it undergoes dynamic changes during remodeling concomitant to alterations in the cell-ECM interactions. Here we discuss recent data addressing the critical role of the widely expressed ECM protein, matrilin-2 (Matn2 in the timely onset of differentiation and regeneration processes in myogenic, neural and other tissues and in tumorigenesis. As a multiadhesion adaptor protein, it interacts with other ECM proteins and integrins. Matn2 promotes neurite outgrowth, Schwann cell migration, neuromuscular junction formation, skeletal muscle and liver regeneration and skin wound healing. Matn2 deposition by myoblasts is crucial for the timely induction of the global switch toward terminal myogenic differentiation during muscle regeneration by affecting transforming growth factor beta/bone morphogenetic protein 7/Smad and other signal transduction pathways. Depending on the type of tissue and the pathomechanism, Matn2 can also promote or suppress tumor growth.

Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

Energy Technology Data Exchange (ETDEWEB)

Alcaraz, Jordi; Xu, Ren; Mori, Hidetoshi; Nelson, Celeste M.; Mroue, Rana; Spencer, Virginia A.; Brownfield, Doug; Radisky, Derek C.; Bustamante, Carlos; Bissell, Mina J.

2008-10-20

In the mammary gland, epithelial cells are embedded in a 'soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for {beta}-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of {beta}-casein expression required both laminin signalling and a 'soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of {beta}-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.

Bioengineering vascularized tissue constructs using an injectable cell-laden enzymatically crosslinked collagen hydrogel derived from dermal extracellular matrix.

Science.gov (United States)

Kuo, Kuan-Chih; Lin, Ruei-Zeng; Tien, Han-Wen; Wu, Pei-Yun; Li, Yen-Cheng; Melero-Martin, Juan M; Chen, Ying-Chieh

2015-11-01

Tissue engineering promises to restore or replace diseased or damaged tissue by creating functional and transplantable artificial tissues. The development of artificial tissues with large dimensions that exceed the diffusion limitation will require nutrients and oxygen to be delivered via perfusion instead of diffusion alone over a short time period. One approach to perfusion is to vascularize engineered tissues, creating a de novo three-dimensional (3D) microvascular network within the tissue construct. This significantly shortens the time of in vivo anastomosis, perfusion and graft integration with the host. In this study, we aimed to develop injectable allogeneic collagen-phenolic hydroxyl (collagen-Ph) hydrogels that are capable of controlling a wide range of physicochemical properties, including stiffness, water absorption and degradability. We tested whether collagen-Ph hydrogels could support the formation of vascularized engineered tissue graft by human blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSC) in vivo. First, we studied the growth of adherent ECFCs and MSCs on or in the hydrogels. To examine the potential formation of functional vascular networks in vivo, a liquid pre-polymer solution of collagen-Ph containing human ECFCs and MSCs, horseradish peroxidase and hydrogen peroxide was injected into the subcutaneous space or abdominal muscle defect of an immunodeficient mouse before gelation, to form a 3D cell-laden polymerized construct. These results showed that extensive human ECFC-lined vascular networks can be generated within 7 days, the engineered vascular density inside collagen-Ph hydrogel constructs can be manipulated through refinable mechanical properties and proteolytic degradability, and these networks can form functional anastomoses with the existing vasculature to further support the survival of host muscle tissues. Finally, optimized conditions of the cell-laden collagen

Extracellular matrix scaffolds for cartilage and bone regeneration

NARCIS (Netherlands)

Benders, K.E.M.; van Weeren, P.R.; Badylak, S.F.; Saris, Daniël B.F.; Dhert, W.J.A.; Malda, J.

2013-01-01

Regenerative medicine approaches based on decellularized extracellular matrix (ECM) scaffolds and tissues are rapidly expanding. The rationale for using ECM as a natural biomaterial is the presence of bioactive molecules that drive tissue homeostasis and regeneration. Moreover, appropriately

Quantitative Visualization of Dynamic Tracer Transportation in the Extracellular Space of Deep Brain Regions Using Tracer-Based Magnetic Resonance Imaging

Science.gov (United States)

Hou, Jin; Wang, Wei; Quan, Xianyue; Liang, Wen; Li, Zhiming; Han, Hongbin; Chen, Deji

2017-01-01

Background This study assessed an innovative tracer-based magnetic resonance imaging (MRI) system to visualize the dynamic transportation of tracers in regions of deep brain extracellular space (ECS) and to measure transportation ability and ECS structure. Material/Methods Gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) was the chosen tracer and was injected into the caudate nucleus and thalamus. Real-time dynamic transportation of Gd-DTPA in ECS was observed and the results were verified by laser scanning confocal microscopy. Using Transwell assay across the blood-brain barrier, a modified diffusion equation was further simplified. Effective diffusion coefficient D* and tortuosity λ were calculated. Immunohistochemical staining and Western blot analysis were used to investigate the extracellular matrix contributing to ECS structure. Results Tracers injected into the caudate nucleus were transported to the ipsilateral frontal and temporal cortices away from the injection points, while both of them injected into the thalamus were only distributed on site. Although the caudate nucleus was closely adjacent to the thalamus, tracer transportation between partitions was not observed. In addition, D* and the λ showed statistically significant differences between partitions. ECS was shown to be a physiologically partitioned system, and its division is characterized by the unique distribution territory and transportation ability of substances located in it. Versican and Tenascin R are possible contributors to the tortuosity of ECS. Conclusions Tracer-based MRI will improve our understanding of the brain microenvironment, improve the techniques for local delivery of drugs, and highlight brain tissue engineering fields in the future. PMID:28866708

Quantitative Visualization of Dynamic Tracer Transportation in the Extracellular Space of Deep Brain Regions Using Tracer-Based Magnetic Resonance Imaging.

Science.gov (United States)

Hou, Jin; Wang, Wei; Quan, Xianyue; Liang, Wen; Li, Zhiming; Chen, Deji; Han, Hongbin

2017-09-03

BACKGROUND This study assessed an innovative tracer-based magnetic resonance imaging (MRI) system to visualize the dynamic transportation of tracers in regions of deep brain extracellular space (ECS) and to measure transportation ability and ECS structure. MATERIAL AND METHODS Gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) was the chosen tracer and was injected into the caudate nucleus and thalamus. Real-time dynamic transportation of Gd-DTPA in ECS was observed and the results were verified by laser scanning confocal microscopy. Using Transwell assay across the blood-brain barrier, a modified diffusion equation was further simplified. Effective diffusion coefficient D* and tortuosity λ were calculated. Immunohistochemical staining and Western blot analysis were used to investigate the extracellular matrix contributing to ECS structure. RESULTS Tracers injected into the caudate nucleus were transported to the ipsilateral frontal and temporal cortices away from the injection points, while both of them injected into the thalamus were only distributed on site. Although the caudate nucleus was closely adjacent to the thalamus, tracer transportation between partitions was not observed. In addition, D* and the λ showed statistically significant differences between partitions. ECS was shown to be a physiologically partitioned system, and its division is characterized by the unique distribution territory and transportation ability of substances located in it. Versican and Tenascin R are possible contributors to the tortuosity of ECS. CONCLUSIONS Tracer-based MRI will improve our understanding of the brain microenvironment, improve the techniques for local delivery of drugs, and highlight brain tissue engineering fields in the future.

Extracellular matrix fluctuations during early embryogenesis

International Nuclear Information System (INIS)

Szabó, A; Rupp, P A; Rongish, B J; Little, C D; Czirók, A

2011-01-01

Extracellular matrix (ECM) movements and rearrangements were studied in avian embryos during early stages of development. We show that the ECM moves as a composite material, whereby distinct molecular components as well as spatially separated layers exhibit similar displacements. Using scanning wide field and confocal microscopy we show that the velocity field of ECM displacement is smooth in space and that ECM movements are correlated even at locations separated by several hundred micrometers. Velocity vectors, however, strongly fluctuate in time. The autocorrelation time of the velocity fluctuations is less than a minute. Suppression of the fluctuations yields a persistent movement pattern that is shared among embryos at equivalent stages of development. The high resolution of the velocity fields allows a detailed spatio-temporal characterization of important morphogenetic processes, especially tissue dynamics surrounding the embryonic organizer (Hensen's node)

Xenogenic extracellular matrices as potential biomaterials for interposition grafting in urological surgery.

LENUS (Irish Health Repository)

Davis, N F

2012-01-31

PURPOSE: The field of tissue engineering focuses on developing strategies for reconstructing injured, diseased, and congenitally absent tissues and organs. During the last decade urologists have benefited from remodeling and regenerative properties of bioscaffolds derived from xenogenic extracellular matrices. We comprehensively reviewed the current literature on structural and functional characteristics of xenogenic extracellular matrix grafting since it was first described in urological surgery. We also reviewed the clinical limitations, and assessed the potential for safe and effective urological application of extracellular matrix grafting in place of autogenous tissue. MATERIALS AND METHODS: We performed literature searches for English language publications using the PubMed(R) and MEDLINE(R) databases. Keywords included "xenogenic," "extracellular matrix" and "genitourinary tract applications." A total of 112 articles were scrutinized, of which 50 were suitable for review based on clinical relevance and importance of content. RESULTS: Since the mid 1990s xenogenic extracellular matrices have been used to successfully treat a number of pathological conditions that affect the upper and lower genitourinary tract. They are typically prepared from porcine organs such as small intestine and bladder. These organs are harvested and subjected to decellularization and sterilization techniques before surgical implantation. Bioinductive growth factors that are retained during the preparation process induce constructive tissue remodeling as the extracellular matrix is simultaneously degraded and excreted. However, recent documented concerns over durability, decreased mechanical strength and residual porcine DNA after preparation techniques have temporarily hampered the potential of extracellular matrices as a reliable replacement for genitourinary tract structures. CONCLUSIONS: Extracellular matrices are a useful alternative for successfully treating a number of urological

Binding of matrix metalloproteinase inhibitors to extracellular matrix: 3D-QSAR analysis.

Science.gov (United States)

Zhang, Yufen; Lukacova, Viera; Bartus, Vladimir; Nie, Xiaoping; Sun, Guorong; Manivannan, Ethirajan; Ghorpade, Sandeep R; Jin, Xiaomin; Manyem, Shankar; Sibi, Mukund P; Cook, Gregory R; Balaz, Stefan

2008-10-01

Binding to the extracellular matrix, one of the most abundant human protein complexes, significantly affects drug disposition. Specifically, the interactions with extracellular matrix determine the free concentrations of small molecules acting in tissues, including signaling peptides, inhibitors of tissue remodeling enzymes such as matrix metalloproteinases, and other drug candidates. The nature of extracellular matrix binding was elucidated for 63 matrix metalloproteinase inhibitors, for which the association constants to an extracellular matrix mimic were reported here. The data did not correlate with lipophilicity as a common determinant of structure-nonspecific, orientation-averaged binding. A hypothetical structure of the binding site of the solidified extracellular matrix surrogate was analyzed using the Comparative Molecular Field Analysis, which needed to be applied in our multi-mode variant. This fact indicates that the compounds bind to extracellular matrix in multiple modes, which cannot be considered as completely orientation-averaged and exhibit structural dependence. The novel comparative molecular field analysis models, exhibiting satisfactory descriptive and predictive abilities, are suitable for prediction of the extracellular matrix binding for the untested chemicals, which are within applicability domains. The results contribute to a better prediction of the pharmacokinetic parameters such as the distribution volume and the tissue-blood partition coefficients, in addition to a more imminent benefit for the development of more effective matrix metalloproteinase inhibitors.

Development of schizogenous intercellular spaces in plants

Directory of Open Access Journals (Sweden)

Kimitsune eIshizaki

2015-07-01

Full Text Available Gas exchange is essential for multicellular organisms. In contrast to the circulatory systems of animals, land plants have tissues with intercellular spaces (ICSs, called aerenchyma, that are critical for efficient gas exchange. Plants form ICSs by two different mechanisms: schizogeny, where localized cell separation creates spaces; and lysogeny, where cells die to create intercellular spaces. In schizogenous ICS formation, specific molecular mechanisms regulate the sites of cell separation and coordinate extensive reorganization of cell walls. Emerging evidence suggests the involvement of extracellular signaling, mediated by peptide ligands and leucine-rich repeat receptor-like kinases, in the regulation of cell wall remodeling during cell separation. Recent work on the liverwort Marchantia polymorpha has demonstrated a critical role for a plasma membrane-associated plant U-box E3 ubiquitin ligase in ICS formation. In this review, I discuss the mechanism of schizogenous ICS formation, focusing on the potential role of extracellular signaling in the regulation of cell separation.

Feasibility of autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffold for cartilage tissue engineering.

Science.gov (United States)

Tang, Cheng; Xu, Yan; Jin, Chengzhe; Min, Byoung-Hyun; Li, Zhiyong; Pei, Xuan; Wang, Liming

2013-12-01

Extracellular matrix (ECM) materials are widely used in cartilage tissue engineering. However, the current ECM materials are unsatisfactory for clinical practice as most of them are derived from allogenous or xenogenous tissue. This study was designed to develop a novel autologous ECM scaffold for cartilage tissue engineering. The autologous bone marrow mesenchymal stem cell-derived ECM (aBMSC-dECM) membrane was collected and fabricated into a three-dimensional porous scaffold via cross-linking and freeze-drying techniques. Articular chondrocytes were seeded into the aBMSC-dECM scaffold and atelocollagen scaffold, respectively. An in vitro culture and an in vivo implantation in nude mice model were performed to evaluate the influence on engineered cartilage. The current results showed that the aBMSC-dECM scaffold had a good microstructure and biocompatibility. After 4 weeks in vitro culture, the engineered cartilage in the aBMSC-dECM scaffold group formed thicker cartilage tissue with more homogeneous structure and higher expressions of cartilaginous gene and protein compared with the atelocollagen scaffold group. Furthermore, the engineered cartilage based on the aBMSC-dECM scaffold showed better cartilage formation in terms of volume and homogeneity, cartilage matrix content, and compressive modulus after 3 weeks in vivo implantation. These results indicated that the aBMSC-dECM scaffold could be a successful novel candidate scaffold for cartilage tissue engineering. © 2013 Wiley Periodicals, Inc. and International Center for Artificial Organs and Transplantation.

A genetically encoded ratiometric sensor to measure extracellular pH in microdomains bounded by basolateral membranes of epithelial cells.

Science.gov (United States)

Urra, Javier; Sandoval, Moisés; Cornejo, Isabel; Barros, L Felipe; Sepúlveda, Francisco V; Cid, L Pablo

2008-10-01

Extracellular pH, especially in relatively inaccessible microdomains between cells, affects transport membrane protein activity and might have an intercellular signaling role. We have developed a genetically encoded extracellular pH sensor capable of detecting pH changes in basolateral spaces of epithelial cells. It consists of a chimerical membrane protein displaying concatenated enhanced variants of cyan fluorescence protein (ECFP) and yellow fluorescence protein (EYFP) at the external aspect of the cell surface. The construct, termed pHCECSensor01, was targeted to basolateral membranes of Madin-Darby canine kidney (MDCK) cells by means of a sequence derived from the aquaporin AQP4. The fusion of pH-sensitive EYFP with pH-insensitive ECFP allows ratiometric pH measurements. The titration curve of pHCECSensor01 in vivo had a pK (a) value of 6.5 +/- 0.04. Only minor effects of extracellular chloride on pHCECSensor01 were observed around the physiological concentrations of this anion. In MDCK cells, the sensor was able to detect changes in pH secondary to H(+) efflux into the basolateral spaces elicited by an ammonium prepulse or lactate load. This genetically encoded sensor has the potential to serve as a noninvasive tool for monitoring changes in extracellular pH microdomains in epithelial and other tissues in vivo.

Extracellular matrix components direct porcine muscle stem cell behavior

International Nuclear Information System (INIS)

Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J.

2010-01-01

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.

Extracellular matrix components direct porcine muscle stem cell behavior

Energy Technology Data Exchange (ETDEWEB)

Wilschut, Karlijn J. [Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM, Utrecht (Netherlands); Haagsman, Henk P. [Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL, Utrecht (Netherlands); Roelen, Bernard A.J., E-mail: b.a.j.roelen@uu.nl [Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM, Utrecht (Netherlands)

2010-02-01

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.

Assessing the transport rate of hyperpolarized pyruvate and lactate from the intra- to the extracellular space.

Science.gov (United States)

Reineri, Francesca; Daniele, Valeria; Cavallari, Eleonora; Aime, Silvio

2016-08-01

The use of [1-(13) C]pyruvate hyperpolarized by means of dynamic nuclear polarization provides a direct way to track the metabolic transformations of this metabolite in vivo and in cell cultures. The identification of the intra- and extracellular contributions to the (13) C NMR resonances is not straightforward. In order to obtain information about the rate of pyruvate and lactate transport through the cellular membrane, we set up a method that relies on the sudden 'quenching' of the extracellular metabolites' signal. The paramagnetic Gd-tetraazacyclododecane triacetic acid (Gd-DO3A) complex was used to dramatically decrease the longitudinal relaxation time constants of the (13) C-carboxylate resonances of both pyruvate and lactate. When Gd-DO3A was added to an MCF-7 cellular culture, which had previously received a dose of hyperpolarized [1-(13) C]pyruvate, the contributions of the extracellular pyruvate and lactate signals were deleted. From the analysis of the decay curves of the (13) C-carboxylate resonances of pyruvate and lactate it was possible to extract information about the exchange rate of the two metabolites across the cellular membrane. In particular, it was found that, in the reported experimental conditions, the lactate transport from the intra- to the extracellular space is not much lower than the rate of lactate formation. The method reported herein is non-destructive and it could be translated to in vivo studies. It opens a route for the use of hyperpolarized pyruvate to assess altered activity of carboxylate transporter proteins that may occur in pathological conditions. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Co(III)EDTA as extra-cellular marker in μPIXE-analysis of rat cardiomyocytes

International Nuclear Information System (INIS)

Quaedackers, J.A.; Queens, R.M.G.J.; Mutsaers, P.H.A.; Voigt, M.J.A. de; Vusse, G.J. van der

1998-01-01

In previous studies no clear difference was found between the intra- and extra-cellular compartment in nuclear microprobe elemental distribution maps of freeze-dried cryo sections of heart tissue. Probably due to artefacts during the preparation of these samples, the intra-cellular and the extra-cellular content of elements are mixed up. In this article a method, using NaCo(III)EDTA as an extra-cellular marker, was applied to deconvolute the total ion content in an extra- and intra-cellular contribution. This method was both applied to normoxic heart tissue and low-flow ischemic heart tissue. Intra-cellular ion concentrations calculated from the corrected ion contents of the normoxic tissue agrees well with literature values. Moreover a clear elevation of the intra-cellular sodium and chlorine concentration was found in low-flow ischemic tissue. (orig.)

Distinction between the extracellular matrix of the nucleus pulposus and hyaline cartilage: a requisite for tissue engineering of intervertebral disc

OpenAIRE

Mwale F.; Roughley P.; Antoniou J.

2004-01-01

Tissue engineering of intervertebral discs (IVD) using mesenchymal stem cells (MSCs) induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. However, since there is no unique marker characteristic of discs and since hyaline cartilage and immature nucleus pulposus (NP) possess similar macromolecules in their extracellular matrix, it is currently difficult to recognize MSC conversion to a disc cell. This study was performed to c...

Saturated fatty acid palmitate induces extracellular release of histone H3: A possible mechanistic basis for high-fat diet-induced inflammation and thrombosis

Energy Technology Data Exchange (ETDEWEB)

Shrestha, Chandan [Department of Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Department of Laboratory and Vascular Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Ito, Takashi [Department of Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Kawahara, Ko-ichi [Department of Biomedical Engineering, Osaka Institute of Technology, Osaka (Japan); Shrestha, Binita; Yamakuchi, Munekazu; Hashiguchi, Teruto [Department of Laboratory and Vascular Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Maruyama, Ikuro, E-mail: rinken@m3.kufm.kagoshima-u.ac.jp [Department of Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan)

2013-08-09

Highlights: •High-fat diet feeding and palmitate induces the release of nuclear protein histone H3. •ROS production and JNK signaling mediates the release of histone H3. •Extracellular histones induces proinflammatory and procoagulant response. -- Abstract: Chronic low-grade inflammation is a key contributor to high-fat diet (HFD)-related diseases, such as type 2 diabetes, non-alcoholic steatohepatitis, and atherosclerosis. The inflammation is characterized by infiltration of inflammatory cells, particularly macrophages, into obese adipose tissue. However, the molecular mechanisms by which a HFD induces low-grade inflammation are poorly understood. Here, we show that histone H3, a major protein component of chromatin, is released into the extracellular space when mice are fed a HFD or macrophages are stimulated with the saturated fatty acid palmitate. In a murine macrophage cell line, RAW 264.7, palmitate activated reactive oxygen species (ROS) production and JNK signaling. Inhibitors of these pathways dampened palmitate-induced histone H3 release, suggesting that the extracellular release of histone H3 was mediated, in part, through ROS and JNK signaling. Extracellular histone activated endothelial cells toexpress the adhesion molecules ICAM-1 and VCAM-1 and the procoagulant molecule tissue factor, which are known to contribute to inflammatory cell recruitment and thrombosis. These results suggest the possible contribution of extracellular histone to the pathogenesis of HFD-induced inflammation and thrombosis.

Saturated fatty acid palmitate induces extracellular release of histone H3: A possible mechanistic basis for high-fat diet-induced inflammation and thrombosis

International Nuclear Information System (INIS)

Shrestha, Chandan; Ito, Takashi; Kawahara, Ko-ichi; Shrestha, Binita; Yamakuchi, Munekazu; Hashiguchi, Teruto; Maruyama, Ikuro

2013-01-01

Highlights: •High-fat diet feeding and palmitate induces the release of nuclear protein histone H3. •ROS production and JNK signaling mediates the release of histone H3. •Extracellular histones induces proinflammatory and procoagulant response. -- Abstract: Chronic low-grade inflammation is a key contributor to high-fat diet (HFD)-related diseases, such as type 2 diabetes, non-alcoholic steatohepatitis, and atherosclerosis. The inflammation is characterized by infiltration of inflammatory cells, particularly macrophages, into obese adipose tissue. However, the molecular mechanisms by which a HFD induces low-grade inflammation are poorly understood. Here, we show that histone H3, a major protein component of chromatin, is released into the extracellular space when mice are fed a HFD or macrophages are stimulated with the saturated fatty acid palmitate. In a murine macrophage cell line, RAW 264.7, palmitate activated reactive oxygen species (ROS) production and JNK signaling. Inhibitors of these pathways dampened palmitate-induced histone H3 release, suggesting that the extracellular release of histone H3 was mediated, in part, through ROS and JNK signaling. Extracellular histone activated endothelial cells toexpress the adhesion molecules ICAM-1 and VCAM-1 and the procoagulant molecule tissue factor, which are known to contribute to inflammatory cell recruitment and thrombosis. These results suggest the possible contribution of extracellular histone to the pathogenesis of HFD-induced inflammation and thrombosis

Targeting Heparin to Collagen within Extracellular Matrix Significantly Reduces Thrombogenicity and Improves Endothelialization of Decellularized Tissues.

Science.gov (United States)

Jiang, Bin; Suen, Rachel; Wertheim, Jason A; Ameer, Guillermo A

2016-12-12

Thrombosis within small-diameter vascular grafts limits the development of bioartificial, engineered vascular conduits, especially those derived from extracellular matrix (ECM). Here we describe an easy-to-implement strategy to chemically modify vascular ECM by covalently linking a collagen binding peptide (CBP) to heparin to form a heparin derivative (CBP-heparin) that selectively binds a subset of collagens. Modification of ECM with CBP-heparin leads to increased deposition of functional heparin (by ∼7.2-fold measured by glycosaminoglycan composition) and a corresponding reduction in platelet binding (>70%) and whole blood clotting (>80%) onto the ECM. Furthermore, addition of CBP-heparin to the ECM stabilizes long-term endothelial cell attachment to the lumen of ECM-derived vascular conduits, potentially through recruitment of heparin-binding growth factors that ultimately improve the durability of endothelialization in vitro. Overall, our findings provide a simple yet effective method to increase deposition of functional heparin on the surface of ECM-based vascular grafts and thereby minimize thrombogenicity of decellularized tissue, overcoming a significant challenge in tissue engineering of bioartificial vessels and vascularized organs.

Ischiofemoral space on MRI in an asymptomatic population: Normative width measurements and soft tissue signal variations

International Nuclear Information System (INIS)

Maras Oezdemir, Zeynep; Goermeli, Cemile Ayse; Sagir Kahraman, Ayseguel; Aydingoez, Uestuen

2015-01-01

To make normative width measurements of the ischiofemoral (IF) space in an asymptomatic population and to record soft tissue MRI signal variations within the IF space in order to determine whether such variations are associated with IF space dimensions. Normative width measurements of the IF space were prospectively made in 418 hips on 1.5 T MR images of 209 asymptomatic volunteers. Quantitative and qualitative assessments of the IF soft tissues including the quadratus femoris (QF) muscle were also made. The mean IF space width was 2.56 ± 0.75 cm (right, 2.60 ± 0.75 cm; left, 2.53 ± 0.75 cm). Soft tissue MRI signal abnormalities were present within the IF space in 19 (9.1 %) of 209 volunteers. Soft tissue abnormalities within the IF space included oedema (3/209, 1.4 %) of the QF and/or surrounding soft tissue, and only fatty infiltration (16/209, 7.7 %) of the QF. Bilateral IF spaces are asymmetrical in asymptomatic persons. There is ≥10 % of width difference between right and left IF spaces in approximately half of asymptomatic individuals. Fatty infiltration and oedema can be present at the IF space in a small portion of the asymptomatic population, who also have narrower IF spaces than those without soft tissue MRI signal abnormalities. (orig.)

Extracellular distribution of radiolabel obscures specific binding of diethylstilbestrol in mouse skeletal muscle

International Nuclear Information System (INIS)

Gruber, B.; Blix, P.M.; Cohen, L.

1985-01-01

The extracellular distribution of 3 H-diethylstilbestrol ( 3 H-DES) in mouse skeletal muscle was assessed following intraperitoneal injection. Total muscle extracellular space was measured with 14 C-inulin, and the vascular space with 125 I-albumin. A significant difference in the distribution of native 3 H-DES and its metabolites in muscle and blood was found. This could only be explained if these compounds distributed with the albumin space and not the inulin space

Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates.

Science.gov (United States)

Bayne, E K; Anderson, M J; Fambrough, D M

1984-10-01

Monoclonal antibodies recognizing laminin, heparan sulfate proteoglycan, fibronectin, and two apparently novel connective tissue components have been used to examine the organization of extracellular matrix of skeletal muscle in vivo and in vitro. Four of the five monoclonal antibodies are described for the first time here. Immunocytochemical experiments with frozen-sectioned muscle demonstrated that both the heparan sulfate proteoglycan and laminin exhibited staining patterns identical to that expected for components of the basal lamina. In contrast, the remaining matrix constituents were detected in all regions of muscle connective tissue: the endomysium, perimysium, and epimysium. Embryonic muscle cells developing in culture elaborated an extracellular matrix, each antigen exhibiting a unique distribution. Of particular interest was the organization of extracellular matrix on myotubes: the build-up of matrix components was most apparent in plaques overlying clusters of an integral membrane protein, the acetylcholine receptor (AChR). The heparan sulfate proteoglycan was concentrated at virtually all AChR clusters and showed a remarkable level of congruence with receptor organization; laminin was detected at 70-95% of AChR clusters but often was not completely co-distributed with AChR within the cluster; fibronectin and the two other extracellular matrix antigens occurred at approximately 20, 8, and 2% of the AChR clusters, respectively, and showed little or no congruence with AChR. From observations on the distribution of extracellular matrix components in tissue cultured fibroblasts and myogenic cells, several ideas about the organization of extracellular matrix are suggested. (a) Congruence between AChR clusters and heparan sulfate proteoglycan suggests the existence of some linkage between the two molecules, possibly important for regulation of AChR distribution within the muscle membrane. (b) The qualitatively different patterns of extracellular matrix

The mechanical properties of human adipose tissues and their relationships to the structure and composition of the extracellular matrix.

Science.gov (United States)

Alkhouli, Nadia; Mansfield, Jessica; Green, Ellen; Bell, James; Knight, Beatrice; Liversedge, Neil; Tham, Ji Chung; Welbourn, Richard; Shore, Angela C; Kos, Katarina; Winlove, C Peter

2013-12-01

Adipose tissue (AT) expansion in obesity is characterized by cellular growth and continuous extracellular matrix (ECM) remodeling with increased fibrillar collagen deposition. It is hypothesized that the matrix can inhibit cellular expansion and lipid storage. Therefore, it is important to fully characterize the ECM's biomechanical properties and its interactions with cells. In this study, we characterize and compare the mechanical properties of human subcutaneous and omental tissues, which have different physiological functions. AT was obtained from 44 subjects undergoing surgery. Force/extension and stress/relaxation data were obtained. The effects of osmotic challenge were measured to investigate the cellular contribution to tissue mechanics. Tissue structure and its response to tensile strain were determined using nonlinear microscopy. AT showed nonlinear stress/strain characteristics of up to a 30% strain. Comparing paired subcutaneous and omental samples (n = 19), the moduli were lower in subcutaneous: initial 1.6 ± 0.8 (means ± SD) and 2.9 ± 1.5 kPa (P = 0.001), final 11.7 ± 6.4 and 32 ± 15.6 kPa (P matrix fibers. These results suggest that subcutaneous AT has greater capacity for expansion and recovery from mechanical deformation than omental AT.

Collective cell behavior on basement membranes floating in space

Science.gov (United States)

Ellison, Sarah; Bhattacharjee, Tapomoy; Morley, Cameron; Sawyer, W.; Angelini, Thomas

The basement membrane is an essential part of the polarity of endothelial and epithelial tissues. In tissue culture and organ-on-chip devices, monolayer polarity can be established by coating flat surfaces with extracellular matrix proteins and tuning the trans-substrate permeability. In epithelial 3D culture, spheroids spontaneously establish inside-out polarity, morphing into hollow shell-like structures called acini, generating their own basement membrane on the inner radius of the shell. However, 3D culture approaches generally lack the high degree of control provided by the 2D culture plate or organ-on-chip devices, making it difficult to create more faithful in vitro tissue models with complex surface curvature and morphology. Here we present a method for 3D printing complex basement membranes covered in cells. We 3D print collagen-I and Matrigel into a 3D growth medium made from jammed microgels. This soft, yielding material allows extracellular matrix to be formed as complex surfaces and shapes, floating in space. We then distribute MCF10A epithelial cells across the polymerized surface. We envision employing this strategy to study 3D collective cell behavior in numerous model tissue layers, beyond this simple epithelial model.

Distinction between the extracellular matrix of the nucleus pulposus and hyaline cartilage: a requisite for tissue engineering of intervertebral disc.

Science.gov (United States)

Mwale, F; Roughley, P; Antoniou, J

2004-12-15

Tissue engineering of intervertebral discs (IVD) using mesenchymal stem cells (MSCs) induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. However, since there is no unique marker characteristic of discs and since hyaline cartilage and immature nucleus pulposus (NP) possess similar macromolecules in their extracellular matrix, it is currently difficult to recognize MSC conversion to a disc cell. This study was performed to compare the proteoglycan to collagen ratio (measured as GAG to hydroxyproline ratio) in the NP of normal disc to that of the hyaline cartilage of the endplate within the same group of individuals and test the hypothesis that this ratio can be used for in vivo studies to distinguish between a normal NP and hyaline cartilage phenotype. Whole human lumbar spine specimens from fresh cadavers, ranging in age from 12 weeks to 79 years, were used to harvest the IVDs and adjacent endplates. The GAG to hydroxyproline ratio within the NP of young adults is approximately 27:1, whereas the ratio within the hyaline cartilage endplate of the same aged individuals is about 2:1. The production of an extracellular matrix with a high proteoglycan to collagen ratio can be used in vivo to distinguish NP cells from chondrocytes, and could help in identifying a NP-like phenotype in vivo as opposed to a chondrocyte when MSCs are induced to differentiate for tissue engineering of a disc.

Distinction between the extracellular matrix of the nucleus pulposus and hyaline cartilage: a requisite for tissue engineering of intervertebral disc

Directory of Open Access Journals (Sweden)

Mwale F.

2004-12-01

Full Text Available Tissue engineering of intervertebral discs (IVD using mesenchymal stem cells (MSCs induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. However, since there is no unique marker characteristic of discs and since hyaline cartilage and immature nucleus pulposus (NP possess similar macromolecules in their extracellular matrix, it is currently difficult to recognize MSC conversion to a disc cell. This study was performed to compare the proteoglycan to collagen ratio (measured as GAG to hydroxyproline ratio in the NP of normal disc to that of the hyaline cartilage of the endplate within the same group of individuals and test the hypothesis that this ratio can be used for in vivo studies to distinguish between a normal NP and hyaline cartilage phenotype. Whole human lumbar spine specimens from fresh cadavers, ranging in age from 12 weeks to 79 years, were used to harvest the IVDs and adjacent endplates. The GAG to hydroxyproline ratio within the NP of young adults is approximately 27:1, whereas the ratio within the hyaline cartilage endplate of the same aged individuals is about 2:1. The production of an extracellular matrix with a high proteoglycan to collagen ratio can be used in vivo to distinguish NP cells from chondrocytes, and could help in identifying a NP-like phenotype in vivo as opposed to a chondrocyte when MSCs are induced to differentiate for tissue engineering of a disc.

Regeneration of Cartilage in Human Knee Osteoarthritis with Autologous Adipose Tissue-Derived Stem Cells and Autologous Extracellular Matrix

Directory of Open Access Journals (Sweden)

Jaewoo Pak

2016-08-01

Full Text Available This clinical case series demonstrates that percutaneous injections of autologous adipose tissue-derived stem cells (ADSCs and homogenized extracellular matrix (ECM in the form of adipose stromal vascular fraction (SVF, along with hyaluronic acid (HA and platelet-rich plasma (PRP activated by calcium chloride, could regenerate cartilage-like tissue in human knee osteoarthritis (OA patients. Autologous lipoaspirates were obtained from adipose tissue of the abdominal origin. Afterward, the lipoaspirates were minced to homogenize the ECM. These homogenized lipoaspirates were then mixed with collagenase and incubated. The resulting mixture of ADSCs and ECM in the form of SVF was injected, along with HA and PRP activated by calcium chloride, into knees of three Korean patients with OA. The same affected knees were reinjected weekly with additional PRP activated by calcium chloride for 3 weeks. Pretreatment and post-treatment magnetic resonance imaging (MRI data, functional rating index, range of motion (ROM, and pain score data were then analyzed. All patients' MRI data showed cartilage-like tissue regeneration. Along with MRI evidence, the measured physical therapy outcomes in terms of ROM, subjective pain, and functional status were all improved. This study demonstrates that percutaneous injection of ADSCs with ECM contained in autologous adipose SVF, in conjunction with HA and PRP activated by calcium chloride, is a safe and potentially effective minimally invasive therapy for OA of human knees.

Intermolecular interactions of thrombospondins drive their accumulation in extracellular matrix

OpenAIRE

Kim, Dae Joong; Christofidou, Elena D.; Keene, Douglas R.; Hassan Milde, Marwah; Adams, Josephine C.

2015-01-01

Thrombospondins participate in many aspects of tissue organization in adult tissue homeostasis, and their dysregulation contributes to pathological processes such as fibrosis and tumor progression. The incorporation of thrombospondins into extracellular matrix (ECM) as discrete puncta has been documented in various tissue and cell biological contexts, yet the underlying mechanisms remain poorly understood. We find that collagen fibrils are disorganized in multiple tissues of Thbs1 −/− mice. I...

Extracellular matrix-derived hydrogels for dental stem cell delivery

OpenAIRE

Viswanath, Aiswarya; Vanacker, Julie; Germain, Loic; Leprince, Julien G.; Diogenes, Anibal; Shakesheff, Kevin M.; White, Lisa J.; des Rieux, Anne

2016-01-01

Decellularised mammalian extracellular matrices (ECM) have been widely accepted as an ideal substrate for repair and remodelling of numerous tissues in clinical and pre-clinical studies. Recent studies have demonstrated the ability of ECM scaffolds derived from site-specific homologous tissues to direct cell differentiation. The present study investigated the suitability of hydrogels derived from different source tissues: bone, spinal cord and dentine, as suitable carriers to deliver human ap...

Extracellular matrix fragmentation in young, healthy cartilaginous tissues.

Science.gov (United States)

Craddock, R J; Hodson, N W; Ozols, M; Shearer, T; Hoyland, J A; Sherratt, M J

2018-02-09

Although the composition and structure of cartilaginous tissues is complex, collagen II fibrils and aggrecan are the most abundant assemblies in both articular cartilage (AC) and the nucleus pulposus (NP) of the intervertebral disc (IVD). Whilst structural heterogeneity of intact aggrecan ( containing three globular domains) is well characterised, the extent of aggrecan fragmentation in healthy tissues is poorly defined. Using young, yet skeletally mature (18-30 months), bovine AC and NP tissues, it was shown that, whilst the ultrastructure of intact aggrecan was tissue-dependent, most molecules (AC: 95 %; NP: 99.5 %) were fragmented (lacking one or more globular domains). Fragments were significantly smaller and more structurally heterogeneous in the NP compared with the AC (molecular area; AC: 8543 nm2; NP: 4625 nm2; p tissue-invariant. Molecular fragmentation is considered indicative of a pathology; however, these young, skeletally mature tissues were histologically and mechanically (reduced modulus: AC: ≈ 500 kPa; NP: ≈ 80 kPa) comparable to healthy tissues and devoid of notable gelatinase activity (compared with rat dermis). As aggrecan fragmentation was prevalent in neonatal bovine AC (99.5 % fragmented, molecular area: 5137 nm2) as compared with mature AC (95.0 % fragmented, molecular area: 8667 nm2), it was hypothesised that targeted proteolysis might be an adaptive process that modified aggrecan packing (as simulated computationally) and, hence, tissue charge density, mechanical properties and porosity. These observations provided a baseline against which pathological and/or age-related fragmentation of aggrecan could be assessed and suggested that new strategies might be required to engineer constructs that mimic the mechanical properties of native cartilaginous tissues.

[The connective tissues, from the origin of the concept to its "Maturation" to extracellular matrix. Application to ocular tissues. Contribution to the history of medical sciences].

Science.gov (United States)

Labat-Robert, J; Robert, L; Pouliquen, Y

2011-06-01

The "Tissue" concept emerged apparently in the medical literature at about the French revolution, during the second half of the 18(th) century. It was found in the texts written by the physicians of Béarn and Montpellier, the Bordeu-s and also by the famous physician, Felix Vicq d'Azyr, the last attending physician of the queen Marie-Antoinette, "Bordeu et al. (1775) et Pouliquen (2009)". It was elaborated into a coherent doctrine somewhat later by Xavier Bichat, considered as the founder of modern pathological anatomy, Bichat. With the advent of histochemistry, from the beginning of the 20(th) century, several of the principal macromolecular components of connective tissues, collagens, elastin, "acid mucopolysaccharides" (later glycosaminoglycans and proteoglycans) and finally structural glycoproteins were characterized. These constituents of connective tissues were then designated as components of the extracellular matrix (ECM), closely associated to the cellular components of these tissues by adhesive (structural) glycoproteins as fibronectin, several others and cell receptors, "recognising" ECM-components as integrins, the elastin-receptor and others. This molecular arrangement fastens cells to the ECM-components they synthesize and mediates the exchange of informations between the cells to the ECM (inside-out) and also from the ECM-components to the cells (outside-in). This macromolecular arrangement is specific for each tissue as a result of the differentiation of their cellular components. It is also the basis and condition of the fulfillment of the specific functions of differentiated tissues. This is a short description of the passage of the "tissue" concept from its vague origin towards its precise identification at the cellular and molecular level up to the recognition of its functional importance and its establishment as an autonomous science. This can be considered as a new example of the importance of metaphors for the progress of science, Keller

Chitosan microspheres with an extracellular matrix-mimicking nanofibrous structure as cell-carrier building blocks for bottom-up cartilage tissue engineering

Science.gov (United States)

Zhou, Yong; Gao, Huai-Ling; Shen, Li-Li; Pan, Zhao; Mao, Li-Bo; Wu, Tao; He, Jia-Cai; Zou, Duo-Hong; Zhang, Zhi-Yuan; Yu, Shu-Hong

2015-12-01

Scaffolds for tissue engineering (TE) which closely mimic the physicochemical properties of the natural extracellular matrix (ECM) have been proven to advantageously favor cell attachment, proliferation, migration and new tissue formation. Recently, as a valuable alternative, a bottom-up TE approach utilizing cell-loaded micrometer-scale modular components as building blocks to reconstruct a new tissue in vitro or in vivo has been proved to demonstrate a number of desirable advantages compared with the traditional bulk scaffold based top-down TE approach. Nevertheless, micro-components with an ECM-mimicking nanofibrous structure are still very scarce and highly desirable. Chitosan (CS), an accessible natural polymer, has demonstrated appealing intrinsic properties and promising application potential for TE, especially the cartilage tissue regeneration. According to this background, we report here the fabrication of chitosan microspheres with an ECM-mimicking nanofibrous structure for the first time based on a physical gelation process. By combining this physical fabrication procedure with microfluidic technology, uniform CS microspheres (CMS) with controlled nanofibrous microstructure and tunable sizes can be facilely obtained. Especially, no potentially toxic or denaturizing chemical crosslinking agent was introduced into the products. Notably, in vitro chondrocyte culture tests revealed that enhanced cell attachment and proliferation were realized, and a macroscopic 3D geometrically shaped cartilage-like composite can be easily constructed with the nanofibrous CMS (NCMS) and chondrocytes, which demonstrate significant application potential of NCMS as the bottom-up cell-carrier components for cartilage tissue engineering.Scaffolds for tissue engineering (TE) which closely mimic the physicochemical properties of the natural extracellular matrix (ECM) have been proven to advantageously favor cell attachment, proliferation, migration and new tissue formation

Extracellular matrix fragmentation in young, healthy cartilaginous tissues

Directory of Open Access Journals (Sweden)

RJ Craddock

2018-02-01

Full Text Available Although the composition and structure of cartilaginous tissues is complex, collagen II fibrils and aggrecan are the most abundant assemblies in both articular cartilage (AC and the nucleus pulposus (NP of the intervertebral disc (IVD. Whilst structural heterogeneity of intact aggrecan ( containing three globular domains is well characterised, the extent of aggrecan fragmentation in healthy tissues is poorly defined. Using young, yet skeletally mature (18-30 months, bovine AC and NP tissues, it was shown that, whilst the ultrastructure of intact aggrecan was tissue-dependent, most molecules (AC: 95 %; NP: 99.5 % were fragmented (lacking one or more globular domains. Fragments were significantly smaller and more structurally heterogeneous in the NP compared with the AC (molecular area; AC: 8543 nm2; NP: 4625 nm2; p < 0.0001. In contrast, fibrillar collagen appeared structurally intact and tissue-invariant. Molecular fragmentation is considered indicative of a pathology; however, these young, skeletally mature tissues were histologically and mechanically (reduced modulus: AC: ≈ 500 kPa; NP: ≈ 80 kPa comparable to healthy tissues and devoid of notable gelatinase activity (compared with rat dermis. As aggrecan fragmentation was prevalent in neonatal bovine AC (99.5 % fragmented, molecular area: 5137 nm2 as compared with mature AC (95.0 % fragmented, molecular area: 8667 nm2, it was hypothesised that targeted proteolysis might be an adaptive process that modified aggrecan packing (as simulated computationally and, hence, tissue charge density, mechanical properties and porosity. These observations provided a baseline against which pathological and/or age-related fragmentation of aggrecan could be assessed and suggested that new strategies might be required to engineer constructs that mimic the mechanical properties of native cartilaginous tissues.

Extracellular matrix organization in various regions of rat brain grey matter.

Science.gov (United States)

Brückner, G; Härtig, W; Kacza, J; Seeger, J; Welt, K; Brauer, K

1996-05-01

Previous studies revealed the concentration of extracellular matrix proteoglycans in the so-called perineuronal nets on the one hand and in certain zones of the neuropil on the other. This nonhomogeneous distribution suggested a non-random chemical and spatial heterogeneity of the extracellular space. In the present investigation, regions dominated by one of both distribution patterns, i.e. piriform and parietal cortex, reticular thalamic nucleus, medial septum/diagonal band complex and cerebellar nuclei, were selected for correlative light and electron microscopic analysis. The labelling was performed by the use of the N-acetylgalactosamine-binding plant lectin Wisteria floribunda agglutinin visualized by peroxidase staining and additionally by photoconversion of red carbocyanine fluorescence labelling for electron microscopy. The intense labelling of the neuropil of a superficial piriform region, presumably identical with sublayer Ia, was confined to a fine meshwork spreading over the extracellular space between non-myelinated axons, dendrites and glial profiles. In the reticular thalamic nucleus the neuronal cell bodies were embedded in zones of labelled neuropil. In contrast to these patterns, the labelled extracellular matrix in different cortical layers and in the other subcortical regions was concentrated in perineuronal nets as large accumulations at surface areas of the neuronal perikarya and dendrites and the attached presynaptic boutons. Astrocytic processes usually were separated from the neuronal surface by the interposed extracellular material. Despite a great variability, the width of the extracellular space containing the labelled matrix components in all perineuronal nets appeared to be considerably larger than that in the labelled zones of neuropil and the non-labelled microenvironment of other neurons. Our results support the view that differences expressed in topographical and spatial peculiarities of the extracellular matrix constituents are

Modeling extracellular electrical stimulation: I. Derivation and interpretation of neurite equations.

Science.gov (United States)

Meffin, Hamish; Tahayori, Bahman; Grayden, David B; Burkitt, Anthony N

2012-12-01

Neuroprosthetic devices, such as cochlear and retinal implants, work by directly stimulating neurons with extracellular electrodes. This is commonly modeled using the cable equation with an applied extracellular voltage. In this paper a framework for modeling extracellular electrical stimulation is presented. To this end, a cylindrical neurite with confined extracellular space in the subthreshold regime is modeled in three-dimensional space. Through cylindrical harmonic expansion of Laplace's equation, we derive the spatio-temporal equations governing different modes of stimulation, referred to as longitudinal and transverse modes, under types of boundary conditions. The longitudinal mode is described by the well-known cable equation, however, the transverse modes are described by a novel ordinary differential equation. For the longitudinal mode, we find that different electrotonic length constants apply under the two different boundary conditions. Equations connecting current density to voltage boundary conditions are derived that are used to calculate the trans-impedance of the neurite-plus-thin-extracellular-sheath. A detailed explanation on depolarization mechanisms and the dominant current pathway under different modes of stimulation is provided. The analytic results derived here enable the estimation of a neurite's membrane potential under extracellular stimulation, hence bypassing the heavy computational cost of using numerical methods.

Compression instrument for tissue experiments (cite) at the meso-scale: device validation - biomed 2011.

Science.gov (United States)

Evans, Douglas W; Rajagopalan, Padma; Devita, Raffaella; Sparks, Jessica L

2011-01-01

Liver sinusoidal endothelial cells (LSECs) are the primary site of numerous transport and exchange processes essential for liver function. LSECs rest on a sparse extracellular matrix layer housed in the space of Disse, a 0.5-1LSECs from hepatocytes. To develop bioengineered liver tissue constructs, it is important to understand the mechanical interactions among LSECs, hepatocytes, and the extracellular matrix in the space of Disse. Currently the mechanical properties of space of Disse matrix are not well understood. The objective of this study was to develop and validate a device for performing mechanical tests at the meso-scale (100nm-100m), to enable novel matrix characterization within the space of Disse. The device utilizes a glass micro-spherical indentor attached to a cantilever made from a fiber optic cable. The 3-axis translation table used to bring the specimen in contact with the indentor and deform the cantilever. A position detector monitors the location of a laser passing through the cantilever and allows for the calculation of subsequent tissue deformation. The design allows micro-newton and nano-newton stress-strain tissue behavior to be quantified. To validate the device accuracy, 11 samples of silicon rubber in two formulations were tested to experimentally confirm their Young's moduli. Prior macroscopic unconfined compression tests determined the formulations of EcoFlex030 (n-6) and EcoFlex010 (n-5) to posses Young's moduli of 92.67+-6.22 and 43.10+-3.29 kPa respectively. Optical measurements taken utilizing CITE's position control and fiber optic cantilever found the moduli to be 106.4 kPa and 47.82 kPa.

Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase.

Directory of Open Access Journals (Sweden)

Patrizia Pellegatti

2008-07-01

Full Text Available There is growing awareness that tumour cells build up a "self-advantageous" microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP.Measurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours.Our results show that ATP in the tumour interstitium is in the hundreds micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.

Tissue equivalent detector measurements on Mir space station. Comparison with other data

Energy Technology Data Exchange (ETDEWEB)

Bottollier-Depois, J.F. [CEA Centre d`Etudes de Fontenay-aux-Roses, 92 (France). Dept. de Protection de la Sante de l`Homme et de Dosimetrie; Siegrist, M. [Centre National d`Etudes Spatiales (CNES), 31 - Toulouse (France); Duvivier, E.; Almarcha, B. [STEEL Technologies, Mazeres sur Salat (France); Dachev, T.P.; Semkova, J.V. [Bulgarian Academy of Sciences, Sofia (Bulgaria). Central Lab. of Solar Energy and New Energy Sources; Petrov, V.M.; Bengin, V.; Koslova, S.B. [Institute of Biomedical Problems, Moscow (Russian Federation)

1995-12-31

The measurement of the dose received by the cosmonauts, due to cosmic radiations, during a space mission is an important parameter to estimate the radiological risk. Tissue equivalent measurements of radiation environment inside the MIR space station were performed continuously since July 1992. Interesting results about radiation measurements show (a) the South Atlantic Anomaly (SAA) crossing, (c) the increase of radiation near the poles and (d) the effects of solar eruptions. These data are compared with solid state detector (SSD) and other tissue equivalent proportional counter (TEPC) results. (authors). 4 refs., 7 figs.

Tissue equivalent detector measurements on Mir space station. Comparison with other data

International Nuclear Information System (INIS)

Bottollier-Depois, J.F.; Duvivier, E.; Almarcha, B.; Dachev, T.P.; Semkova, J.V.

1995-01-01

The measurement of the dose received by the cosmonauts, due to cosmic radiations, during a space mission is an important parameter to estimate the radiological risk. Tissue equivalent measurements of radiation environment inside the MIR space station were performed continuously since July 1992. Interesting results about radiation measurements show (a) the South Atlantic Anomaly (SAA) crossing, (c) the increase of radiation near the poles and (d) the effects of solar eruptions. These data are compared with solid state detector (SSD) and other tissue equivalent proportional counter (TEPC) results. (authors). 4 refs., 7 figs

An investigation of the influence of extracellular matrix anisotropy and cell–matrix interactions on tissue architecture

KAUST Repository

Dyson, R. J.

2015-09-02

© 2015 Springer-Verlag Berlin Heidelberg Mechanical interactions between cells and the fibrous extracellular matrix (ECM) in which they reside play a key role in tissue development. Mechanical cues from the environment (such as stress, strain and fibre orientation) regulate a range of cell behaviours, including proliferation, differentiation and motility. In turn, the ECM structure is affected by cells exerting forces on the matrix which result in deformation and fibre realignment. In this paper we develop a mathematical model to investigate this mechanical feedback between cells and the ECM. We consider a three-phase mixture of collagen, culture medium and cells, and formulate a system of partial differential equations which represents conservation of mass and momentum for each phase. This modelling framework takes into account the anisotropic mechanical properties of the collagen gel arising from its fibrous microstructure. We also propose a cell–collagen interaction force which depends upon fibre orientation and collagen density. We use a combination of numerical and analytical techniques to study the influence of cell–ECM interactions on pattern formation in tissues. Our results illustrate the wide range of structures which may be formed, and how those that emerge depend upon the importance of cell–ECM interactions.

An investigation of the influence of extracellular matrix anisotropy and cell–matrix interactions on tissue architecture

KAUST Repository

Dyson, R. J.; Green, J. E. F.; Whiteley, J. P.; Byrne, H. M.

2015-01-01

© 2015 Springer-Verlag Berlin Heidelberg Mechanical interactions between cells and the fibrous extracellular matrix (ECM) in which they reside play a key role in tissue development. Mechanical cues from the environment (such as stress, strain and fibre orientation) regulate a range of cell behaviours, including proliferation, differentiation and motility. In turn, the ECM structure is affected by cells exerting forces on the matrix which result in deformation and fibre realignment. In this paper we develop a mathematical model to investigate this mechanical feedback between cells and the ECM. We consider a three-phase mixture of collagen, culture medium and cells, and formulate a system of partial differential equations which represents conservation of mass and momentum for each phase. This modelling framework takes into account the anisotropic mechanical properties of the collagen gel arising from its fibrous microstructure. We also propose a cell–collagen interaction force which depends upon fibre orientation and collagen density. We use a combination of numerical and analytical techniques to study the influence of cell–ECM interactions on pattern formation in tissues. Our results illustrate the wide range of structures which may be formed, and how those that emerge depend upon the importance of cell–ECM interactions.

Serum HER 2 extracellular domain level is correlated with tissue HER 2 status in metastatic gastric or gastro-oesophageal junction adenocarcinoma.

Directory of Open Access Journals (Sweden)

Shu-Qin Dai

Full Text Available BACKGROUND: To explore the association between serum human epidermal growth factor receptor 2 (HER 2 extracellular domain (ECD levels and tissue HER 2 status in metastatic gastric cancer. PATIENTS AND METHODS: HER 2 status was retrospectively analyzed in 219 advanced gastric or gastroesophageal junction (GEJ patients. Serum HER 2 ECD was measured by chemiluminescent assay and tissue HER 2 was assessed by fluorescent in situ hybridisation (FISH and immunohistochemistry (IHC assay. RESULTS: Significant associations were found between serum HER 2 ECD levels and tissue HER 2 status. Twenty-four patients had HER 2 ECD levels >16.35 ng/mL, which has a sensitivity of 51.4% and a specificity of 97.3% to predict tissue HER 2 status. When the cut-off value was increased to 22 ng/mL, then all 12 patients with serum HER 2 ECD levels>22 ng/mL were tissue HER 2 positive, corresponding to a specificity of 100% and a sensitivity of 32.4%. High serum HER 2 ECD levels were strongly associated with the intestinal histological type (Lauren's classification, liver metastasis, multiple metastasis (>2 and increased LDH levels, but not with overall survival. CONCLUSIONS: The high specificity of the serum HER 2 ECD assay in predicting tissue HER 2 status suggests its potential as a surrogate marker of the HER 2 status in gastric cancer.

The effect of extracellular calcium and inorganic phosphate on the growth and osteogenic differentiation of mesenchymal stem cells in vitro: implication for bone tissue engineering

Energy Technology Data Exchange (ETDEWEB)

Liu Yukan; Lu Qiaozhi; Ji Huijiao; Zhao Xiaoli; Zhang Ming [College of Life Science, ZheJiang University, Hangzhou 310058 (China); Pei Rui [XiJing Hospital, Fourth Military Medical University, Xi An 710032 (China); Zhou Guoshun [Huzhou Central Hospital, Huzhou, Zhejiang Province 313000 (China); Tang, Rui Kang, E-mail: Lyk_cn@yahoo.com.c [Department of Chemistry, College of Science, ZheJiang University, Hangzhou 310027 (China)

2009-04-15

The aim of this study is to demonstrate the effect of extracellular calcium ion (Ca{sup 2+}) and inorganic phosphate (Pi) concentrations on the growth and differentiation of bone-marrow-derived mesenchymal stem cells (MSCs), which is essential to understand the interaction between calcium phosphate ceramic (CPC) scaffolds and seeded cells during the construction of tissue-engineered bones. MSCs were separated from rabbits and cultured in media with different concentrations of Ca{sup 2+} and Pi supplements. Their proliferation, apoptosis, mineralization and osteogenic differentiation were determined by the MTT assay, TUNEL assay, Vonkossa stain and RT-PCR examination. A two-way ANOVA calculation with comparisons of estimated marginal means by LSD was used for statistical analysis. Results showed that the optimal extracellular Ca{sup 2+} and Pi concentrations for the cells to proliferate and differentiate were 1.8 mM and 0.09 mM, respectively, which are the concentrations supplied in many commonly used culture media such as DMEM and alpha-MEM. Cell proliferation and differentiation decreased significantly with greater or lower concentrations of the Pi supplement. Greater Pi concentrations also led to significant cell apoptosis. Greater Ca{sup 2+} concentrations did not change cell proliferation but significantly inhibited cell differentiation. In addition, greater Ca{sup 2+} concentrations could significantly enhance cell mineralization. In conclusion, extracellular Ca{sup 2+} and Pi significantly influence the growth and osteogenic differentiation of MSCs. It is important to take the cellular effect of Ca{sup 2+} and Pi into consideration when designing or constructing scaffolds for bone tissue engineering with CPC.

Biodistribution, Uptake and Effects Caused by Cancer-Derived Extracellular Vesicles

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Lilite Sadovska

2015-03-01

Full Text Available Extracellular vesicles (EVs have recently emerged as important mediators of intercellular communication. They are released in the extracellular space by a variety of normal and cancerous cell types and have been found in all human body fluids. Cancer-derived EVs have been shown to carry lipids, proteins, mRNAs, non-coding and structural RNAs and even extra-chromosomal DNA, which can be taken up by recipient cells and trigger diverse physiological and pathological responses. An increasing body of evidence suggests that cancer-derived EVs mediate paracrine signalling between cancer cells. This leads to the increased invasiveness, proliferation rate and chemoresistance, as well as the acquisition of the cancer stem cell phenotype. This stimulates angiogenesis and the reprogramming of normal stromal cells into cancer-promoting cell types. Furthermore, cancer-derived EVs contribute to the formation of the pre-metastatic niche and modulation of anti-tumour immune response. However, as most of these data are obtained by in vitro studies, it is not entirely clear which of these effects are recapitulated in vivo . In the current review, we summarize studies that assess the tissue distribution, trafficking, clearance and uptake of cancer-derived EVs in vivo and discuss the impact they have, both locally and systemically.

Brain infection with Staphylococcus aureus leads to high extracellular levels of glutamate, aspartate, γ-aminobutyric acid, and zinc.

Science.gov (United States)

Hassel, Bjørnar; Dahlberg, Daniel; Mariussen, Espen; Goverud, Ingeborg Løstegaard; Antal, Ellen-Ann; Tønjum, Tone; Maehlen, Jan

2014-12-01

Staphylococcal brain infections may cause mental deterioration and epileptic seizures, suggesting interference with normal neurotransmission in the brain. We injected Staphylococcus aureus into rat striatum and found an initial 76% reduction in the extracellular level of glutamate as detected by microdialysis at 2 hr after staphylococcal infection. At 8 hr after staphylococcal infection, however, the extracellular level of glutamate had increased 12-fold, and at 20 hr it had increased >30-fold. The extracellular level of aspartate and γ-aminobutyric acid (GABA) also increased greatly. Extracellular Zn(2+) , which was estimated at ∼2.6 µmol/liter in the control situation, was increased by 330% 1-2.5 hr after staphylococcal infection and by 100% at 8 and 20 hr. The increase in extracellular glutamate, aspartate, and GABA appeared to reflect the degree of tissue damage. The area of tissue damage greatly exceeded the area of staphylococcal infiltration, pointing to soluble factors being responsible for cell death. However, the N-methyl-D-aspartate receptor antagonist MK-801 ameliorated neither tissue damage nor the increase in extracellular neuroactive amino acids, suggesting the presence of neurotoxic factors other than glutamate and aspartate. In vitro staphylococci incubated with glutamine and glucose formed glutamate, so bacteria could be an additional source of infection-related glutamate. We conclude that the dramatic increase in the extracellular concentration of neuroactive amino acids and zinc could interfere with neurotransmission in the surrounding brain tissue, contributing to mental deterioration and a predisposition to epileptic seizures, which are often seen in brain abscess patients. © 2014 Wiley Periodicals, Inc.

Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies.

Science.gov (United States)

Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Collin, Estelle; Rochev, Yury; Rodriguez, Brian J; Gorelov, Alexander; Dillon, Simon; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I

2015-03-04

Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro.

Decreased extracellular pH inhibits osteogenesis through proton-sensing GPR4-mediated suppression of yes-associated protein.

Science.gov (United States)

Tao, Shi-Cong; Gao, You-Shui; Zhu, Hong-Yi; Yin, Jun-Hui; Chen, Yi-Xuan; Zhang, Yue-Lei; Guo, Shang-Chun; Zhang, Chang-Qing

2016-06-03

The pH of extracellular fluids is a basic property of the tissue microenvironment and is normally maintained at 7.40 ± 0.05 in humans. Many pathological circumstances, such as ischemia, inflammation, and tumorigenesis, result in the reduction of extracellular pH in the affected tissues. In this study, we reported that the osteogenic differentiation of BMSCs was significantly inhibited by decreases in the extracellular pH. Moreover, we demonstrated that proton-sensing GPR4 signaling mediated the proton-induced inhibitory effects on the osteogenesis of BMSCs. Additionally, we found that YAP was the downstream effector of GPR4 signaling. Our findings revealed that the extracellular pH modulates the osteogenic responses of BMSCs by regulating the proton-sensing GPR4-YAP pathway.

The Impact of Alpha-Syntrophin Deletion on the Changes in Tissue Structure and Extracellular Diffusion Associated with Cell Swelling under Physiological and Pathological Conditions

Czech Academy of Sciences Publication Activity Database

Dmytrenko, Lesia; Cicanič, Michal; Anděrová, Miroslava; Voříšek, Ivan; Ottersen, O. P.; Syková, Eva; Vargová, Lýdia

2013-01-01

Roč. 8, č. 7 (2013), e68044 E-ISSN 1932-6203 R&D Projects: GA ČR GA309/09/1597; GA ČR(CZ) GAP303/11/2378; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:68378041 Keywords : extracellular space * diffusion parameters * cell swelling Subject RIV: FH - Neurology Impact factor: 3.534, year: 2013

Extracellular Matrix components regulate cellular polarity and tissue structure in the developing and mature Retina

Directory of Open Access Journals (Sweden)

Shweta Varshney

2015-01-01

Full Text Available While genetic networks and other intrinsic mechanisms regulate much of retinal development, interactions with the extracellular environment shape these networks and modify their output. The present review has focused on the role of one family of extracellular matrix molecules and their signaling pathways in retinal development. In addition to their effects on the developing retina, laminins play a role in maintaining Müller cell polarity and compartmentalization, thereby contributing to retinal homeostasis. This article which is intended for the clinical audience, reviews the fundamentals of retinal development, extracellular matrix organization and the role of laminins in retinal development. The role of laminin in cortical development is also briefly discussed.

Distribution volumes of macromolecules in human ovarian and endometrial cancers--effects of extracellular matrix structure.

Science.gov (United States)

Haslene-Hox, Hanne; Oveland, Eystein; Woie, Kathrine; Salvesen, Helga B; Tenstad, Olav; Wiig, Helge

2015-01-01

Elements of the extracellular matrix (ECM), notably collagen and glucosaminoglycans, will restrict part of the space available for soluble macromolecules simply because the molecules cannot occupy the same space. This phenomenon may influence macromolecular drug uptake. To study the influence of steric and charge effects of the ECM on the distribution volumes of macromolecules in human healthy and malignant gynecologic tissues we used as probes 15 abundant plasma proteins quantified by high-resolution mass spectrometry. The available distribution volume (VA) of albumin was increased in ovarian carcinoma compared with healthy ovarian tissue. Furthermore, VA of plasma proteins between 40 and 190 kDa decreased with size for endometrial carcinoma and healthy ovarian tissue, but was independent of molecular weight for the ovarian carcinomas. An effect of charge on distribution volume was only found in healthy ovaries, which had lower hydration and high collagen content, indicating that a condensed interstitium increases the influence of negative charges. A number of earlier suggested biomarker candidates were detected in increased amounts in malignant tissue, e.g., stathmin and spindlin-1, showing that interstitial fluid, even when unfractionated, can be a valuable source for tissue-specific proteins. We demonstrate that the distribution of abundant plasma proteins in the interstitium can be elucidated by mass spectrometry methods and depends markedly on hydration and ECM structure. Our data can be used in modeling of drug uptake, and give indications on ECM components to be targeted to increase the uptake of macromolecular substances. Copyright © 2015 the American Physiological Society.

Brain washing : Transport of cerebral extracellular fluids and solutes

NARCIS (Netherlands)

Bedussi, B.

2017-01-01

Regulation of extracellular volume and fluid composition provides a robust microenvironment for brain cells. In peripheral tissue, fluid surplus and solutes are removed from the interstitium via drainage into lymphatic channels. Since the central nervous system lacks a proper lymphatic vasculature,

Effect of Urea and Thiourea on Generation of Xenogeneic Extracellular Matrix Scaffolds for Tissue Engineering

Science.gov (United States)

Wong, Maelene L.; Wong, Janelle L.; Horn, Rebecca M.; Sannajust, Kimberley C.; Rice, Dawn A.

2016-01-01

Effective solubilization of proteins by chaotropes in proteomic applications motivates their use in solubilization-based antigen removal/decellularization strategies. A high urea concentration has previously been reported to significantly reduce lipophilic antigen content of bovine pericardium (BP); however, structure and function of the resultant extracellular matrix (ECM) scaffold were compromised. It has been recently demonstrated that in vivo ECM scaffold fate is determined by two primary outcome measures as follows: (1) sufficient reduction in antigen content to avoid graft-specific adaptive immune responses and (2) maintenance of native ECM structural proteins to avoid graft-specific innate responses. In this work, we assessed residual antigenicity, ECM architecture, ECM content, thermal stability, and tensile properties of BP subjected to a gradient of urea concentrations to determine whether an intermediate concentration exists at which both antigenicity and structure–function primary outcome measures for successful in vivo scaffold outcome can simultaneously be achieved. Alteration in tissue structure–function properties at various urea concentrations with decreased effectiveness for antigen removal makes use of urea-mediated antigen removal unlikely to be suitable for functional scaffold generation. PMID:27230226

A human pericardium biopolymeric scaffold for autologous heart valve tissue engineering: cellular and extracellular matrix structure and biomechanical properties in comparison with a normal aortic heart valve.

Science.gov (United States)

Straka, Frantisek; Schornik, David; Masin, Jaroslav; Filova, Elena; Mirejovsky, Tomas; Burdikova, Zuzana; Svindrych, Zdenek; Chlup, Hynek; Horny, Lukas; Daniel, Matej; Machac, Jiri; Skibová, Jelena; Pirk, Jan; Bacakova, Lucie

2018-04-01

The objective of our study was to compare the cellular and extracellular matrix (ECM) structure and the biomechanical properties of human pericardium (HP) with the normal human aortic heart valve (NAV). HP tissues (from 12 patients) and NAV samples (from 5 patients) were harvested during heart surgery. The main cells in HP were pericardial interstitial cells, which are fibroblast-like cells of mesenchymal origin similar to the valvular interstitial cells in NAV tissue. The ECM of HP had a statistically significantly (p structures of the two tissues, the dense part of fibrous HP (49 ± 2%) and the lamina fibrosa of NAV (47 ± 4%), was similar. In both tissues, the secant elastic modulus (Es) was significantly lower in the transversal direction (p structure and has the biomechanical properties required for a tissue from which an autologous heart valve replacement may be constructed.

Single proteins that serve linked functions in intracellular and extracellular microenvironments

Energy Technology Data Exchange (ETDEWEB)

Radisky, Derek C.; Stallings-Mann, Melody; Hirai, Yohei; Bissell, Mina J.

2009-06-03

Maintenance of organ homeostasis and control of appropriate response to environmental alterations requires intimate coordination of cellular function and tissue organization. An important component of this coordination may be provided by proteins that can serve distinct, but linked, functions on both sides of the plasma membrane. Here we present a novel hypothesis in which non-classical secretion can provide a mechanism through which single proteins can integrate complex tissue functions. Single genes can exert a complex, dynamic influence through a number of different processes that act to multiply the function of the gene product(s). Alternative splicing can create many different transcripts that encode proteins of diverse, even antagonistic, function from a single gene. Posttranslational modifications can alter the stability, activity, localization, and even basic function of proteins. A protein can exist in different subcellular localizations. More recently, it has become clear that single proteins can function both inside and outside the cell. These proteins often lack defined secretory signal sequences, and transit the plasma membrane by mechanisms separate from the classical ER/Golgi secretory process. When examples of such proteins are examined individually, the multifunctionality and lack of a signal sequence are puzzling - why should a protein with a well known function in one context function in such a distinct fashion in another? We propose that one reason for a single protein to perform intracellular and extracellular roles is to coordinate organization and maintenance of a global tissue function. Here, we describe in detail three specific examples of proteins that act in this fashion, outlining their specific functions in the extracellular space and in the intracellular space, and we discuss how these functions may be linked. We present epimorphin/syntaxin-2, which may coordinate morphogenesis of secretory organs (as epimorphin) with control of

Laser surface modification of decellularized extracellular cartilage matrix for cartilage tissue engineering.

Science.gov (United States)

Goldberg-Bockhorn, Eva; Schwarz, Silke; Subedi, Rachana; Elsässer, Alexander; Riepl, Ricarda; Walther, Paul; Körber, Ludwig; Breiter, Roman; Stock, Karl; Rotter, Nicole

2018-02-01

The implantation of autologous cartilage as the gold standard operative procedure for the reconstruction of cartilage defects in the head and neck region unfortunately implicates a variety of negative effects at the donor site. Tissue-engineered cartilage appears to be a promising alternative. However, due to the complex requirements, the optimal material is yet to be determined. As demonstrated previously, decellularized porcine cartilage (DECM) might be a good option to engineer vital cartilage. As the dense structure of DECM limits cellular infiltration, we investigated surface modifications of the scaffolds by carbon dioxide (CO 2 ) and Er:YAG laser application to facilitate the migration of chondrocytes inside the scaffold. After laser treatment, the scaffolds were seeded with human nasal septal chondrocytes and analyzed with respect to cell migration and formation of new extracellular matrix proteins. Histology, immunohistochemistry, SEM, and TEM examination revealed an increase of the scaffolds' surface area with proliferation of cell numbers on the scaffolds for both laser types. The lack of cytotoxic effects was demonstrated by standard cytotoxicity testing. However, a thermal denaturation area seemed to hinder the migration of the chondrocytes inside the scaffolds, even more so after CO 2 laser treatment. Therefore, the Er:YAG laser seemed to be better suitable. Further modifications of the laser adjustments or the use of alternative laser systems might be advantageous for surface enlargement and to facilitate migration of chondrocytes into the scaffold in one step.

Extrasynaptic transmission and the diffusion parameters of the extracellular space

Czech Academy of Sciences Publication Activity Database

Syková, Eva; Vargová, Lýdia

2008-01-01

Roč. 52, 1-2 (2008), s. 5-13 ISSN 0197-0186 R&D Projects: GA MŠk(CZ) LC554 Grant - others:GA ČR(CZ) GA309/04/0753 Institutional research plan: CEZ:AV0Z50390512 Keywords : Diffusion * Extracellular volume * Magnetic resonance Subject RIV: FH - Neurology Impact factor: 3.228, year: 2008

Group B Streptococcus Induces Neutrophil Recruitment to Gestational Tissues and Elaboration of Extracellular Traps and Nutritional Immunity.

Science.gov (United States)

Kothary, Vishesh; Doster, Ryan S; Rogers, Lisa M; Kirk, Leslie A; Boyd, Kelli L; Romano-Keeler, Joann; Haley, Kathryn P; Manning, Shannon D; Aronoff, David M; Gaddy, Jennifer A

2017-01-01

Streptococcus agalactiae , or Group B Streptococcus (GBS), is a gram-positive bacterial pathogen associated with infection during pregnancy and is a major cause of morbidity and mortality in neonates. Infection of the extraplacental membranes surrounding the developing fetus, a condition known as chorioamnionitis, is characterized histopathologically by profound infiltration of polymorphonuclear cells (PMNs, neutrophils) and greatly increases the risk for preterm labor, stillbirth, or neonatal GBS infection. The advent of animal models of chorioamnionitis provides a powerful tool to study host-pathogen relationships in vivo and ex vivo . The purpose of this study was to evaluate the innate immune response elicited by GBS and evaluate how antimicrobial strategies elaborated by these innate immune cells affect bacteria. Our work using a mouse model of GBS ascending vaginal infection during pregnancy reveals that clinically isolated GBS has the capacity to invade reproductive tissues and elicit host immune responses including infiltration of PMNs within the choriodecidua and placenta during infection, mirroring the human condition. Upon interacting with GBS, murine neutrophils elaborate DNA-containing extracellular traps, which immobilize GBS and are studded with antimicrobial molecules including lactoferrin. Exposure of GBS to holo- or apo-forms of lactoferrin reveals that the iron-sequestration activity of lactoferrin represses GBS growth and viability in a dose-dependent manner. Together, these data indicate that the mouse model of ascending infection is a useful tool to recapitulate human models of GBS infection during pregnancy. Furthermore, this work reveals that neutrophil extracellular traps ensnare GBS and repress bacterial growth via deposition of antimicrobial molecules, which drive nutritional immunity via metal sequestration strategies.

Extracellular Matrix Hydrogel Derived from Human Umbilical Cord as a Scaffold for Neural Tissue Repair and Its Comparison with Extracellular Matrix from Porcine Tissues

Czech Academy of Sciences Publication Activity Database

Kočí, Zuzana; Výborný, Karel; Dubišová, Jana; Vacková, Irena; Jäger, Aleš; Lunov, Oleg; Jiráková, Klára; Kubinová, Šárka

2017-01-01

Roč. 23, č. 6 (2017), s. 333-345 ISSN 1937-3384 R&D Projects: GA ČR(CZ) GA15-01396S; GA MŠk(CZ) LO1309; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) EF15_003/0000419 Grant - others:AV ČR(CZ) Fellowship J. E. Purkyně Institutional support: RVO:68378041 ; RVO:68378271 Keywords : extracellular matrix * hydrogel * umbilical cord Subject RIV: FH - Neurology; EB - Genetics ; Molecular Biology (FZU-D) OBOR OECD: Neurosciences (including psychophysiology; Biophysics (FZU-D)

Extracellular matrix component signaling in cancer

DEFF Research Database (Denmark)

Multhaupt, Hinke A. B.; Leitinger, Birgit; Gullberg, Donald

2016-01-01

Cell responses to the extracellular matrix depend on specific signaling events. These are important from early development, through differentiation and tissue homeostasis, immune surveillance, and disease pathogenesis. Signaling not only regulates cell adhesion cytoskeletal organization and motil...... as well as matrix constitution and protein crosslinking. Here we summarize roles of the three major matrix receptor types, with emphasis on how they function in tumor progression. [on SciFinder(R)]...

Dynamic culture of a thermosensitive collagen hydrogel as an extracellular matrix improves the construction of tissue-engineered peripheral nerve.

Science.gov (United States)

Huang, Lanfeng; Li, Rui; Liu, Wanguo; Dai, Jin; Du, Zhenwu; Wang, Xiaonan; Ma, Jianchao; Zhao, Jinsong

2014-07-15

Tissue engineering technologies offer new treatment strategies for the repair of peripheral nerve injury, but cell loss between seeding and adhesion to the scaffold remains inevitable. A thermosensitive collagen hydrogel was used as an extracellular matrix in this study and combined with bone marrow mesenchymal stem cells to construct tissue-engineered peripheral nerve composites in vitro. Dynamic culture was performed at an oscillating frequency of 0.5 Hz and 35° swing angle above and below the horizontal plane. The results demonstrated that bone marrow mesenchymal stem cells formed membrane-like structures around the poly-L-lactic acid scaffolds and exhibited regular alignment on the composite surface. Collagen was used to fill in the pores, and seeded cells adhered onto the poly-L-lactic acid fibers. The DNA content of the bone marrow mesenchymal stem cells was higher in the composites constructed with a thermosensitive collagen hydrogel compared with that in collagen I scaffold controls. The cellular DNA content was also higher in the thermosensitive collagen hydrogel composites constructed with the thermosensitive collagen hydrogel in dynamic culture than that in static culture. These results indicate that tissue-engineered composites formed with thermosensitive collagen hydrogel in dynamic culture can maintain larger numbers of seeded cells by avoiding cell loss during the initial adhesion stage. Moreover, seeded cells were distributed throughout the material.

CELLULAR CONTROL OF CONNECTIVE TISSUE MATRIX TENSION†

Science.gov (United States)

Langevin, Helene M.; Nedergaard, Maiken; Howe, Alan

2013-01-01

The biomechanical behavior of connective tissue in response to stretching is generally attributed to the molecular composition and organization of its extracellular matrix. It also is becoming apparent that fibroblasts play an active role in regulating connective tissue tension. In response to static stretching of the tissue, fibroblasts expand within minutes by actively remodeling their cytoskeleton. This dynamic change in fibroblast shape contributes to the drop in tissue tension that occurs during viscoelastic relaxation. We propose that this response of fibroblasts plays a role in regulating extracellular fluid flow into the tissue, and protects against swelling when the matrix is stretched. This article reviews the evidence supporting possible mechanisms underlying this response including autocrine purinergic signaling. We also discuss fibroblast regulation of connective tissue tension with respect to lymphatic flow, immune function and cancer. PMID:23444198

Cellular control of connective tissue matrix tension.

Science.gov (United States)

Langevin, Helene M; Nedergaard, Maiken; Howe, Alan K

2013-08-01

The biomechanical behavior of connective tissue in response to stretching is generally attributed to the molecular composition and organization of its extracellular matrix. It also is becoming apparent that fibroblasts play an active role in regulating connective tissue tension. In response to static stretching of the tissue, fibroblasts expand within minutes by actively remodeling their cytoskeleton. This dynamic change in fibroblast shape contributes to the drop in tissue tension that occurs during viscoelastic relaxation. We propose that this response of fibroblasts plays a role in regulating extracellular fluid flow into the tissue, and protects against swelling when the matrix is stretched. This article reviews the evidence supporting possible mechanisms underlying this response including autocrine purinergic signaling. We also discuss fibroblast regulation of connective tissue tension with respect to lymphatic flow, immune function, and cancer. Copyright © 2013 Wiley Periodicals, Inc.

Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid.

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Hyong Woo Choi

2016-03-01

Full Text Available Damage-associated molecular pattern molecules (DAMPs signal the presence of tissue damage to induce immune responses in plants and animals. Here, we report that High Mobility Group Box 3 (HMGB3 is a novel plant DAMP. Extracellular HMGB3, through receptor-like kinases BAK1 and BKK1, induced hallmark innate immune responses, including i MAPK activation, ii defense-related gene expression, iii callose deposition, and iv enhanced resistance to Botrytis cinerea. Infection by necrotrophic B. cinerea released HMGB3 into the extracellular space (apoplast. Silencing HMGBs enhanced susceptibility to B. cinerea, while HMGB3 injection into apoplast restored resistance. Like its human counterpart, HMGB3 binds salicylic acid (SA, which results in inhibition of its DAMP activity. An SA-binding site mutant of HMGB3 retained its DAMP activity, which was no longer inhibited by SA, consistent with its reduced SA-binding activity. These results provide cross-kingdom evidence that HMGB proteins function as DAMPs and that SA is their conserved inhibitor.

Biochemical and biomechanical properties of the pacemaking sinoatrial node extracellular matrix are distinct from contractile left ventricular matrix.

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Jessica M Gluck

Full Text Available Extracellular matrix plays a role in differentiation and phenotype development of its resident cells. Although cardiac extracellular matrix from the contractile tissues has been studied and utilized in tissue engineering, extracellular matrix properties of the pacemaking sinoatrial node are largely unknown. In this study, the biomechanical properties and biochemical composition and distribution of extracellular matrix in the sinoatrial node were investigated relative to the left ventricle. Extracellular matrix of the sinoatrial node was found to be overall stiffer than that of the left ventricle and highly heterogeneous with interstitial regions composed of predominantly fibrillar collagens and rich in elastin. The extracellular matrix protein distribution suggests that resident pacemaking cardiomyocytes are enclosed in fibrillar collagens that can withstand greater tensile strength while the surrounding elastin-rich regions may undergo deformation to reduce the mechanical strain in these cells. Moreover, basement membrane-associated adhesion proteins that are ligands for integrins were of low abundance in the sinoatrial node, which may decrease force transduction in the pacemaking cardiomyocytes. In contrast to extracellular matrix of the left ventricle, extracellular matrix of the sinoatrial node may reduce mechanical strain and force transduction in pacemaking cardiomyocytes. These findings provide the criteria for a suitable matrix scaffold for engineering biopacemakers.

Increased tenascin C and Toll-like receptor 4 levels in visceral adipose tissue as a link between inflammation and extracellular matrix remodeling in obesity.

Science.gov (United States)

Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Rotellar, Fernando; Valentí, Victor; Silva, Camilo; Gil, María J; Salvador, Javier; Frühbeck, Gema

2012-10-01

Obesity is associated with an altered inflammatory and extracellular matrix (ECM) profile. Tenascin C (TNC) is an ECM glycoprotein with proinflammatory effects. We aimed to explore the expression levels of TNC in adipose tissue analyzing the contribution of adipocytes and stromovascular fraction cells (SVFC) as well as its impact on inflammation and ECM regulation. We also analyzed the effect of the stimulation with TNF-α and lipopolysaccharide (LPS) on both SVFC and adipocytes. Samples obtained from 75 subjects were used in the study. Expression levels of TNC, TLR4, MMP2, and MMP9 were analyzed in visceral adipose tissue (VAT) as well as in both adipocytes and SVFC. In addition, Tnc expression was measured in two mice models of obesity. We show, for the first time, that VAT expression levels of TNC are increased in normoglycemic and type 2 diabetic obese patients (Pobese patients with nonalcoholic steatohepatitis (Pobesity were significantly increased (Pexogenous TNC induced (Pobesity via visceral adipose tissue inflammation representing a link with ECM remodeling.

EXTRACELLULAR VESICLES: CLASSIFICATION, FUNCTIONS AND CLINICAL RELEVANCE

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A. V. Oberemko

2014-12-01

Full Text Available This review presents a generalized definition of vesicles as bilayer extracellular organelles of all celular forms of life: not only eu-, but also prokaryotic. The structure and composition of extracellular vesicles, history of research, nomenclature, their impact on life processes in health and disease are discussed. Moreover, vesicles may be useful as clinical instruments for biomarkers, and they are promising as biotechnological drug. However, many questions in this area are still unresolved and need to be addressed in the future. The most interesting from the point of view of practical health care represents a direction to study the effect of exosomes and microvesicles in the development and progression of a particular disease, the possibility of adjusting the pathological process by means of extracellular vesicles of a particular type, acting as an active ingredient. Relevant is the further elucidation of the role and importance of exosomes to the surrounding cells, tissues and organs at the molecular level, the prospects for the use of non-cellular vesicles as biomarkers of disease.

Investigation of periodontal tissue during a long space flights

Science.gov (United States)

Solovyeva, Zoya; Viacheslav, Ilyin; Skedina, Marina

Previous studies conducted on the International Space Station found that upon completion of the space flight there are significant changes in the local immunity and periodontal microflora of astronauts. Also research in ground-based experiments that simulate space flight factors showed that prolonged hypokinesia antiorthostatic leads to impaired functional indicators of the periodontal vascular system, an unidirectional change from the microbiota and the immune system. That results in the appearance and progressive increase of the parodontial pathogenic bacteria and increase of the content of immunoglobulins in the oral fluid. All these changes are classified as risk factors for the development of inflammatory periodontal diseases in astronauts. However, the studies were unable to determine whether the changes result from a long space flight and the peculiarities of formation the local immunity and periodontal microbiota during the space flight, or they are one of the specific manifestations of the readaptationary post-flight condition of the body. In this regard, the planned research in a long space flight suggests: to use the means of microbial control, which can retain of the anaerobes periodontal microbiota sampling directly in the space flight; to assess the specificity of changes of the periodontal immune status under the influence of the space flight factors, and to assess the state of microcirculation of periodontal tissue in astronauts. A comprehensive study of the reaction of dentition during the space flight will make it possible to study the pathogenesis of changes for developing an adequate prevention aimed at optimizing the state of dentition of the astronauts.

Integration of concepts: cardiac extracellular matrix remodeling after myocardial infarction

NARCIS (Netherlands)

Cleutjens, Jack P. M.; Creemers, Esther E. J. M.

2002-01-01

The cardiac extracellular matrix consists of a three-dimensional structural network of interstitial collagens to which other matrix components are attached. The main physiological functions of this network are to retain tissue integrity and cardiac pump function. Collagen deposition is controlled

Managing Brain Extracellular K(+) during Neuronal Activity

DEFF Research Database (Denmark)

Larsen, Brian Roland; Stoica, Anca; MacAulay, Nanna

2016-01-01

characteristics required to fulfill their distinct physiological roles in clearance of K(+) from the extracellular space in the face of neuronal activity. Understanding the nature, impact and effects of the various Na(+)/K(+)-ATPase isoform combinations in K(+) management in the central nervous system might...... understanding of the pathological events occurring during disease....

Group B Streptococcus Induces Neutrophil Recruitment to Gestational Tissues and Elaboration of Extracellular Traps and Nutritional Immunity

Science.gov (United States)

Kothary, Vishesh; Doster, Ryan S.; Rogers, Lisa M.; Kirk, Leslie A.; Boyd, Kelli L.; Romano-Keeler, Joann; Haley, Kathryn P.; Manning, Shannon D.; Aronoff, David M.; Gaddy, Jennifer A.

2017-01-01

Streptococcus agalactiae, or Group B Streptococcus (GBS), is a gram-positive bacterial pathogen associated with infection during pregnancy and is a major cause of morbidity and mortality in neonates. Infection of the extraplacental membranes surrounding the developing fetus, a condition known as chorioamnionitis, is characterized histopathologically by profound infiltration of polymorphonuclear cells (PMNs, neutrophils) and greatly increases the risk for preterm labor, stillbirth, or neonatal GBS infection. The advent of animal models of chorioamnionitis provides a powerful tool to study host-pathogen relationships in vivo and ex vivo. The purpose of this study was to evaluate the innate immune response elicited by GBS and evaluate how antimicrobial strategies elaborated by these innate immune cells affect bacteria. Our work using a mouse model of GBS ascending vaginal infection during pregnancy reveals that clinically isolated GBS has the capacity to invade reproductive tissues and elicit host immune responses including infiltration of PMNs within the choriodecidua and placenta during infection, mirroring the human condition. Upon interacting with GBS, murine neutrophils elaborate DNA-containing extracellular traps, which immobilize GBS and are studded with antimicrobial molecules including lactoferrin. Exposure of GBS to holo- or apo-forms of lactoferrin reveals that the iron-sequestration activity of lactoferrin represses GBS growth and viability in a dose-dependent manner. Together, these data indicate that the mouse model of ascending infection is a useful tool to recapitulate human models of GBS infection during pregnancy. Furthermore, this work reveals that neutrophil extracellular traps ensnare GBS and repress bacterial growth via deposition of antimicrobial molecules, which drive nutritional immunity via metal sequestration strategies. PMID:28217556

Oxygen and Glucose Deprivation Induces Bergmann Glia Membrane Depolarization and Ca2+ Rises Mainly Mediated by K+ and ATP Increases in the Extracellular Space

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Romain Helleringer

2017-11-01

Full Text Available During brain ischemia, intense energy deficiency induces a complex succession of events including pump failure, acidosis and exacerbated glutamate release. In the cerebellum, glutamate is the principal mediator of Purkinje neuron anoxic depolarization during episodes of oxygen and glucose deprivation (OGD. Here, the impact of OGD is studied in Bergmann glia, specialized astrocytes closely associated to Purkinje neurons. Patch clamp experiments reveal that during OGD Bergmann glial cells develop a large depolarizing current that is not mediated by glutamate and purinergic receptors but is mainly due to the accumulation of K+ in the extracellular space. Furthermore, we also found that increases in the intracellular Ca2+ concentration appear in Bergmann glia processes several minutes following OGD. These elevations require, in an early phase, Ca2+ mobilization from internal stores via P2Y receptor activation, and, over longer periods, Ca2+ entry through store-operated calcium channels. Our results suggest that increases of K+ and ATP concentrations in the extracellular space are primordial mediators of the OGD effects on Bergmann glia. In the cerebellum, glial responses to energy deprivation-triggering events are therefore highly likely to follow largely distinct rules from those of their neuronal counterparts.

Evaluation of Bioelectrical Impedance Spectroscopy for the Assessment of Extracellular Body Water

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Sören Weyer

2012-01-01

Full Text Available This study evaluates bioelectrical impedance spectroscopy (BIS measurements to detect body fluid status. The multifrequency impedance measurements were performed in five female pigs. Animals were connected to an extracorporeal membrane oxygenation device during a lung disease experiment and fluid balance was recorded. Every 15 min the amount of fluid infusion and the weight of the urine drainage bag was recorded. From the fluid intake and output, the fluid balance was calculated. These data were compared with values calculated from a mathematical model, based on the extracellular tissue resistance and the Hanai Mixture theory. The extracellular tissue resistance was also measured with BIS. These experimental results strongly support the feasibility and clinical value of BIS for in vivo assessment of the hydration status.

Extracellular matrix proteins: a positive feedback loop in lung fibrosis?

NARCIS (Netherlands)

Blaauboer, M.E.; van Boeijen, F.R.; Emson, C.L.; Turner, S.M.; Zandieh-Doulabi, B.; Hanemaaijer, R.; Smit, T.H.; Stoop, R.; Everts, V.

2014-01-01

Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the

Extracellular matrix proteins: A positive feedback loop in lung fibrosis?

NARCIS (Netherlands)

Blaauboer, M.E.; Boeijen, F.R.; Emson, C.L.; Turner, S.M.; Zandieh-Doulabi, B.; Hanemaaijer, R.; Smit, T.H.; Stoop, R.; Everts, V.

2014-01-01

Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the

Neoproteoglycans in tissue engineering

Science.gov (United States)

Weyers, Amanda; Linhardt, Robert J.

2014-01-01

Proteoglycans, comprised of a core protein to which glycosaminoglycan chains are covalently linked, are an important structural and functional family of macromolecules found in the extracellular matrix. Advances in our understanding of biological interactions have lead to a greater appreciation for the need to design tissue engineering scaffolds that incorporate mimetics of key extracellular matrix components. A variety of synthetic and semisynthetic molecules and polymers have been examined by tissue engineers that serve as structural, chemical and biological replacements for proteoglycans. These proteoglycan mimetics have been referred to as neoproteoglycans and serve as functional and therapeutic replacements for natural proteoglycans that are often unavailable for tissue engineering studies. Although neoproteoglycans have important limitations, such as limited signaling ability and biocompatibility, they have shown promise in replacing the natural activity of proteoglycans through cell and protein binding interactions. This review focuses on the recent in vivo and in vitro tissue engineering applications of three basic types of neoproteoglycan structures, protein–glycosaminoglycan conjugates, nano-glycosaminoglycan composites and polymer–glycosaminoglycan complexes. PMID:23399318

Astrocytes and extracellular matrix in extrasynaptic volume transmission

Czech Academy of Sciences Publication Activity Database

Vargová, Lýdia; Syková, Eva

2014-01-01

Roč. 369, č. 1654 (2014) ISSN 0962-8436 R&D Projects: GA ČR GA13-11867S; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:68378041 Keywords : extracellular space * diffusion * astrocytes Subject RIV: FH - Neurology Impact factor: 7.055, year: 2014

Autonomous informational stability in connective tissues.

Science.gov (United States)

Brand, R A

1992-02-01

No coherent theories currently explain connective tissue stability (i.e. 'memory') as well as spatial and temporal adaptability in the face of continual flux of its constituents. Furthermore, explanations of stability based exclusively upon DNA raise certain inherent problems, particularly with the spatial concordance of somatic tissues. As an alternative explanation, it is hypothesized that while connective tissue cells produce extracellular protein precursors through DNA-dependent processes, the assembly, location, orientation and configuration of the extracellular macromolecules as well as their degree of cell attachment depend primarily upon local micro-environmental conditions and/or self-organization rather than strictly cellular processes. The resulting extracellular matrix (ECM) serves as a time- and spatially-variable filter about each cell to afford a relatively consistent micro-environment for all similar cells, regardless of the more variable macro-environment. By insuring a consistent set of signals to the cell, the filter provides a non-genetic memory complementary to genetic memory. The half-lives of constituent molecules define the duration of the filter, allowing the filter to adapt to new environmental demands, yet to maintain a consistent milieu for the cell. The cell/matrix construct permits local, self-optimizing, non-deterministic tissue autonomy obviating the need to postulate certain intricate mechanisms coordinating spatial morphology and temporal behavior.

Trash or Treasure: extracellular microRNAs and cell-to-cell communication

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Nobuyoshi eKosaka

2013-09-01

Full Text Available Circulating RNAs in human body fluids are promising candidates for diagnostic purposes. However, the biological significance of circulating RNAs remains elusive. Recently, small non-coding RNAs, microRNAs (miRNAs, were isolated from multiple human body fluids, and these circulating miRNAs have been implicated as novel disease biomarkers. Concurrently, miRNAs were also identified in the extracellular space associated with extracellular vesicles (EVs, which are small membrane vesicles secreted from various types of cells. The function of these secreted miRNAs has been revealed in several papers. Circulating miRNAs have been experimentally found to be associated with EVs, however, other types of extracellular miRNAs were also described. This review discusses studies related to extracellular miRNAs, including circulating miRNAs and secreted miRNAs, to highlight the importance of studying not only secreted miRNAs but also circulating miRNAs to determine the contribution of extracellular miRNAs especially in cancer development.

Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo.

Science.gov (United States)

Westman, Johannes; Papareddy, Praveen; Dahlgren, Madelene W; Chakrakodi, Bhavya; Norrby-Teglund, Anna; Smeds, Emanuel; Linder, Adam; Mörgelin, Matthias; Johansson-Lindbom, Bengt; Egesten, Arne; Herwald, Heiko

2015-12-01

The innate immune system relies to a great deal on the interaction of pattern recognition receptors with pathogen- or damage-associated molecular pattern molecules. Extracellular histones belong to the latter group and their release has been described to contribute to the induction of systemic inflammatory reactions. However, little is known about their functions in the early immune response to an invading pathogen. Here we show that extracellular histones specifically target monocytes in human blood and this evokes the mobilization of the chemotactic chemokines CXCL9 and CXCL10 from these cells. The chemokine induction involves the toll-like receptor 4/myeloid differentiation factor 2 complex on monocytes, and is under the control of interferon-γ. Consequently, subcutaneous challenge with extracellular histones results in elevated levels of CXCL10 in a murine air pouch model and an influx of leukocytes to the site of injection in a TLR4 dependent manner. When analyzing tissue biopsies from patients with necrotizing fasciitis caused by Streptococcus pyogenes, extracellular histone H4 and CXCL10 are immunostained in necrotic, but not healthy tissue. Collectively, these results show for the first time that extracellular histones have an important function as chemoattractants as their local release triggers the recruitment of immune cells to the site of infection.

3D Space Radiation Transport in a Shielded ICRU Tissue Sphere

Science.gov (United States)

Wilson, John W.; Slaba, Tony C.; Badavi, Francis F.; Reddell, Brandon D.; Bahadori, Amir A.

2014-01-01

A computationally efficient 3DHZETRN code capable of simulating High Charge (Z) and Energy (HZE) and light ions (including neutrons) under space-like boundary conditions with enhanced neutron and light ion propagation was recently developed for a simple homogeneous shield object. Monte Carlo benchmarks were used to verify the methodology in slab and spherical geometry, and the 3D corrections were shown to provide significant improvement over the straight-ahead approximation in some cases. In the present report, the new algorithms with well-defined convergence criteria are extended to inhomogeneous media within a shielded tissue slab and a shielded tissue sphere and tested against Monte Carlo simulation to verify the solution methods. The 3D corrections are again found to more accurately describe the neutron and light ion fluence spectra as compared to the straight-ahead approximation. These computationally efficient methods provide a basis for software capable of space shield analysis and optimization.

Tissue engineering

CERN Document Server

Fisher, John P; Bronzino, Joseph D

2007-01-01

Increasingly viewed as the future of medicine, the field of tissue engineering is still in its infancy. As evidenced in both the scientific and popular press, there exists considerable excitement surrounding the strategy of regenerative medicine. To achieve its highest potential, a series of technological advances must be made. Putting the numerous breakthroughs made in this field into a broad context, Tissue Engineering disseminates current thinking on the development of engineered tissues. Divided into three sections, the book covers the fundamentals of tissue engineering, enabling technologies, and tissue engineering applications. It examines the properties of stem cells, primary cells, growth factors, and extracellular matrix as well as their impact on the development of tissue engineered devices. Contributions focus on those strategies typically incorporated into tissue engineered devices or utilized in their development, including scaffolds, nanocomposites, bioreactors, drug delivery systems, and gene t...

A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue.

Science.gov (United States)

Azaripour, Adriano; Lagerweij, Tonny; Scharfbillig, Christina; Jadczak, Anna Elisabeth; Willershausen, Brita; Van Noorden, Cornelis J F

2016-08-01

For 3-dimensional (3D) imaging of a tissue, 3 methodological steps are essential and their successful application depends on specific characteristics of the type of tissue. The steps are 1° clearing of the opaque tissue to render it transparent for microscopy, 2° fluorescence labeling of the tissues and 3° 3D imaging. In the past decades, new methodologies were introduced for the clearing steps with their specific advantages and disadvantages. Most clearing techniques have been applied to the central nervous system and other organs that contain relatively low amounts of connective tissue including extracellular matrix. However, tissues that contain large amounts of extracellular matrix such as dermis in skin or gingiva are difficult to clear. The present survey lists methodologies that are available for clearing of tissues for 3D imaging. We report here that the BABB method using a mixture of benzyl alcohol and benzyl benzoate and iDISCO using dibenzylether (DBE) are the most successful methods for clearing connective tissue-rich gingiva and dermis of skin for 3D histochemistry and imaging of fluorescence using light-sheet microscopy. Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.

Next Generation Tissue Engineering of Orthopedic Soft Tissue-to-Bone Interfaces

Science.gov (United States)

Boys, Alexander J.; McCorry, Mary Clare; Rodeo, Scott; Bonassar, Lawrence J.; Estroff, Lara A.

2017-01-01

Soft tissue-to-bone interfaces are complex structures that consist of gradients of extracellular matrix materials, cell phenotypes, and biochemical signals. These interfaces, called entheses for ligaments, tendons, and the meniscus, are crucial to joint function, transferring mechanical loads and stabilizing orthopedic joints. When injuries occur to connected soft tissue, the enthesis must be re-established to restore function, but due to structural complexity, repair has proven challenging. Tissue engineering offers a promising solution for regenerating these tissues. This prospective review discusses methodologies for tissue engineering the enthesis, outlined in three key design inputs: materials processing methods, cellular contributions, and biochemical factors. PMID:29333332

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

Science.gov (United States)

Chiavegatti, T; Costa, V L; Araújo, M S; Godinho, R O

2007-01-01

Background and purpose: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. Experimental approach: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Key results: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5′-AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5′-AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Conclusions and implications: Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine. PMID:18157164

Extracellular deoxyribonuclease production by periodontal bacteria.

Science.gov (United States)

Palmer, L J; Chapple, I L C; Wright, H J; Roberts, A; Cooper, P R

2012-08-01

Whilst certain bacteria have long been known to secrete extracellular deoxyribonuclease (DNase), the purpose in microbial physiology was unclear. Recently, however, this enzyme has been demonstrated to confer enhanced virulence, enabling bacteria to evade the host's immune defence of extruded DNA/chromatin filaments, termed neutrophil extracellular traps (NETs). As NETs have recently been identified in infected periodontal tissue, the aim of this study was to screen periodontal bacteria for extracellular DNase activity. To determine whether DNase activity was membrane bound or secreted, 34 periodontal bacteria were cultured in broth and on agar plates. Pelleted bacteria and supernatants from broth cultures were analysed for their ability to degrade DNA, with relative activity levels determined using an agarose gel electrophoresis assay. Following culture on DNA-supplemented agar, expression was determined by the presence of a zone of hydrolysis and DNase activity related to colony size. Twenty-seven bacteria, including red and orange complex members Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, Prevotella intermedia, Streptococcus constellatus, Campylobacter rectus and Prevotella nigrescens, were observed to express extracellular DNase activity. Differences in DNase activity were noted, however, when bacteria were assayed in different culture states. Analysis of the activity of secreted DNase from bacterial broth cultures confirmed their ability to degrade NETs. The present study demonstrates, for the first time, that DNase activity is a relatively common property of bacteria associated with advanced periodontal disease. Further work is required to determine the importance of this bacterial DNase activity in the pathogenesis of periodontitis. © 2011 John Wiley & Sons A/S.

An Update to Space Biomedical Research: Tissue Engineering in Microgravity Bioreactors

Directory of Open Access Journals (Sweden)

Abolfazl Barzegari

2012-03-01

Full Text Available Introduction: The severe need for constructing replacement tissues in organ transplantation has necessitated the development of tissue engineering approaches and bioreactors that can bring these approaches to reality. The inherent limitations of conventional bioreactors in generating realistic tissue constructs led to the devise of the microgravity tissue engineering that uses Rotating Wall Vessel (RWV bioreactors initially developed by NASA. Methods: In this review article, we intend to highlight some major advances and accomplishments in the rapidly-growing field of tissue engineering that could not be achieved without using microgravity. Results: Research is now focused on assembly of 3 dimensional (3D tissue fragments from various cell types in human body such as chondrocytes, osteoblasts, embryonic and mesenchymal stem cells, hepatocytes and pancreas islet cells. Hepatocytes cultured under microgravity are now being used in extracorporeal bioartificial liver devices. Tissue constructs can be used not only in organ replacement therapy, but also in pharmaco-toxicology and food safety assessment. 3D models of various cancers may be used in studying cancer development and biology or in high-throughput screening of anticancer drug candidates. Finally, 3D heterogeneous assemblies from cancer/immune cells provide models for immunotherapy of cancer. Conclusion: Tissue engineering in (simulated microgravity has been one of the stunning impacts of space research on biomedical sciences and their applications on earth.

Three-dimensional bioprinting of thick vascularized tissues

Science.gov (United States)

Kolesky, David B.; Homan, Kimberly A.; Skylar-Scott, Mark A.; Lewis, Jennifer A.

2016-03-01

The advancement of tissue and, ultimately, organ engineering requires the ability to pattern human tissues composed of cells, extracellular matrix, and vasculature with controlled microenvironments that can be sustained over prolonged time periods. To date, bioprinting methods have yielded thin tissues that only survive for short durations. To improve their physiological relevance, we report a method for bioprinting 3D cell-laden, vascularized tissues that exceed 1 cm in thickness and can be perfused on chip for long time periods (>6 wk). Specifically, we integrate parenchyma, stroma, and endothelium into a single thick tissue by coprinting multiple inks composed of human mesenchymal stem cells (hMSCs) and human neonatal dermal fibroblasts (hNDFs) within a customized extracellular matrix alongside embedded vasculature, which is subsequently lined with human umbilical vein endothelial cells (HUVECs). These thick vascularized tissues are actively perfused with growth factors to differentiate hMSCs toward an osteogenic lineage in situ. This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.

The effect of low-dose neutron irradiation on extracellular matrix

International Nuclear Information System (INIS)

Chen Tiehe; Lu Yongjie; Chai Mingsheng; Peng Wulin; Yang Yifang; Pan Yan; Chen Jinguo

2003-01-01

Projective: To study the effect of neutron irradiation on extracellular matrix. Methods: 120 male wistar rats were divided into four groups at random, and then exposed to neutron of 252 Cf-source at the doses of 0, 0.29, 0.62 and 1.20 Gy, respectively. After the exposure of 3 days, 1 month and 2 months, the rats were sacrificed and lung tissue specimens stored at -30 degree C. Hyaluronan, laminin, type III procollagen and type IV collagen in the lung tissue were detected by the method of radioimmunoassay. Results: The differences of the levels of hyaluronan in lung tissue among the groups were unsignificant. The levels of laminin in 0.29, 0.62 and 1.20 Gy groups after the 3-day exposure were remarkably different to those of the control group, and unable to recover completely even 2 months after the exposure. The levels of type IV collagen in higher three irradiated groups were all higher, but not significantly. The levels of type III procollagen in the early stage after exposure were higher, and later they lowered. Conclusion: The levels of some components of extracellular matrix in the lung tissue of rat can be changed by low-dose of neutron irradiation, but their variational modes and degrees depend on the dose of neutron irradiation and the length of period after exposure

Maintenance of the Extracellular Matrix in Rat Anterior Pituitary Gland: Identification of Cells Expressing Tissue Inhibitors of Metalloproteinases.

Science.gov (United States)

Azuma, Morio; Tofrizal, Alimuddin; Maliza, Rita; Batchuluun, Khongorzul; Ramadhani, Dini; Syaidah, Rahimi; Tsukada, Takehiro; Fujiwara, Ken; Kikuchi, Motoshi; Horiguchi, Kotaro; Yashiro, Takashi

2015-12-25

The extracellular matrix (ECM) is important in creating cellular environments in tissues. Recent studies have demonstrated that ECM components are localized in anterior pituitary cells and affect cell activity. Thus, clarifying the mechanism responsible for ECM maintenance would improve understanding of gland function. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of matrix metalloproteinases and participate in ECM degradation. In this study, we investigated whether cells expressing TIMPs are present in rat anterior pituitary gland. Reverse transcription polymerase chain reaction was used to analyze expression of the TIMP family (TIMP1-4), and cells producing TIMPs in the gland were identified by using in situ hybridization. Expression of TIMP1, TIMP2, and TIMP3 mRNAs was detected, and the TIMP-expressing cells were located in the gland. The TIMP-expressing cells were also investigated by means of double-staining with in situ hybridization and immunohistochemical techniques. Double-staining revealed that TIMP1 mRNA was expressed in folliculostellate cells. TIMP2 mRNA was detected in folliculostellate cells, prolactin cells, and thyroid-stimulating hormone cells. TIMP3 mRNA was identified in endothelial cells, pericytes, novel desmin-immunopositive perivascular cells, and folliculostellate cells. These findings indicate that TIMP1-, TIMP2-, and TIMP3-expressing cells are present in rat anterior pituitary gland and that they are involved in maintaining ECM components.

Maintenance of the Extracellular Matrix in Rat Anterior Pituitary Gland: Identification of Cells Expressing Tissue Inhibitors of Metalloproteinases

International Nuclear Information System (INIS)

Azuma, Morio; Tofrizal, Alimuddin; Maliza, Rita; Batchuluun, Khongorzul; Ramadhani, Dini; Syaidah, Rahimi; Tsukada, Takehiro; Fujiwara, Ken; Kikuchi, Motoshi; Horiguchi, Kotaro; Yashiro, Takashi

2015-01-01

The extracellular matrix (ECM) is important in creating cellular environments in tissues. Recent studies have demonstrated that ECM components are localized in anterior pituitary cells and affect cell activity. Thus, clarifying the mechanism responsible for ECM maintenance would improve understanding of gland function. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of matrix metalloproteinases and participate in ECM degradation. In this study, we investigated whether cells expressing TIMPs are present in rat anterior pituitary gland. Reverse transcription polymerase chain reaction was used to analyze expression of the TIMP family (TIMP1-4), and cells producing TIMPs in the gland were identified by using in situ hybridization. Expression of TIMP1, TIMP2, and TIMP3 mRNAs was detected, and the TIMP-expressing cells were located in the gland. The TIMP-expressing cells were also investigated by means of double-staining with in situ hybridization and immunohistochemical techniques. Double-staining revealed that TIMP1 mRNA was expressed in folliculostellate cells. TIMP2 mRNA was detected in folliculostellate cells, prolactin cells, and thyroid-stimulating hormone cells. TIMP3 mRNA was identified in endothelial cells, pericytes, novel desmin-immunopositive perivascular cells, and folliculostellate cells. These findings indicate that TIMP1-, TIMP2-, and TIMP3-expressing cells are present in rat anterior pituitary gland and that they are involved in maintaining ECM components

The interplay between tissue growth and scaffold degradation in engineered tissue constructs

KAUST Repository

O’ Dea, R. D.; Osborne, J. M.; El Haj, A. J.; Byrne, H. M.; Waters, S. L.

2012-01-01

of the engineered tissue are appropriate for the in vivo environment. Achieving this goal will require detailed understanding of the interplay between cell proliferation, extracellular matrix (ECM) deposition and scaffold degradation. In this paper, we use a

Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate

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Villa-Bellosta, Ricardo; Hamczyk, Magda R.; Andrés, Vicente

2017-01-01

Purpose Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues. Methods and results High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate. Conclusion We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification. PMID:28362852

Association of Bordetella dermonecrotic toxin with the extracellular matrix

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Miyake Masami

2010-09-01

Full Text Available Abstract Background Bordetella dermonecrotic toxin (DNT causes the turbinate atrophy in swine atrophic rhinitis, caused by a Bordetella bronchiseptica infection of pigs, by inhibiting osteoblastic differentiation. The toxin is not actively secreted from the bacteria, and is presumed to be present in only small amounts in infected areas. How such small amounts can affect target tissues is unknown. Results Fluorescence microscopy revealed that DNT associated with a fibrillar structure developed on cultured cells. A cellular component cross-linked with DNT conjugated with a cross-linker was identified as fibronectin by mass spectrometry. Colocalization of the fibronectin network on the cells with DNT was also observed by fluorescence microscope. Several lines of evidence suggested that DNT interacts with fibronectin not directly, but through another cellular component that remains to be identified. The colocalization was observed in not only DNT-sensitive cells but also insensitive cells, indicating that the fibronectin network neither serves as a receptor for the toxin nor is involved in the intoxicating procedures. The fibronectin network-associated toxin was easily liberated when the concentration of toxin in the local environment decreased, and was still active. Conclusions Components in the extracellular matrix are known to regulate activities of various growth factors by binding and liberating them in response to alterations in the extracellular environment. Similarly, the fibronectin-based extracellular matrix may function as a temporary storage system for DNT, enabling small amounts of the toxin to efficiently affect target tissues or cells.

Biomaterials for Tissue Engineering

Science.gov (United States)

Lee, Esther J.; Kasper, F. Kurtis; Mikos, Antonios G.

2013-01-01

Biomaterials serve as an integral component of tissue engineering. They are designed to provide architectural framework reminiscent of native extracellular matrix in order to encourage cell growth and eventual tissue regeneration. Bone and cartilage represent two distinct tissues with varying compositional and mechanical properties. Despite these differences, both meet at the osteochondral interface. This article presents an overview of current biomaterials employed in bone and cartilage applications, discusses some design considerations, and alludes to future prospects within this field of research. PMID:23820768

Cathepsin K in Lymphangioleiomyomatosis: LAM Cell-Fibroblast Interactions Enhance Protease Activity by Extracellular Acidification.

Science.gov (United States)

Dongre, Arundhati; Clements, Debbie; Fisher, Andrew J; Johnson, Simon R

2017-08-01

Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells and fibroblasts form lung nodules and it is hypothesized that LAM nodule-derived proteases cause cyst formation and tissue damage. On protease gene expression profiling in whole lung tissue, cathepsin K gene expression was 40-fold overexpressed in LAM compared with control lung tissue (P ≤ 0.0001). Immunohistochemistry confirmed cathepsin K protein was expressed in LAM but not control lungs. Cathepsin K gene expression and protein and protease activity were detected in LAM-associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immunoreactivity predominantly co-localized with LAM-associated fibroblasts. In vitro, fibroblast extracellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced at pH 7 and 6. 621-101 cells reduced extracellular pH with acidification dependent on 621-101 mechanistic target of rapamycin activity and net hydrogen ion exporters, particularly sodium bicarbonate co-transporters and carbonic anhydrases, which were also expressed in LAM lung tissue. In LAM cell-fibroblast co-cultures, acidification paralleled cathepsin K activity, and both were reduced by sodium bicarbonate co-transporter (P ≤ 0.0001) and carbonic anhydrase inhibitors (P = 0.0021). Our findings suggest that cathepsin K activity is dependent on LAM cell-fibroblast interactions, and inhibitors of extracellular acidification may be potential therapies for LAM. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Fibronectin distribution in epithelial and associated tissues of the rat

DEFF Research Database (Denmark)

Couchman, J R; Gibson, W T; Thom, D

1979-01-01

Specific antiserum was used to investigate the distribution of the extracellular glycoprotein, fibronectin, in rat skin and tongue tissue by light and electron microscopy with immunofluorescence and immunoperoxidase techniques. We conclude that fibronectin is absent from stable, differentiated...... parts of tissues, such as the sebaceous glands or the matrix, medulla, cortex, and cuticles of the hair and the inner and outer root sheaths, or even in tissues in which there is some cell movement, such as the epidermis. It is, however, characteristic of sites at which cell division is occurring...... in contact with an extracellular scaffolding, such as basement membrane or loose connective tissue. Conspicuous examples were in the glassy membrane and connective tissue sheath associated with the follicular epithelium, the basement membrane underlying vascular endothelial cells, the connective tissues...

Region-Specific Effect of the Decellularized Meniscus Extracellular Matrix on Mesenchymal Stem Cell-Based Meniscus Tissue Engineering.

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Shimomura, Kazunori; Rothrauff, Benjamin B; Tuan, Rocky S

2017-03-01

The meniscus is the most commonly injured knee structure, and surgical repair is often ineffective. Tissue engineering-based repair or regeneration may provide a needed solution. Decellularized, tissue-derived extracellular matrices (ECMs) have received attention for their potential use as tissue-engineered scaffolds. In considering meniscus-derived ECMs (mECMs) for meniscus tissue engineering, it is noteworthy that the inner and outer regions of the meniscus have different structural and biochemical features, potentially directing the differentiation of cells toward region-specific phenotypes. To investigate the applicability of mECMs for meniscus tissue engineering by specifically comparing region-dependent effects of mECMs on 3-dimensional constructs seeded with human bone marrow mesenchymal stem cells (hBMSCs). Controlled laboratory study. Bovine menisci were divided into inner and outer halves and were minced, treated with Triton X-100 and DNase, and extracted with urea. Then, hBMSCs (1 × 10 6 cells/mL) were encapsulated in a photo-cross-linked 10% polyethylene glycol diacrylate scaffold containing mECMs (60 μg/mL) derived from either the inner or outer meniscus, with an ECM-free scaffold as a control. The cell-seeded constructs were cultured with chondrogenic medium containing recombinant human transforming growth factor β3 (TGF-β3) and were analyzed for expression of meniscus-associated genes as well as for the collagen (hydroxyproline) and glycosaminoglycan content as a function of time. Decellularization was verified by the absence of 4',6-diamidino-2-phenylindole (DAPI)-stained cell nuclei and a reduction in the DNA content. Quantitative real-time polymerase chain reaction showed that collagen type I expression was significantly higher in the outer mECM group than in the other groups, while collagen type II and aggrecan expression was highest in the inner mECM group. The collagen (hydroxyproline) content was highest in the outer mECM group, while the

The extracellular redox state modulates mitochondrial function, gluconeogenesis, and glycogen synthesis in murine hepatocytes.

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Nocito, Laura; Kleckner, Amber S; Yoo, Elsia J; Jones Iv, Albert R; Liesa, Marc; Corkey, Barbara E

2015-01-01

Circulating redox state changes, determined by the ratio of reduced/oxidized pairs of different metabolites, have been associated with metabolic diseases. However, the pathogenic contribution of these changes and whether they modulate normal tissue function is unclear. As alterations in hepatic gluconeogenesis and glycogen metabolism are hallmarks that characterize insulin resistance and type 2 diabetes, we tested whether imposed changes in the extracellular redox state could modulate these processes. Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB)/acetoacetate (Acoc), reduced glutathione (GSH)/oxidized glutathione (GSSG), and cysteine/cystine. Exposure to a more oxidized ratio via extracellular βOHB/Acoc, GSH/GSSG, and cysteine/cystine in hepatocytes from fed mice increased intracellular hydrogen peroxide without causing oxidative damage. On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(P)H and maximal mitochondrial respiratory capacity in hepatocytes. Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis. In hepatocytes from fasted mice, a more extracellular reduced state of βOHB/Acoc led to increased alanine-stimulated gluconeogenesis and enhanced glycogen synthesis capacity from added glucose. Thus, we demonstrated for the first time that the extracellular redox state regulates the major metabolic functions of the liver and involves changes in intracellular NADH, hydrogen peroxide, and mitochondrial respiration. Because redox state in the blood can be communicated to all metabolically sensitive tissues, this work confirms the hypothesis that circulating redox state may be an important regulator of whole body metabolism and contribute to alterations associated with metabolic diseases.

The extracellular redox state modulates mitochondrial function, gluconeogenesis, and glycogen synthesis in murine hepatocytes.

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Laura Nocito

Full Text Available Circulating redox state changes, determined by the ratio of reduced/oxidized pairs of different metabolites, have been associated with metabolic diseases. However, the pathogenic contribution of these changes and whether they modulate normal tissue function is unclear. As alterations in hepatic gluconeogenesis and glycogen metabolism are hallmarks that characterize insulin resistance and type 2 diabetes, we tested whether imposed changes in the extracellular redox state could modulate these processes. Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB/acetoacetate (Acoc, reduced glutathione (GSH/oxidized glutathione (GSSG, and cysteine/cystine. Exposure to a more oxidized ratio via extracellular βOHB/Acoc, GSH/GSSG, and cysteine/cystine in hepatocytes from fed mice increased intracellular hydrogen peroxide without causing oxidative damage. On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(PH and maximal mitochondrial respiratory capacity in hepatocytes. Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis. In hepatocytes from fasted mice, a more extracellular reduced state of βOHB/Acoc led to increased alanine-stimulated gluconeogenesis and enhanced glycogen synthesis capacity from added glucose. Thus, we demonstrated for the first time that the extracellular redox state regulates the major metabolic functions of the liver and involves changes in intracellular NADH, hydrogen peroxide, and mitochondrial respiration. Because redox state in the blood can be communicated to all metabolically sensitive tissues, this work confirms the hypothesis that circulating redox state may be an important regulator of whole body metabolism and contribute to alterations associated with metabolic diseases.

ISEV position paper: extracellular vesicle RNA analysis and bioinformatics

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Andrew F. Hill

2013-12-01

Full Text Available Extracellular vesicles (EVs are the collective term for the various vesicles that are released by cells into the extracellular space. Such vesicles include exosomes and microvesicles, which vary by their size and/or protein and genetic cargo. With the discovery that EVs contain genetic material in the form of RNA (evRNA has come the increased interest in these vesicles for their potential use as sources of disease biomarkers and potential therapeutic agents. Rapid developments in the availability of deep sequencing technologies have enabled the study of EV-related RNA in detail. In October 2012, the International Society for Extracellular Vesicles (ISEV held a workshop on “evRNA analysis and bioinformatics.” Here, we report the conclusions of one of the roundtable discussions where we discussed evRNA analysis technologies and provide some guidelines to researchers in the field to consider when performing such analysis.

Synthetic osteogenic extracellular matrix formed by coated silicon dioxide nanosprings

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Hass Jamie L

2012-01-01

Full Text Available Abstract Background The design of biomimetic materials that parallel the morphology and biology of extracellular matrixes is key to the ability to grow functional tissues in vitro and to enhance the integration of biomaterial implants into existing tissues in vivo. Special attention has been put into mimicking the nanostructures of the extracellular matrix of bone, as there is a need to find biomaterials that can enhance the bonding between orthopedic devices and this tissue. Methods We have tested the ability of normal human osteoblasts to propagate and differentiate on silicon dioxide nanosprings, which can be easily grown on practically any surface. In addition, we tested different metals and metal alloys as coats for the nanosprings in tissue culture experiments with bone cells. Results Normal human osteoblasts grown on coated nanosprings exhibited an enhanced rate of propagation, differentiation into bone forming cells and mineralization. While osteoblasts did not attach effectively to bare nanowires grown on glass, these cells propagated successfully on nanosprings coated with titanium oxide and gold. We observed a 270 fold increase in the division rate of osteoblasts when grow on titanium/gold coated nanosprings. This effect was shown to be dependent on the nanosprings, as the coating by themselves did not alter the growth rate of osteoblast. We also observed that titanium/zinc/gold coated nanosprings increased the levels of osteoblast production of alkaline phosphatase seven folds. This result indicates that osteoblasts grown on this metal alloy coated nanosprings are differentiating to mature bone making cells. Consistent with this hypothesis, we showed that osteoblasts grown on the same metal alloy coated nanosprings have an enhanced ability to deposit calcium salt. Conclusion We have established that metal/metal alloy coated silicon dioxide nanosprings can be used as a biomimetic material paralleling the morphology and biology of

Methods to isolate extracellular vesicles for diagnosis

Science.gov (United States)

Kang, Hyejin; Kim, Jiyoon; Park, Jaesung

2017-12-01

Extracellular vesicles (EVs) are small membrane-bound bodies that are released into extracellular space by diverse cells, and are found in body fluids like blood, urine and saliva. EVs contain RNA, DNA and proteins, which can be biomarkers for diagnosis. EVs can be obtained by minimally-invasive biopsy, so they are useful in disease diagnosis. High yield and purity contribute to precise diagnosis of disease, but damaged EVs and impurities can cause confu sed results. However, EV isolation methods have different yields and purities. Furthermore, the isolation method that is most suitable to maximize EV recovery efficiency depends on the experimental conditions. This review focuses on merits and demerits of several types of EV isolation methods, and provides examples of how to diagnose disease by exploiting information obtained by analysis of EVs.

The matrikine N-α-PGP couples extracellular matrix fragmentation to endothelial permeability

NARCIS (Netherlands)

Hahn, Cornelia S; Scott, David W; Xu, Xin; Roda, Mojtaba Abdul; Payne, Gregory A; Wells, J Michael; Viera, Liliana; Winstead, Colleen J; Bratcher, Preston; Sparidans, Rolf W; Redegeld, Frank A; Jackson, Patricia L; Folkerts, Gert; Blalock, J Edwin; Patel, Rakesh P; Gaggar, Amit

2015-01-01

The compartmentalization and transport of proteins and solutes across the endothelium is a critical biologic function altered during inflammation and disease, leading to pathology in multiple disorders. The impact of tissue damage and subsequent extracellular matrix (ECM) fragmentation in regulating

Fabrication method, structure, mechanical, and biological properties of decellularized extracellular matrix for replacement of wide bone tissue defects.

Science.gov (United States)

Anisimova, N Y; Kiselevsky, M V; Sukhorukova, I V; Shvindina, N V; Shtansky, D V

2015-09-01

The present paper was focused on the development of a new method of decellularized extracellular matrix (DECM) fabrication via a chemical treatment of a native bone tissue. Particular attention was paid to the influence of chemical treatment on the mechanical properties of native bones, sterility, and biological performance in vivo using the syngeneic heterotopic and orthotopic implantation models. The obtained data indicated that after a chemical decellularization treatment in 4% aqueous sodium chlorite, no noticeable signs of the erosion of compact cortical bone surface or destruction of trabeculae of spongy bone in spinal channel were observed. The histological studies showed that the chemical treatment resulted in the decellularization of both bone and cartilage tissues. The DECM samples demonstrated no signs of chemical and biological degradation in vivo. Thorough structural characterization revealed that after decellularization, the mineral frame retained its integrity with the organic phase; however clotting and destruction of organic molecules and fibers were observed. FTIR studies revealed several structural changes associated with the destruction of organic molecules, although all organic components typical of intact bone were preserved. The decellularization-induced structural changes in the collagen constituent resulted changed the deformation under compression mechanism: from the major fracture by crack propagation throughout the sample to the predominantly brittle fracture. Although the mechanical properties of radius bones subjected to decellularization were observed to degrade, the mechanical properties of ulna bones in compression and humerus bones in bending remained unchanged. The compressive strength of both the intact and decellularized ulna bones was 125-130 MPa and the flexural strength of humerus bones was 156 and 145 MPa for the intact and decellularized samples, respectively. These results open new avenues for the use of DECM samples as

3D cell printing of in vitro stabilized skin model and in vivo pre-vascularized skin patch using tissue-specific extracellular matrix bioink: A step towards advanced skin tissue engineering.

Science.gov (United States)

Kim, Byoung Soo; Kwon, Yang Woo; Kong, Jeong-Sik; Park, Gyu Tae; Gao, Ge; Han, Wonil; Kim, Moon-Bum; Lee, Hyungseok; Kim, Jae Ho; Cho, Dong-Woo

2018-06-01

3D cell-printing technique has been under spotlight as an appealing biofabrication platform due to its ability to precisely pattern living cells in pre-defined spatial locations. In skin tissue engineering, a major remaining challenge is to seek for a suitable source of bioink capable of supporting and stimulating printed cells for tissue development. However, current bioinks for skin printing rely on homogeneous biomaterials, which has several shortcomings such as insufficient mechanical properties and recapitulation of microenvironment. In this study, we investigated the capability of skin-derived extracellular matrix (S-dECM) bioink for 3D cell printing-based skin tissue engineering. S-dECM was for the first time formulated as a printable material and retained the major ECM compositions of skin as well as favorable growth factors and cytokines. This bioink was used to print a full thickness 3D human skin model. The matured 3D cell-printed skin tissue using S-dECM bioink was stabilized with minimal shrinkage, whereas the collagen-based skin tissue was significantly contracted during in vitro tissue culture. This physical stabilization and the tissue-specific microenvironment from our bioink improved epidermal organization, dermal ECM secretion, and barrier function. We further used this bioink to print 3D pre-vascularized skin patch able to promote in vivo wound healing. In vivo results revealed that endothelial progenitor cells (EPCs)-laden 3D-printed skin patch together with adipose-derived stem cells (ASCs) accelerates wound closure, re-epithelization, and neovascularization as well as blood flow. We envision that the results of this paper can provide an insightful step towards the next generation source for bioink manufacturing. Copyright © 2018 Elsevier Ltd. All rights reserved.

Autocrine signal transmission with extracellular ligand degradation

International Nuclear Information System (INIS)

Muratov, C B; Posta, F; Shvartsman, S Y

2009-01-01

Traveling waves of cell signaling in epithelial layers orchestrate a number of important processes in developing and adult tissues. These waves can be mediated by positive feedback autocrine loops, a mode of cell signaling where binding of a diffusible extracellular ligand to a cell surface receptor can lead to further ligand release. We formulate and analyze a biophysical model that accounts for ligand-induced ligand release, extracellular ligand diffusion and ligand–receptor interaction. We focus on the case when the main mode for ligand degradation is extracellular and analyze the problem with the sharp threshold positive feedback nonlinearity. We derive expressions that link the speed of propagation and other characteristics of traveling waves to the parameters of the biophysical processes, such as diffusion rates, receptor expression level, etc. Analyzing the derived expressions we found that traveling waves in such systems can exhibit a number of unusual properties, e.g. non-monotonic dependence of the speed of propagation on ligand diffusivity. Our results for the fully developed traveling fronts can be used to analyze wave initiation from localized perturbations, a scenario that frequently arises in the in vitro models of epithelial wound healing, and guide future modeling studies of cell communication in epithelial layers

Autocrine signal transmission with extracellular ligand degradation

Science.gov (United States)

Muratov, C B; Posta, F; Shvartsman, S Y

2009-03-01

Traveling waves of cell signaling in epithelial layers orchestrate a number of important processes in developing and adult tissues. These waves can be mediated by positive feedback autocrine loops, a mode of cell signaling where binding of a diffusible extracellular ligand to a cell surface receptor can lead to further ligand release. We formulate and analyze a biophysical model that accounts for ligand-induced ligand release, extracellular ligand diffusion and ligand-receptor interaction. We focus on the case when the main mode for ligand degradation is extracellular and analyze the problem with the sharp threshold positive feedback nonlinearity. We derive expressions that link the speed of propagation and other characteristics of traveling waves to the parameters of the biophysical processes, such as diffusion rates, receptor expression level, etc. Analyzing the derived expressions we found that traveling waves in such systems can exhibit a number of unusual properties, e.g. non-monotonic dependence of the speed of propagation on ligand diffusivity. Our results for the fully developed traveling fronts can be used to analyze wave initiation from localized perturbations, a scenario that frequently arises in the in vitro models of epithelial wound healing, and guide future modeling studies of cell communication in epithelial layers.

Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres.

Science.gov (United States)

Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

2016-01-01

Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.

Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs in 3D Collagen Microspheres.

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Sejin Han

Full Text Available Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.

Three-Dimensional Microstructure of Biological Tissues during Freezing and Thawing

Science.gov (United States)

Ishiguro, Hiroshi; Horimizu, Takashi; Kataori, Akinobu; Kajigaya, Hiroshi

Three-dimensional behavior of ice crystals and cells during the freezing and thawing of biological tissues was investigated microscopically in real time by using a confocal laser scanning microscope(CLSM) and a fluorescent dye, acridine orange (AO). Fresh tender meat (2nd pectoral muscles) of chicken was stained with the AO in physiological saline to distinguish ice crystals and cells by their different colors, and then frozen and thawed under two different thermal protocols: a) slow-cooling and rapid-warming and b) rapid-cooling and rapid-warming. The CLSM noninvasively produced optical tomograms of the tissues to clarify the pattern of freezing, morphology of ice crystals in the tissues, and the interaction between ice crystals and cells. Also, the tissues were morphologically investigated by pathological means after the freezing and thawing. Typical freezing pattern during the slow-cooling was extracellular-freezing, and those during the rapid-cooling were extracellular-freezing and intracellular freezing with a lot of fine ice crystals in the cells. Cracks caused by the extracellular and intracellular ice crystals remained in the muscle tissues after the thawing. The results obtained by using the CLSM/dye method were consistent with pathologically morphological changes in the tissues through freezing and thawing.

SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity.

Science.gov (United States)

Barker, Thomas H; Baneyx, Gretchen; Cardó-Vila, Marina; Workman, Gail A; Weaver, Matt; Menon, Priya M; Dedhar, Shoukat; Rempel, Sandra A; Arap, Wadih; Pasqualini, Renata; Vogel, Viola; Sage, E Helene

2005-10-28

SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.

Recombinant heparan sulfate for use in tissue engineering applications

DEFF Research Database (Denmark)

Whitelock, J.; Ma, J.L.; Davies, N.

2008-01-01

Background: Heparan sulfate (HS) is an important component of many extracellular matrices that interacts with mitogens and morphogens to guide and control tissue and organ development. These interactions are controlled by its structure, which varies when produced by different cell types and diffe......Background: Heparan sulfate (HS) is an important component of many extracellular matrices that interacts with mitogens and morphogens to guide and control tissue and organ development. These interactions are controlled by its structure, which varies when produced by different cell types...

Managing brain extracellular K+ during neuronal activity: The physiological role of the Na+/K+-ATPase subunit isoforms

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Brian Roland eLarsen

2016-04-01

Full Text Available AbstractDuring neuronal activity in the brain, extracellular K+ rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K+ is the Na+/K+-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na+/K+-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K+ from neurons, whereas the neurons themselves become the primary K+ absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na+/K+-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K+ from the extracellular space in the face of neuronal activity.Understanding the nature, impact and effects of the various Na+/K+-ATPase isoform combinations in K+ management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2 and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood. This review addresses aspects of the Na+/K+-ATPase in the regulation of extracellular K+ in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease.

CELLULAR CONTROL OF CONNECTIVE TISSUE MATRIX TENSION†

OpenAIRE

Langevin, Helene M.; Nedergaard, Maiken; Howe, Alan

2013-01-01

The biomechanical behavior of connective tissue in response to stretching is generally attributed to the molecular composition and organization of its extracellular matrix. It also is becoming apparent that fibroblasts play an active role in regulating connective tissue tension. In response to static stretching of the tissue, fibroblasts expand within minutes by actively remodeling their cytoskeleton. This dynamic change in fibroblast shape contributes to the drop in tissue tension that occur...

Host-Parasite Interaction: Parasite-Derived and -Induced Proteases That Degrade Human Extracellular Matrix

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Carolina Piña-Vázquez

2012-01-01

Full Text Available Parasitic protozoa are among the most important pathogens worldwide. Diseases such as malaria, leishmaniasis, amoebiasis, giardiasis, trichomoniasis, and trypanosomiasis affect millions of people. Humans are constantly threatened by infections caused by these pathogens. Parasites engage a plethora of surface and secreted molecules to attach to and enter mammalian cells. The secretion of lytic enzymes by parasites into host organs mediates critical interactions because of the invasion and destruction of interstitial tissues, enabling parasite migration to other sites within the hosts. Extracellular matrix is a complex, cross-linked structure that holds cells together in an organized assembly and that forms the basement membrane lining (basal lamina. The extracellular matrix represents a major barrier to parasites. Therefore, the evolution of mechanisms for connective-tissue degradation may be of great importance for parasite survival. Recent advances have been achieved in our understanding of the biochemistry and molecular biology of proteases from parasitic protozoa. The focus of this paper is to discuss the role of protozoan parasitic proteases in the degradation of host ECM proteins and the participation of these molecules as virulence factors. We divide the paper into two sections, extracellular and intracellular protozoa.

Cancer-derived extracellular vesicles: friend and foe of tumour immunosurveillance.

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Dörsam, Bastian; Reiners, Kathrin S; von Strandmann, Elke Pogge

2018-01-05

Extracellular vesicles (EVs) are important players of intercellular signalling mechanisms, including communication with and among immune cells. EVs can affect the surrounding tissue as well as peripheral cells. Recently, EVs have been identified to be involved in the aetiology of several diseases, including cancer. Tumour cell-released EVs or exosomes have been shown to promote a tumour-supporting environment in non-malignant tissue and, thus, benefit metastasis. The underlying mechanisms are numerous: loss of antigen expression, direct suppression of immune effector cells, exchange of nucleic acids, alteration of the recipient cells' transcription and direct suppression of immune cells. Consequently, tumour cells can subvert the host's immune detection as well as suppress the immune system. On the contrary, recent studies reported the existence of EVs able to activate immune cells, thus promoting the tumour-directed immune response. In this article, the immunosuppressive capabilities of EVs, on the one hand, and their potential use in immunoactivation and therapeutic potential, on the other hand, are discussed.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'. © 2017 The Authors.

Altered extracellular matrix remodeling and angiogenesis in sponge granulomas of thrombospondin 2-null mice.

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Kyriakides, T R; Zhu, Y H; Yang, Z; Huynh, G; Bornstein, P

2001-10-01

The matricellular angiogenesis inhibitor, thrombospondin (TSP) 2, has been shown to be an important modulator of wound healing and the foreign body response. Specifically, TSP2-null mice display improved healing with minimal scarring and form well-vascularized foreign body capsules. In this study we performed subcutaneous implantation of sponges and investigated the resulting angiogenic and fibrogenic responses. Histological and immunohistochemical analysis of sponges, excised at 7, 14, and 21 days after implantation, revealed significant differences between TSP2-null and wild-type mice. Most notably, TSP2-null mice exhibited increased angiogenesis and fibrotic encapsulation of the sponge. However, invasion of dense tissue was compromised, even though its overall density was increased. Furthermore, histomorphometry and biochemical assays demonstrated a significant increase in the extracellular distribution of matrix metalloproteinase (MMP) 2, but no change in the levels of active transforming growth factor-beta(1). The alterations in neovascularization, dense tissue invasion, and MMP2 in TSP2-null mice coincided with the deposition of TSP2 in the extracellular matrix of wild-type animals. These observations support the proposed role of TSP2 as a modulator of angiogenesis and matrix remodeling during tissue repair. In addition, they provide in vivo evidence for a newly proposed function of TSP2 as a modulator of extracellular MMP2 levels.

Multistability in a neuron model with extracellular potassium dynamics

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Wu, Xing-Xing; Shuai, J. W.

2012-06-01

Experiments show a primary role of extracellular potassium concentrations in neuronal hyperexcitability and in the generation of epileptiform bursting and depolarization blocks without synaptic mechanisms. We adopt a physiologically relevant hippocampal CA1 neuron model in a zero-calcium condition to better understand the function of extracellular potassium in neuronal seizurelike activities. The model neuron is surrounded by interstitial space in which potassium ions are able to accumulate. Potassium currents, Na+-K+ pumps, glial buffering, and ion diffusion are regulatory mechanisms of extracellular potassium. We also consider a reduced model with a fixed potassium concentration. The bifurcation structure and spiking frequency of the two models are studied. We show that, besides hyperexcitability and bursting pattern modulation, the potassium dynamics can induce not only bistability but also tristability of different firing patterns. Our results reveal the emergence of the complex behavior of multistability due to the dynamical [K+]o modulation on neuronal activities.

Human adipose tissue mesenchymal stromal cells and their extracellular vesicles act differentially on lung mechanics and inflammation in experimental allergic asthma.

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de Castro, Ligia Lins; Xisto, Debora Gonçalves; Kitoko, Jamil Zola; Cruz, Fernanda Ferreira; Olsen, Priscilla Christina; Redondo, Patricia Albuquerque Garcia; Ferreira, Tatiana Paula Teixeira; Weiss, Daniel Jay; Martins, Marco Aurélio; Morales, Marcelo Marcos; Rocco, Patricia Rieken Macedo

2017-06-24

Asthma is a chronic inflammatory disease that can be difficult to treat due to its complex pathophysiology. Most current drugs focus on controlling the inflammatory process, but are unable to revert the changes of tissue remodeling. Human mesenchymal stromal cells (MSCs) are effective at reducing inflammation and tissue remodeling; nevertheless, no study has evaluated the therapeutic effects of extracellular vesicles (EVs) obtained from human adipose tissue-derived MSCs (AD-MSC) on established airway remodeling in experimental allergic asthma. C57BL/6 female mice were sensitized and challenged with ovalbumin (OVA). Control (CTRL) animals received saline solution using the same protocol. One day after the last challenge, each group received saline, 10 5 human AD-MSCs, or EVs (released by 10 5  AD-MSCs). Seven days after treatment, animals were anesthetized for lung function assessment and subsequently euthanized. Bronchoalveolar lavage fluid (BALF), lungs, thymus, and mediastinal lymph nodes were harvested for analysis of inflammation. Collagen fiber content of airways and lung parenchyma were also evaluated. In OVA animals, AD-MSCs and EVs acted differently on static lung elastance and on BALF regulatory T cells, CD3 + CD4 + T cells, and pro-inflammatory mediators (interleukin [IL]-4, IL-5, IL-13, and eotaxin), but similarly reduced eosinophils in lung tissue, collagen fiber content in airways and lung parenchyma, levels of transforming growth factor-β in lung tissue, and CD3 + CD4 + T cell counts in the thymus. No significant changes were observed in total cell count or percentage of CD3 + CD4 + T cells in the mediastinal lymph nodes. In this immunocompetent mouse model of allergic asthma, human AD-MSCs and EVs effectively reduced eosinophil counts in lung tissue and BALF and modulated airway remodeling, but their effects on T cells differed in lung and thymus. EVs may hold promise for asthma; however, further studies are required to elucidate the different

Extracellular Vesicles as Biomarkers and Therapeutics in Dermatology: A Focus on Exosomes.

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McBride, Jeffrey D; Rodriguez-Menocal, Luis; Badiavas, Evangelos V

2017-08-01

Extracellular vesicles (exosomes, microvesicles, and apoptotic bodies) are ubiquitous in human tissues, circulation, and body fluids. Of these vesicles, exosomes are of growing interest among investigators across multiple fields, including dermatology. The characteristics of exosomes, their associated cargo (nucleic acids, proteins, and lipids), and downstream functions are vastly different, depending on the cell origin. Here, we review concepts in extracellular vesicle biology, with a focus on exosomes, highlighting recent studies in the field of dermatology. Furthermore, we highlight emerging technical issues associated with isolating and measuring exosomes. Extracellular vesicles, including exosomes, have immediate potential for serving as biomarkers and therapeutics in dermatology over the next decade. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Tissue localization and extracellular matrix degradation by PI, PII and PIII snake venom metalloproteinases: clues on the mechanisms of venom-induced hemorrhage.

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Cristina Herrera

2015-04-01

Full Text Available Snake venom hemorrhagic metalloproteinases (SVMPs of the PI, PII and PIII classes were compared in terms of tissue localization and their ability to hydrolyze basement membrane components in vivo, as well as by a proteomics analysis of exudates collected in tissue injected with these enzymes. Immunohistochemical analyses of co-localization of these SVMPs with type IV collagen revealed that PII and PIII enzymes co-localized with type IV collagen in capillaries, arterioles and post-capillary venules to a higher extent than PI SVMP, which showed a more widespread distribution in the tissue. The patterns of hydrolysis by these three SVMPs of laminin, type VI collagen and nidogen in vivo greatly differ, whereas the three enzymes showed a similar pattern of degradation of type IV collagen, supporting the concept that hydrolysis of this component is critical for the destabilization of microvessel structure leading to hemorrhage. Proteomic analysis of wound exudate revealed similarities and differences between the action of the three SVMPs. Higher extent of proteolysis was observed for the PI enzyme regarding several extracellular matrix components and fibrinogen, whereas exudates from mice injected with PII and PIII SVMPs had higher amounts of some intracellular proteins. Our results provide novel clues for understanding the mechanisms by which SVMPs induce damage to the microvasculature and generate hemorrhage.

Basic Components of Connective Tissues and Extracellular Matrix

DEFF Research Database (Denmark)

Halper, Jaroslava; Kjær, Michael

2014-01-01

of specific organ systems, but they also provide a scaffold for elastogenesis in elastic tissues. Fibrillin is important for the assembly of elastin into elastic fibers. Mutations in the fibrillin-1 gene are closely associated with Marfan syndrome. Fibulins are tightly connected with basement membranes...

Suramin Inhibits Osteoarthritic Cartilage Degradation by Increasing Extracellular Levels of Chondroprotective Tissue Inhibitor of Metalloproteinases 3.

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Chanalaris, Anastasios; Doherty, Christine; Marsden, Brian D; Bambridge, Gabriel; Wren, Stephen P; Nagase, Hideaki; Troeberg, Linda

2017-10-01

Osteoarthritis is a common degenerative joint disease for which no disease-modifying drugs are currently available. Attempts to treat the disease with small molecule inhibitors of the metalloproteinases that degrade the cartilage matrix have been hampered by a lack of specificity. We aimed to inhibit cartilage degradation by augmenting levels of the endogenous metalloproteinase inhibitor, tissue inhibitor of metalloproteinases (TIMP)-3, through blocking its interaction with the endocytic scavenger receptor, low-density lipoprotein receptor-related protein 1 (LRP1). We discovered that suramin (C 51 H 40 N 6 O 23 S 6 ) bound to TIMP-3 with a K D value of 1.9 ± 0.2 nM and inhibited its endocytosis via LRP1, thus increasing extracellular levels of TIMP-3 and inhibiting cartilage degradation by the TIMP-3 target enzyme, adamalysin-like metalloproteinase with thrombospondin motifs 5. NF279 (8,8'-[carbonyl bis (imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)] bis -1,3,5-naphthalenetrisulfonic acid hexasodium salt), a structural analog of suramin, has an increased affinity for TIMP-3 and increased ability to inhibit TIMP-3 endocytosis and protect cartilage. Suramin is thus a promising scaffold for the development of novel therapeutics to increase TIMP-3 levels and inhibit cartilage degradation in osteoarthritis. Copyright © 2017 by The Author(s).

Live tissue imaging shows reef corals elevate pH under their calcifying tissue relative to seawater.

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Alexander Venn

Full Text Available The threat posed to coral reefs by changes in seawater pH and carbonate chemistry (ocean acidification raises the need for a better mechanistic understanding of physiological processes linked to coral calcification. Current models of coral calcification argue that corals elevate extracellular pH under their calcifying tissue relative to seawater to promote skeleton formation, but pH measurements taken from the calcifying tissue of living, intact corals have not been achieved to date. We performed live tissue imaging of the reef coral Stylophora pistillata to determine extracellular pH under the calcifying tissue and intracellular pH in calicoblastic cells. We worked with actively calcifying corals under flowing seawater and show that extracellular pH (pHe under the calicoblastic epithelium is elevated by ∼0.5 and ∼0.2 pH units relative to the surrounding seawater in light and dark conditions respectively. By contrast, the intracellular pH (pHi of the calicoblastic epithelium remains stable in the light and dark. Estimates of aragonite saturation states derived from our data indicate the elevation in subcalicoblastic pHe favour calcification and may thus be a critical step in the calcification process. However, the observed close association of the calicoblastic epithelium with the underlying crystals suggests that the calicoblastic cells influence the growth of the coral skeleton by other processes in addition to pHe modification. The procedure used in the current study provides a novel, tangible approach for future investigations into these processes and the impact of environmental change on the cellular mechanisms underpinning coral calcification.

Ultrastructure of the extracellular matrix of bovine dura mater, optic nerve sheath and sclera.

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Raspanti, M; Marchini, M; Della Pasqua, V; Strocchi, R; Ruggeri, A

1992-10-01

The sclera, the outermost sheath of the optic nerve and the dura mater have been investigated histologically and ultrastructurally. Although these tissues appear very similar under the light microscope, being dense connective tissues mainly composed of collagen bundles and a limited amount of cells and elastic fibres, they exhibit subtle differences on electron microscopy. In the dura and sclera collagen appears in the form of large, nonuniform fibrils, similar to those commonly found in tendons, while in the optic nerve sheath the fibrils appear smaller and uniform, similar to those commonly observed in reticular tissues, vessel walls and skin. Freeze-fracture also reveals these fibrils to have different subfibrillar architectures, straight or helical, which correspond to 2 distinct forms of collagen fibril previously described (Raspanti et al. 1989). The other extracellular matrix components also vary with the particular collagen fibril structure. Despite their common embryological derivation, the dura mater, optic nerve sheath and sclera exhibit diversification of their extracellular matrix consistent with the mechanical loads to which these tissues are subjected. Our observations indicate that the outermost sheath of the optic nerve resembles the epineurium of peripheral nerves rather than the dura to which it is commonly likened.

Changes in the extracellular matrix and glycosaminoglycan synthesis during the initiation of regeneration in adult newt forelimbs

International Nuclear Information System (INIS)

Mescher, A.L.; Munaim, S.I.

1986-01-01

The extracellular matrix (ECM) of the distal tissues in a newt limb stump is completely reorganized in the 2-3-week period following amputation. In view of numerous in vitro studies showing that extracellular material influences cellular migration and proliferation, it is likely that the changes in the limb's ECM are important activities in the process leading to regeneration of such limbs. Using biochemical, autoradiographic, and histochemical techniques we studied temporal and spatial differences in the synthesis of glycosaminoglycans (GAGs) during the early, nerve-dependent phase of limb regeneration. Hyaluronic acid synthesis began with the onset of tissue dedifferentiation, became maximal within 1 weeks, and continued throughout the period of active cell proliferation. Chondroitin sulfate synthesis began somewhat later, increased steadily, and reached very high levels during chondrogenesis. During the first 10 days after amputation, distributions of sulfated and nonsulfated GAGs were both uniform throughout dedifferentiating tissues, except for a heavier localization near the bone. Since nerves are necessary to promote the regenerative process, we examined the neural influence on synthesis and accumulation of extracellular GAGs. Denervation decreased GAG production in all parts of the limb stump by approximately 50%. Newt dorsal root ganglia and brain-derived fibroblast growth factor each produced twofold stimulation of GAG synthesis in cultured 7-day regenerates. The latter effect was primarily on synthesis of hyaluronic acid. The results indicate that the trophic action of nerves on amphibian limb regeneration includes a positive influence on synthesis and extracellular accumulation of GAGs

Extracellular Membrane Vesicles and Phytopathogenicity of Acholeplasma laidlawii PG8

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Vladislav M. Chernov

2012-01-01

Full Text Available For the first time, the phytopathogenicity of extracellular vesicles of Acholeplasma laidlawii PG8 (a ubiquitous mycoplasma that is one of the five common species of cell culture contaminants and is a causative agent for phytomycoplasmoses in Oryza sativa L. plants was studied. Data on the ability of extracellular vesicles of Acholeplasma laidlawii PG8 to penetrate from the nutrient medium into overground parts of Oryza sativa L. through the root system and to cause alterations in ultrastructural organization of the plants were presented. As a result of the analysis of ultrathin leaf sections of plants grown in medium with A. laidlawii PG8 vesicles, we detected significant changes in tissue ultrastructure characteristic to oxidative stress in plants as well as their cultivation along with bacterial cells. The presence of nucleotide sequences of some mycoplasma genes within extracellular vesicles of Acholeplasma laidlawii PG8 allowed a possibility to use PCR (with the following sequencing to perform differential detection of cells and bacterial vesicles in samples under study. The obtained data may suggest the ability of extracellular vesicles of the mycoplasma to display in plants the features of infection from the viewpoint of virulence criteria—invasivity, infectivity—and toxigenicity—and to favor to bacterial phytopathogenicity.

Imaging and modeling of collagen architecture in living tissue with polarized light transfer (Conference Presentation)

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Ramella-Roman, Jessica C.; Stoff, Susan; Chue-Sang, Joseph; Bai, Yuqiang

2016-03-01

The extra-cellular space in connective tissue of animals and humans alike is comprised in large part of collagen. Monitoring of collagen arrangement and cross-linking has been utilized to diagnose a variety of medical conditions and guide surgical intervention. For example, collagen monitoring is useful in the assessment and treatment of cervical cancer, skin cancer, myocardial infarction, and non-arteritic anterior ischemic optic neuropathy. We have developed a suite of tools and models based on polarized light transfer for the assessment of collagen presence, cross-linking, and orientation in living tissue. Here we will present some example of such approach applied to the human cervix. We will illustrate a novel Mueller Matrix (MM) imaging system for the study of cervical tissue; furthermore we will show how our model of polarized light transfer through cervical tissue compares to the experimental findings. Finally we will show validation of the methodology through histological results and Second Harmonic imaging microscopy.

Circulating Extracellular microRNA in Systemic Autoimmunity

DEFF Research Database (Denmark)

Heegaard, Niels H. H.; Carlsen, Anting Liu; Skovgaard, Kerstin

2015-01-01

killer cells, neutrophil granulocytes, and monocyte-macrophages. Exploratory studies (only validated in a few cases) also show that specific profiles of circulating miRNAs are associated with different systemic autoimmune diseases including systemic lupus erythematosus (SLE), systemic sclerosis......, extracellular miRNA is protected against degradation by complexation with carrier proteins and/or by being enclosed in subcellular membrane vesicles. This, together with their tissue- and disease-specific expression, has fuelled the interest in using circulating microRNA profiles as harbingers of disease, i.......e., as diagnostic analytes and as clues to dysregulated pathways in disease. Many studies show that inflammation and immune dysregulation, e.g., in autoimmune diseases, are associated with distinct miRNA expression changes in targeted tissues and in innate and adaptive immunity cells such as lymphocytes, natural...

Modelling Brain Tissue using Magnetic Resonance Imaging

DEFF Research Database (Denmark)

Dyrby, Tim Bjørn

2008-01-01

Diffusion MRI, or diffusion weighted imaging (DWI), is a technique that measures the restricted diffusion of water molecules within brain tissue. Different reconstruction methods quantify water-diffusion anisotropy in the intra- and extra-cellular spaces of the neural environment. Fibre tracking...... models then use the directions of greatest diffusion as estimates of white matter fibre orientation. Several fibre tracking algorithms have emerged in the last few years that provide reproducible visualizations of three-dimensional fibre bundles. One class of these algorithms is probabilistic...... the possibility of using high-field experimental MR scanners and long scanning times, thereby significantly improving the signal-to-noise ratio (SNR) and anatomical resolution. Moreover, many of the degrading effects observed in vivo, such as physiological noise, are no longer present. However, the post mortem...

In vivo xenogeneic scaffold fate is determined by residual antigenicity and extracellular matrix preservation

OpenAIRE

Wong, Maelene L.; Wong, Janelle L.; Vapniarsky, Natalia; Griffiths, Leigh G.

2016-01-01

The immunological potential of animal-derived tissues and organs is the critical hurdle to increasing their clinical implementation. Glutaraldehyde-fixation cross-links proteins in xenogeneic tissues (e.g., bovine pericardium) to delay immune rejection, but also compromises the regenerative potential of the resultant biomaterial. Unfixed xenogeneic biomaterials in which xenoantigenicity has been ameliorated and native extracellular matrix (ECM) architecture has been maintained have the potent...

Spatial model of convective solute transport in brain extracellular space does not support a "glymphatic" mechanism.

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Jin, Byung-Ju; Smith, Alex J; Verkman, Alan S

2016-12-01

A "glymphatic system," which involves convective fluid transport from para-arterial to paravenous cerebrospinal fluid through brain extracellular space (ECS), has been proposed to account for solute clearance in brain, and aquaporin-4 water channels in astrocyte endfeet may have a role in this process. Here, we investigate the major predictions of the glymphatic mechanism by modeling diffusive and convective transport in brain ECS and by solving the Navier-Stokes and convection-diffusion equations, using realistic ECS geometry for short-range transport between para-arterial and paravenous spaces. Major model parameters include para-arterial and paravenous pressures, ECS volume fraction, solute diffusion coefficient, and astrocyte foot-process water permeability. The model predicts solute accumulation and clearance from the ECS after a step change in solute concentration in para-arterial fluid. The principal and robust conclusions of the model are as follows: (a) significant convective transport requires a sustained pressure difference of several mmHg between the para-arterial and paravenous fluid and is not affected by pulsatile pressure fluctuations; (b) astrocyte endfoot water permeability does not substantially alter the rate of convective transport in ECS as the resistance to flow across endfeet is far greater than in the gaps surrounding them; and (c) diffusion (without convection) in the ECS is adequate to account for experimental transport studies in brain parenchyma. Therefore, our modeling results do not support a physiologically important role for local parenchymal convective flow in solute transport through brain ECS. © 2016 Jin et al.

Accelerated extracellular matrix turnover during exacerbations of COPD

DEFF Research Database (Denmark)

Sand, Jannie M B; Knox, Alan J; Lange, Peter

2015-01-01

progression. Extracellular matrix (ECM) turnover reflects activity in tissues and consequently assessment of ECM turnover may serve as biomarkers of disease activity. We hypothesized that the turnover of lung ECM proteins were altered during exacerbations of COPD. METHODS: 69 patients with COPD hospitalised...... of circulating fragments of structural proteins, which may serve as markers of disease activity. This suggests that patients with COPD have accelerated ECM turnover during exacerbations which may be related to disease progression....

miR-200–containing extracellular vesicles promote breast cancer cell metastasis

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Le, Minh T.N.; Hamar, Peter; Guo, Changying; Basar, Emre; Perdigão-Henriques, Ricardo; Balaj, Leonora; Lieberman, Judy

2014-01-01

Metastasis is associated with poor prognosis in breast cancer patients. Not all cancer cells within a tumor are capable of metastasizing. The microRNA-200 (miR-200) family, which regulates the mesenchymal-to-epithelial transition, is enriched in the serum of patients with metastatic cancers. Ectopic expression of miR-200 can confer metastatic ability to poorly metastatic tumor cells in some settings. Here, we investigated whether metastatic capability could be transferred between metastatic and nonmetastatic cancer cells via extracellular vesicles. miR-200 was secreted in extracellular vesicles from metastatic murine and human breast cancer cell lines, and miR-200 levels were increased in sera of mice bearing metastatic tumors. In culture, murine and human metastatic breast cancer cell extracellular vesicles transferred miR-200 microRNAs to nonmetastatic cells, altering gene expression and promoting mesenchymal-to-epithelial transition. In murine cancer and human xenograft models, miR-200–expressing tumors and extracellular vesicles from these tumors promoted metastasis of otherwise weakly metastatic cells either nearby or at distant sites and conferred to these cells the ability to colonize distant tissues in a miR-200–dependent manner. Together, our results demonstrate that metastatic capability can be transferred by the uptake of extracellular vesicles. PMID:25401471

Crystallization and preliminary crystallographic analysis of Gibberella zeae extracellular lipase

International Nuclear Information System (INIS)

Sun, Yuna; Li, Ming; Zhang, Yan; Liu, Lifang; Liu, Ye; Liu, Zheng; Li, Xumei; Lou, Zhiyong

2008-01-01

G. zeae extracellular lipase has been overexpressed, purified and crystallized. Diffraction data were collected to 2.8 Å resolution. Fusarium head blight, one of the most destructive crop diseases, is mainly caused by Fusarium graminearum (known in its sexual stage as Gibberella zeae). F. graminearum secretes various extracellular enzymes that have been hypothesized to be involved in host infection. One of the extracellular enzymes secreted by this organism is the G. zeae extracellular lipase (GZEL), which is encoded by the FGL1 gene. In order to solve the crystal structure of GZEL and to gain a better understanding of the biological functions of the protein and of possible inhibitory mechanisms of lipase inhibitors, recombinant GZEL was crystallized at 291 K using PEG 3350 as a precipitant. A data set was collected to 2.8 Å resolution from a single flash-cooled crystal (100 K). The crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 78.4, b = 91.0, c = 195.8 Å, α = β = γ = 90°. The presence of four molecules was assumed per asymmetric unit, which gave a Matthews coefficient of 2.6 Å 3 Da −1

Viable group A streptococci in macrophages during acute soft tissue infection.

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Pontus Thulin

2006-03-01

Full Text Available Group A streptococcal severe soft tissue infections, such as necrotizing fasciitis, are rapidly progressive infections associated with high mortality. Group A streptococcus is typically considered an extracellular pathogen, but has been shown to reside intracellularly in host cells.We characterized in vivo interactions between group A streptococci (GAS and cells involved in innate immune responses, using human biopsies (n = 70 collected from 17 patients with soft tissue infections. Immunostaining and in situ image analysis revealed high amounts of bacteria in the biopsies, even in those collected after prolonged antibiotic therapy. Viability of the streptococci was assessed by use of a bacterial viability stain, which demonstrated viable bacteria in 74% of the biopsies. GAS were present both extracellularly and intracellularly within phagocytic cells, primarily within macrophages. Intracellular GAS were predominantly noted in biopsies from newly involved tissue characterized by lower inflammation and bacterial load, whereas purely extracellular GAS or a combination of intra- and extracellular GAS dominated in severely inflamed tissue. The latter tissue was also associated with a significantly increased amount of the cysteine protease streptococcal pyrogenic exotoxin SpeB. In vitro studies confirmed that macrophages serve as reservoirs for viable GAS, and infection with a speB-deletion mutant produced significantly lower frequencies of cells with viable GAS following infection as compared to the wild-type bacteria.This is the first study to demonstrate that GAS survive intracellularly in macrophages during acute invasive infections. This intracellular presence may have evolved as a mechanism to avoid antibiotic eradication, which may explain our finding that high bacterial load is present even in tissue collected after prolonged intravenous antibiotic therapy. This new insight into the pathogenesis of streptococcal soft tissue infections

Viable Group A Streptococci in Macrophages during Acute Soft Tissue Infection.

Directory of Open Access Journals (Sweden)

2006-01-01

Full Text Available BACKGROUND: Group A streptococcal severe soft tissue infections, such as necrotizing fasciitis, are rapidly progressive infections associated with high mortality. Group A streptococcus is typically considered an extracellular pathogen, but has been shown to reside intracellularly in host cells. METHODS AND FINDINGS: We characterized in vivo interactions between group A streptococci (GAS and cells involved in innate immune responses, using human biopsies (n = 70 collected from 17 patients with soft tissue infections. Immunostaining and in situ image analysis revealed high amounts of bacteria in the biopsies, even in those collected after prolonged antibiotic therapy. Viability of the streptococci was assessed by use of a bacterial viability stain, which demonstrated viable bacteria in 74% of the biopsies. GAS were present both extracellularly and intracellularly within phagocytic cells, primarily within macrophages. Intracellular GAS were predominantly noted in biopsies from newly involved tissue characterized by lower inflammation and bacterial load, whereas purely extracellular GAS or a combination of intra- and extracellular GAS dominated in severely inflamed tissue. The latter tissue was also associated with a significantly increased amount of the cysteine protease streptococcal pyrogenic exotoxin SpeB. In vitro studies confirmed that macrophages serve as reservoirs for viable GAS, and infection with a speB-deletion mutant produced significantly lower frequencies of cells with viable GAS following infection as compared to the wild-type bacteria. CONCLUSIONS: This is the first study to demonstrate that GAS survive intracellularly in macrophages during acute invasive infections. This intracellular presence may have evolved as a mechanism to avoid antibiotic eradication, which may explain our finding that high bacterial load is present even in tissue collected after prolonged intravenous antibiotic therapy. This new insight into the pathogenesis

Immunohistochemical study of extracellular matrices and elastic fibers in a human sternoclavicular joint.

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Shimada, K; Takeshige, N; Moriyama, H; Miyauchi, Y; Shimada, S; Fujimaki, E

1997-12-01

In this study, we clarified the distribution of elastic and oxytalan fibers in a human sternoclavicular joint (SCJ) using a color image system and in extracellular matrices using immunoperoxidase staining. Fine elastic fibers (EFs) were scattered in the fibrous layer of the sternoclavicular disk. This articular disk was composed of a collagenous bundle on the sternum side of the articular disk in the SCJ and cellular components including connective tissue on the clavicular side of the articular disk. The thickness of the disk gradually increased from the inferior to superior portion. Collagen fibers type I, III and V and other extracellular matrices (ECMs) were detected in the hypertrophic zone in the clavicular and sternum side of the SCJ and in the connective tissue of the articulatio condylar. On the cervical surface of the articular disk, cellular activity was higher than on the sternum surface.

Extracellular matrix metalloproteinase inducer (EMMPRIN) remodels the extracellular matrix through enhancing matrix metalloproteinases (MMPs) and inhibiting tissue inhibitors of MMPs expression in HPV-positive cervical cancer cells.

Science.gov (United States)

Xu, Q; Cao, X; Pan, J; Ye, Y; Xie, Y; Ohara, N; Ji, H

2015-01-01

PUPOSE OF INVESTIGATION: To study the expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), and tissue inhibitors of MMP (TIMPs) in uterine cervical cancer cell lines in vitro. EMMPRIN, MMPs, and TIMPs expression were assessed by Western blot and real-time RT-PCR from cervical carcinoma SiHa, HeLa, and C33-A cells. EMMPRIN recombinant significantly increased MMP-2, MMP-9 protein and mRNA expression in SiHa and Hela cells, but not in C33-A cells by Western blot analysis and real-time RT-PCR. EMMPRIN recombinant significantly inhibited TIMP-1 protein and mRNA levels in SiHa and Hela cells, but not in C33-A cells. There was no difference on the TIMP-2 expression in those cells with the treatment of EMMPRIN recombinant. EMMPRIN RNAi decreased MMP-2 and MMP-9 and increased TIMP-1 expression in SiHa and HeLa cells, but not in C33-A cells. There was no change on the expression of TIMP-2 mRNA levels in SiHa, HeLa and C33-A cells transfected with siEMMPRIN. EMMPRIN may induce MMP-2 and MMP-9, and downregulate TIMP-1 in HPV-positive cervical cancer cells in vitro.

High-level secretion of tissue factor-rich extracellular vesicles from ovarian cancer cells mediated by filamin-A and protease-activated receptors.

Science.gov (United States)

Koizume, Shiro; Ito, Shin; Yoshioka, Yusuke; Kanayama, Tomohiko; Nakamura, Yoshiyasu; Yoshihara, Mitsuyo; Yamada, Roppei; Ochiya, Takahiro; Ruf, Wolfram; Miyagi, Etsuko; Hirahara, Fumiki; Miyagi, Yohei

2016-01-01

Thromboembolic events occur frequently in ovarian cancer patients. Tissue factor (TF) is often overexpressed in tumours, including ovarian clear-cell carcinoma (CCC), a subtype with a generally poor prognosis. TF-coagulation factor VII (fVII) complexes on the cell surface activate downstream coagulation mechanisms. Moreover, cancer cells secrete extracellular vesicles (EVs), which act as vehicles for TF. We therefore examined the characteristics of EVs produced by ovarian cancer cells of various histological subtypes. CCC cells secreted high levels of TF within EVs, while the high-TF expressing breast cancer cell line MDA-MB-231 shed fewer TF-positive EVs. We also found that CCC tumours with hypoxic tissue areas synthesised TF and fVII in vivo, rendering the blood of xenograft mice bearing these tumours hypercoagulable compared with mice bearing MDA-MB-231 tumours. Incorporation of TF into EVs and secretion of EVs from CCC cells exposed to hypoxia were both dependent on the actin-binding protein, filamin-A (filA). Furthermore, production of these EVs was dependent on different protease-activated receptors (PARs) on the cell surface. These results show that CCC cells could produce large numbers of TF-positive EVs dependent upon filA and PARs. This phenomenon may be the mechanism underlying the increased incidence of venous thromboembolism in ovarian cancer patients.

High fidelity visualization of cell-to-cell variation and temporal dynamics in nascent extracellular matrix formation.

Science.gov (United States)

McLeod, Claire M; Mauck, Robert L

2016-12-12

Extracellular matrix dynamics are key to tissue morphogenesis, homeostasis, injury, and repair. The spatiotemporal organization of this matrix has profound biological implications, but is challenging to monitor using standard techniques. Here, we address these challenges by using noncanonical amino acid tagging to fluorescently label extracellular matrix synthesized in the presence of bio-orthogonal methionine analogs. This strategy labels matrix proteins with high resolution, without compromising their distribution or mechanical function. We demonstrate that the organization and temporal dynamics of the proteinaceous matrix depend on the biophysical features of the microenvironment, including the biomaterial scaffold and the niche constructed by cells themselves. Pulse labeling experiments reveal that, in immature constructs, nascent matrix is highly fibrous and interdigitates with pre-existing matrix, while in more developed constructs, nascent matrix lacks fibrous organization and is retained in the immediate pericellular space. Inhibition of collagen crosslinking increases matrix synthesis, but compromises matrix organization. Finally, these data demonstrate marked cell-to-cell heterogeneity amongst both chondrocytes and mesenchymal stem cells undergoing chondrogenesis. Collectively, these results introduce fluorescent noncanonical amino acid tagging as a strategy to investigate spatiotemporal matrix organization, and demonstrate its ability to identify differences in phenotype, microenvironment, and matrix assembly at the single cell level.

The extracellular matrix of the lung and its role in edema formation

Directory of Open Access Journals (Sweden)

Paolo Pelosi

2007-06-01

Full Text Available The extracellular matrix is composed of a three-dimensional fiber mesh filled with different macromolecules such as: collagen (mainly type I and III, elastin, glycosaminoglycans, and proteoglycans. In the lung, the extracellular matrix has several functions which provide: 1 mechanical tensile and compressive strength and elasticity, 2 low mechanical tissue compliance contributing to the maintenance of normal interstitial fluid dynamics, 3 low resistive pathway for an effective gas exchange, d control of cell behavior by the binding of growth factors, chemokines, cytokines and the interaction with cell-surface receptors, and e tissue repair and remodeling. Fragmentation and disorganization of extracellular matrix components comprises the protective role of the extracellular matrix, leading to interstitial and eventually severe lung edema. Thus, once conditions of increased microvascular filtration are established, matrix remodeling proceeds fairly rapidly due to the activation of proteases. Conversely, a massive matrix deposition of collagen fiber decreases interstitial compliance and therefore makes the tissue safety factor stronger. As a result, changes in lung extracellular matrix significantly affect edema formation and distribution in the lung.A matriz extracelular é um aglomerado tridimensional demacromoléculas composta por: fibras colágenas (principalmente, tipos I e III, elastina, glicosaminoglicanos e proteoglicanos. No pulmão, a matriz extracelular tem várias funções, tais como: 1 promover estresse tensil e elasticidade tecidual, 2 contribuir para a manutenção da dinâmica de fluidos no interstício, 3 propiciar efetiva troca gasosa, 4 controlar a função celular através de sua ligação com fatores de crescimento, quimiocinas, citocinas e interação com receptores de superfície, e 5 remodelamento e reparo tecidual. A fragmentação e a desorganização da matriz extracelular pode acarretar edema intersticial e

Altered Tracer Distribution and Clearance in the Extracellular Space of the Substantia Nigra in a Rodent Model of Parkinson's Disease

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Yuan Fang

2017-07-01

Full Text Available The relationship between extracellular space (ECS diffusion parameters and brain drug clearance is not well-studied, especially in the context of Parkinson's disease (PD. Therefore, we used a rodent model of PD to explore the distribution and clearance of a magnetic resonance tracer. Forty male Sprague Dawley rats were randomized into four different groups: a PD group, a Madopar group (PD + Madopar treatment, a sham group, and a control group. All rats received an injection of the extracellular tracer gadolinium-diethylene triaminepentacetic acid (Gd-DTPA directly into the substantia nigra (SN. ECS diffusion parameters including the effective diffusion coefficient (D*, clearance coefficient (k', ratio of the maximum distribution volume of the tracer (Vd-max%, and half-life (t1/2 were measured. We found that all parameters were significantly increased in the PD group compared to the other three groups (D*: F = 5.774, p = 0.0025; k': F = 20.00, P < 0.0001; Vd-max%: F = 12.81, P < 0.0001; and t1/2: F = 23.35, P < 0.0001. In conclusion, the PD group exhibited a wider distribution and lower clearance of the tracer compared to the other groups. Moreover, k' was more sensitive than D* for monitoring morphological and functional changes in the ECS in a rodent model of PD.

Chemical Facial Cellulitis Due to Inadvertent Injection of Formalin into Oral Tissue Space.

Science.gov (United States)

Bector, Aditi; Virk, Pawandeep Sandhu; Arakeri, Gururaj

2015-11-05

This paper reports the accidental injection of formalin into oral tissue space, in an 8-year old child resulting in chemical facial necrotizing cellulitis and its management. The common practice of keeping formalin in local anesthesia vials should be avoided by dental clinics, to prevent such unfortunate incidents.

Collagen fiber alignment and biaxial mechanical behavior of porcine urinary bladder derived extracellular matrix

NARCIS (Netherlands)

Gilbert, Thomas W.; Wognum, Silvia; Joyce, Erinn M.; Freytes, Donald O.; Sacks, Michael S.; Badylak, Stephen F.

2008-01-01

The collagen fiber alignment and biomechanical behavior of naturally occurring extracellular matrix (ECM) scaffolds are important considerations for the design of medical devices from these materials. Both should be considered in order to produce a device to meet tissue specific mechanical

Surface modification of polycaprolactone scaffolds fabricated via selective laser sintering for cartilage tissue engineering

International Nuclear Information System (INIS)

Chen, Chih-Hao; Lee, Ming-Yih; Shyu, Victor Bong-Hang; Chen, Yi-Chieh; Chen, Chien-Tzung; Chen, Jyh-Ping

2014-01-01

Surface modified porous polycaprolactone scaffolds fabricated via rapid prototyping techniques were evaluated for cartilage tissue engineering purposes. Polycaprolactone scaffolds manufactured by selective laser sintering (SLS) were surface modified through immersion coating with either gelatin or collagen. Three groups of scaffolds were created and compared for both mechanical and biological properties. Surface modification with collagen or gelatin improved the hydrophilicity, water uptake and mechanical strength of the pristine scaffold. From microscopic observations and biochemical analysis, collagen-modified scaffold was the best for cartilage tissue engineering in terms of cell proliferation and extracellular matrix production. Chondrocytes/collagen-modified scaffold constructs were implanted subdermally in the dorsal spaces of female nude mice. Histological and immunohistochemical staining of the retrieved implants after 8 weeks revealed enhanced cartilage tissue formation. We conclude that collagen surface modification through immersion coating on SLS-manufactured scaffolds is a feasible scaffold for cartilage tissue engineering in craniofacial reconstruction. - Highlights: • Selective laser sintered polycaprolactone scaffolds are prepared. • Scaffolds are surface modified through immersion coating with gelatin or collagen. • Collagen-scaffold is the best for cartilage tissue engineering in vitro. • Chondrocytes/collagen-scaffold reveals enhanced cartilage tissue formation in vivo

Surface modification of polycaprolactone scaffolds fabricated via selective laser sintering for cartilage tissue engineering

Energy Technology Data Exchange (ETDEWEB)

Chen, Chih-Hao [Department of Chemical and Materials Engineering, Chang Gung University, Kweishan, Taoyuan 333, Taiwan, ROC (China); Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Craniofacial Research Center, Chang Gung University, Kweishann, Taoyuan 333, Taiwan, ROC (China); Lee, Ming-Yih [Graduate Institute of Medical Mechatronics, Chang Gung University, Kweishan, Taoyuan 333, Taiwan, ROC (China); Shyu, Victor Bong-Hang; Chen, Yi-Chieh; Chen, Chien-Tzung [Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Craniofacial Research Center, Chang Gung University, Kweishann, Taoyuan 333, Taiwan, ROC (China); Chen, Jyh-Ping, E-mail: jpchen@mail.cgu.edu.tw [Department of Chemical and Materials Engineering, Chang Gung University, Kweishan, Taoyuan 333, Taiwan, ROC (China); Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kweishan, Taoyuan 333, Taiwan, ROC (China)

2014-07-01

Surface modified porous polycaprolactone scaffolds fabricated via rapid prototyping techniques were evaluated for cartilage tissue engineering purposes. Polycaprolactone scaffolds manufactured by selective laser sintering (SLS) were surface modified through immersion coating with either gelatin or collagen. Three groups of scaffolds were created and compared for both mechanical and biological properties. Surface modification with collagen or gelatin improved the hydrophilicity, water uptake and mechanical strength of the pristine scaffold. From microscopic observations and biochemical analysis, collagen-modified scaffold was the best for cartilage tissue engineering in terms of cell proliferation and extracellular matrix production. Chondrocytes/collagen-modified scaffold constructs were implanted subdermally in the dorsal spaces of female nude mice. Histological and immunohistochemical staining of the retrieved implants after 8 weeks revealed enhanced cartilage tissue formation. We conclude that collagen surface modification through immersion coating on SLS-manufactured scaffolds is a feasible scaffold for cartilage tissue engineering in craniofacial reconstruction. - Highlights: • Selective laser sintered polycaprolactone scaffolds are prepared. • Scaffolds are surface modified through immersion coating with gelatin or collagen. • Collagen-scaffold is the best for cartilage tissue engineering in vitro. • Chondrocytes/collagen-scaffold reveals enhanced cartilage tissue formation in vivo.

Chemical facial cellulitis due to inadvertent injection of formalin into oral tissue space

Directory of Open Access Journals (Sweden)

Aditi Bector

2015-12-01

Full Text Available This paper reports the accidental injection of formalin into oral tissue space, in an 8-year old child resulting in chemical facial necrotizing cellulitis and its management. The common practice of keeping formalin in local anesthesia vials should be avoided by dental clinics, to prevent such unfortunate incidents.

Expression of small leucine-rich extracellular matrix proteoglycans biglycan and lumican reveals oral lichen planus malignant potential.

Science.gov (United States)

Lončar-Brzak, Božana; Klobučar, Marko; Veliki-Dalić, Irena; Sabol, Ivan; Kraljević Pavelić, Sandra; Krušlin, Božo; Mravak-Stipetić, Marinka

2018-03-01

The aim of this study was to examine molecular alterations on the protein level in lesions of oral lichen planus (OLP), oral squamous cell carcinoma (OSCC) and healthy mucosa. Global protein profiling methods based on liquid chromatography coupled to mass spectrometry (LC-MS) were used, with a special emphasis on evaluation of deregulated extracellular matrix molecules expression, as well as on analyses of IG2F and IGFR2 expression in healthy mucosa, OLP and OSCC tissues by comparative semi-quantitative immunohistochemistry. Mass spectrometry-based proteomics profiling of healthy mucosa, OLP and OSCC tissues (and accompanied histologically unaltered tissues, respectively) identified 55 extracellular matrix proteins. Twenty among identified proteins were common to all groups of samples. Expression of small leucine-rich extracellular matrix proteoglycans lumican and biglycan was found both in OSCC and OLP and they were validated by Western blot analysis as putative biomarkers. A significant increase (p < 0.05) of biglycan expression in OLP-AT group was determined in comparison with OLP-T group, while lumican showed significant up-regulation (p < 0.05) in OLP-T and OSCC-T groups vs. adjacent and control tissue groups. Biglycan expression was only determined in OSCC-AT group. Immunohistochemical analysis of IGF2 and IG2FR expression revealed no significant difference among groups of samples. Biglycan and lumican were identified as important pathogenesis biomarkers of OLP that point to its malignant potential.

Design properties of hydrogel tissue-engineering scaffolds

Science.gov (United States)

Zhu, Junmin; Marchant, Roger E

2011-01-01

This article summarizes the recent progress in the design and synthesis of hydrogels as tissue-engineering scaffolds. Hydrogels are attractive scaffolding materials owing to their highly swollen network structure, ability to encapsulate cells and bioactive molecules, and efficient mass transfer. Various polymers, including natural, synthetic and natural/synthetic hybrid polymers, have been used to make hydrogels via chemical or physical crosslinking. Recently, bioactive synthetic hydrogels have emerged as promising scaffolds because they can provide molecularly tailored biofunctions and adjustable mechanical properties, as well as an extracellular matrix-like microenvironment for cell growth and tissue formation. This article addresses various strategies that have been explored to design synthetic hydrogels with extracellular matrix-mimetic bioactive properties, such as cell adhesion, proteolytic degradation and growth factor-binding. PMID:22026626

Pathophysiology of neutrophil-mediated extracellular redox reactions.

Science.gov (United States)

Jaganjac, Morana; Cipak, Ana; Schaur, Rudolf Joerg; Zarkovic, Neven

2016-01-01

Neutrophil granulocyte leukocytes (neutrophils) play fundamental role in the innate immune response. In the presence of adequate stimuli, neutrophils release excessive amount of reactive oxygen species (ROS) that may induce cell and tissue injury. Oxidative burst of neutrophils acts as a double-edged sword. It may contribute to the pathology of atherosclerosis and brain injury but is also necessary in resolving infections. Moreover, neutrophil-derived ROS may also have both a tumor promoting and tumor suppressing role. ROS have a specific activities and diffusion distance, which is related to their short lifetime. Therefore, the manner in which ROS will act depends on the cells targeted and the intra- and extracellular levels of individual ROS, which can further cause production of reactive aldehydes like 4-hydroxynonenal (HNE) that act as a second messengers of ROS. In this review we discuss the influence of neutrophil mediated extracellular redox reactions in ischemia reperfusion injury, transplant rejection and chronic diseases (atherosclerosis, inflammatory bowel diseases and cancer). At the end a brief overview of cellular mechanisms to maintain ROS homeostasis is given.

Stem Cell Extracellular Vesicles: Extended Messages of Regeneration

Science.gov (United States)

Riazifar, Milad; Pone, Egest J.; Lötvall, Jan; Zhao, Weian

2017-01-01

Stem cells are critical to maintaining steady-state organ homeostasis and regenerating injured tissues. Recent intriguing reports implicate extracellular vesicles (EVs) as carriers for the distribution of morphogens and growth and differentiation factors from tissue parenchymal cells to stem cells, and conversely, stem cell–derived EVs carrying certain proteins and nucleic acids can support healing of injured tissues. We describe approaches to make use of engineered EVs as technology platforms in therapeutics and diagnostics in the context of stem cells. For some regenerative therapies, natural and engineered EVs from stem cells may be superior to single-molecule drugs, biologics, whole cells, and synthetic liposome or nanoparticle formulations because of the ease of bioengineering with multiple factors while retaining superior biocompatibility and biostability and posing fewer risks for abnormal differentiation or neoplastic transformation. Finally, we provide an overview of current challenges and future directions of EVs as potential therapeutic alternatives to cells for clinical applications. PMID:27814025

Neutral tissue uptake and clearance of iohexol following lumbar myelography in rabbits

International Nuclear Information System (INIS)

Ekholm, S.E.; Foley, M.; Morris, T.W.; Sahler, L.

1985-01-01

The diffusion of water-soluble contrast media (CM) into the extracellular space of the central nervous system following injection into the subarachnoid space has previously been shown. As a result of this, water-soluble CM will come in direct contact with the neurons and may interfere with their normal function. The toxic effects would thus be a result both of the molecular properties of the CM as well as the local tissue concentration. The neuronal tissue uptake and clearance of metrizamide in rabbits following lumbar myelography was described in a previous study by our group. This study indicated some retention of metrizamide in the spinal cord probably as a result of binding of the CM to the cell membrane. The mechanism for this has not yet been shown although it may relate to the binding of metrizamide via its 2-deoxy-D-glucose (2-DG) portion and the specific glucose membrane carrier. The present investigation was performed to evaluate the diffusion kinetics of a new non-ionic CM. With iohexol, which lacks a 2-DG component in its molecule a direct relationship between the neural tissue and CSF concentration was found which seems to follow a simple diffusion model. Since iohexol shows no sign of entrapment in the tissue, the contact time for neurons will be shorter than that seen with metrizamide assuming that their rate of drainage from the SCF is identical. (orig.)

Microstructural inhomogeneity of electrical conductivity in subcutaneous fat tissue.

Directory of Open Access Journals (Sweden)

Ilja L Kruglikov

Full Text Available Microscopic peculiarities stemming from a temperature increase in subcutaneous adipose tissue (sWAT after applying a radio-frequency (RF current, must be strongly dependent on the type of sWAT. This effect is connected with different electrical conductivities of pathways inside (triglycerides in adipocytes and outside (extra-cellular matrix the cells and to the different weighting of these pathways in hypertrophic and hyperplastic types of sWAT. The application of the RF current to hypertrophic sWAT, which normally has a strongly developed extracellular matrix with high concentrations of hyaluronan and collagen in a peri-cellular space of adipocytes, can produce, micro-structurally, a highly inhomogeneous temperature distribution, characterized by strong temperature gradients between the peri-cellular sheath of the extra-cellular matrix around the hypertrophic adipocytes and their volumes. In addition to normal temperature effects, which are generally considered in body contouring, these temperature gradients can produce thermo-mechanical stresses on the cells' surfaces. Whereas these stresses are relatively small under normal conditions and cannot cause any direct fracturing or damage of the cell structure, these stresses can, under some supportive conditions, be theoretically increased by several orders of magnitude, causing the thermo-mechanical cell damage. This effect cannot be realized in sWAT of normal or hyperplastic types where the peri-cellular structures are under-developed. It is concluded that the results of RF application in body contouring procedures must be strongly dependent on the morphological structure of sWAT.

Macroporous Hydrogel Scaffolds for Three-Dimensional Cell Culture and Tissue Engineering.

Science.gov (United States)

Fan, Changjiang; Wang, Dong-An

2017-10-01

Hydrogels have been promising candidate scaffolds for cell delivery and tissue engineering due to their tissue-like physical properties and capability for homogeneous cell loading. However, the encapsulated cells are generally entrapped and constrained in the submicron- or nanosized gel networks, seriously limiting cell growth and tissue formation. Meanwhile, the spatially confined settlement inhibits attachment and spreading of anchorage-dependent cells, leading to their apoptosis. In recent years, macroporous hydrogels have attracted increasing attention in use as cell delivery vehicles and tissue engineering scaffolds. The introduction of macropores within gel scaffolds not only improves their permeability for better nutrient transport but also creates space/interface for cell adhesion, proliferation, and extracellular matrix deposition. Herein, we will first review the development of macroporous gel scaffolds and outline the impact of macropores on cell behaviors. In the first part, the advantages and challenges of hydrogels as three-dimensional (3D) cell culture scaffolds will be described. In the second part, the fabrication of various macroporous hydrogels will be presented. Third, the enhancement of cell activities within macroporous gel scaffolds will be discussed. Finally, several crucial factors that are envisaged to propel the improvement of macroporous gel scaffolds are proposed for 3D cell culture and tissue engineering.

Fetal brain extracellular matrix boosts neuronal network formation in 3D bioengineered model of cortical brain tissue.

Science.gov (United States)

Sood, Disha; Chwalek, Karolina; Stuntz, Emily; Pouli, Dimitra; Du, Chuang; Tang-Schomer, Min; Georgakoudi, Irene; Black, Lauren D; Kaplan, David L

2016-01-01

The extracellular matrix (ECM) constituting up to 20% of the organ volume is a significant component of the brain due to its instructive role in the compartmentalization of functional microdomains in every brain structure. The composition, quantity and structure of ECM changes dramatically during the development of an organism greatly contributing to the remarkably sophisticated architecture and function of the brain. Since fetal brain is highly plastic, we hypothesize that the fetal brain ECM may contain cues promoting neural growth and differentiation, highly desired in regenerative medicine. Thus, we studied the effect of brain-derived fetal and adult ECM complemented with matricellular proteins on cortical neurons using in vitro 3D bioengineered model of cortical brain tissue. The tested parameters included neuronal network density, cell viability, calcium signaling and electrophysiology. Both, adult and fetal brain ECM as well as matricellular proteins significantly improved neural network formation as compared to single component, collagen I matrix. Additionally, the brain ECM improved cell viability and lowered glutamate release. The fetal brain ECM induced superior neural network formation, calcium signaling and spontaneous spiking activity over adult brain ECM. This study highlights the difference in the neuroinductive properties of fetal and adult brain ECM and suggests that delineating the basis for this divergence may have implications for regenerative medicine.

Electrodiffusion Models of Neurons and Extracellular Space Using the Poisson-Nernst-Planck Equations—Numerical Simulation of the Intra- and Extracellular Potential for an Axon Model

Science.gov (United States)

Pods, Jurgis; Schönke, Johannes; Bastian, Peter

2013-01-01

In neurophysiology, extracellular signals—as measured by local field potentials (LFP) or electroencephalography—are of great significance. Their exact biophysical basis is, however, still not fully understood. We present a three-dimensional model exploiting the cylinder symmetry of a single axon in extracellular fluid based on the Poisson-Nernst-Planck equations of electrodiffusion. The propagation of an action potential along the axonal membrane is investigated by means of numerical simulations. Special attention is paid to the Debye layer, the region with strong concentration gradients close to the membrane, which is explicitly resolved by the computational mesh. We focus on the evolution of the extracellular electric potential. A characteristic up-down-up LFP waveform in the far-field is found. Close to the membrane, the potential shows a more intricate shape. A comparison with the widely used line source approximation reveals similarities and demonstrates the strong influence of membrane currents. However, the electrodiffusion model shows another signal component stemming directly from the intracellular electric field, called the action potential echo. Depending on the neuronal configuration, this might have a significant effect on the LFP. In these situations, electrodiffusion models should be used for quantitative comparisons with experimental data. PMID:23823244

Extracellular matrix structure.

Science.gov (United States)

Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

2016-02-01

Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented. Copyright © 2015 Elsevier B.V. All rights reserved.

Blood and interstitial flow in the hierarchical pore space architecture of bone tissue.

Science.gov (United States)

Cowin, Stephen C; Cardoso, Luis

2015-03-18

There are two main types of fluid in bone tissue, blood and interstitial fluid. The chemical composition of these fluids varies with time and location in bone. Blood arrives through the arterial system containing oxygen and other nutrients and the blood components depart via the venous system containing less oxygen and reduced nutrition. Within the bone, as within other tissues, substances pass from the blood through the arterial walls into the interstitial fluid. The movement of the interstitial fluid carries these substances to the cells within the bone and, at the same time, carries off the waste materials from the cells. Bone tissue would not live without these fluid movements. The development of a model for poroelastic materials with hierarchical pore space architecture for the description of blood flow and interstitial fluid flow in living bone tissue is reviewed. The model is applied to the problem of determining the exchange of pore fluid between the vascular porosity and the lacunar-canalicular porosity in bone tissue due to cyclic mechanical loading and blood pressure. These results are basic to the understanding of interstitial flow in bone tissue that, in turn, is basic to understanding of nutrient transport from the vasculature to the bone cells buried in the bone tissue and to the process of mechanotransduction by these cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

Extracellular matrix and growth factor engineering for controlled angiogenesis in regenerative medicine

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Mikaël M Martino

2015-04-01

Full Text Available Blood vessel growth plays a key role in regenerative medicine, both to restore blood supply to ischemic tissues and to ensure rapid vascularization of clinical-size tissue-engineered grafts. For example, vascular endothelial growth factor (VEGF is the master regulator of physiological blood vessel growth and is one of the main molecular targets of therapeutic angiogenesis approaches. However, angiogenesis is a complex process and there is a need to develop rational therapeutic strategies based on a firm understanding of basic vascular biology principles, as evidenced by the disappointing results of initial clinical trials of angiogenic factor delivery. In particular, the spatial localization of angiogenic signals in the extracellular matrix is crucial to ensure the proper assembly and maturation of new vascular structures. Here we discuss the therapeutic implications of matrix interactions of angiogenic factors, with a special emphasis on VEGF, as well as provide an overview of current approaches, based on protein and biomaterial engineering that mimic the regulatory functions of extracellular matrix to optimize the signaling microenvironment of vascular growth factors.

Spatial model of convective solute transport in brain extracellular space does not support a “glymphatic” mechanism

Science.gov (United States)

Jin, Byung-Ju; Smith, Alex J.

2016-01-01

A “glymphatic system,” which involves convective fluid transport from para-arterial to paravenous cerebrospinal fluid through brain extracellular space (ECS), has been proposed to account for solute clearance in brain, and aquaporin-4 water channels in astrocyte endfeet may have a role in this process. Here, we investigate the major predictions of the glymphatic mechanism by modeling diffusive and convective transport in brain ECS and by solving the Navier–Stokes and convection–diffusion equations, using realistic ECS geometry for short-range transport between para-arterial and paravenous spaces. Major model parameters include para-arterial and paravenous pressures, ECS volume fraction, solute diffusion coefficient, and astrocyte foot-process water permeability. The model predicts solute accumulation and clearance from the ECS after a step change in solute concentration in para-arterial fluid. The principal and robust conclusions of the model are as follows: (a) significant convective transport requires a sustained pressure difference of several mmHg between the para-arterial and paravenous fluid and is not affected by pulsatile pressure fluctuations; (b) astrocyte endfoot water permeability does not substantially alter the rate of convective transport in ECS as the resistance to flow across endfeet is far greater than in the gaps surrounding them; and (c) diffusion (without convection) in the ECS is adequate to account for experimental transport studies in brain parenchyma. Therefore, our modeling results do not support a physiologically important role for local parenchymal convective flow in solute transport through brain ECS. PMID:27836940

Cell and Tissue Engineering

CERN Document Server

2012-01-01

“Cell and Tissue Engineering” introduces the principles and new approaches in cell and tissue engineering. It includes both the fundamentals and the current trends in cell and tissue engineering, in a way useful both to a novice and an expert in the field. The book is composed of 13 chapters all of which are written by the leading experts. It is organized to gradually assemble an insight in cell and tissue function starting form a molecular nano-level, extending to a cellular micro-level and finishing at the tissue macro-level. In specific, biological, physiological, biophysical, biochemical, medical, and engineering aspects are covered from the standpoint of the development of functional substitutes of biological tissues for potential clinical use. Topics in the area of cell engineering include cell membrane biophysics, structure and function of the cytoskeleton, cell-extracellular matrix interactions, and mechanotransduction. In the area of tissue engineering the focus is on the in vitro cultivation of ...

Modeling the tumor extracellular matrix: Tissue engineering tools repurposed towards new frontiers in cancer biology.

Science.gov (United States)

Gill, Bartley J; West, Jennifer L

2014-06-27

Cancer progression is mediated by complex epigenetic, protein and structural influences. Critical among them are the biochemical, mechanical and architectural properties of the extracellular matrix (ECM). In recognition of the ECM's important role, cancer biologists have repurposed matrix mimetic culture systems first widely used by tissue engineers as new tools for in vitro study of tumor models. In this review we discuss the pathological changes in tumor ECM, the limitations of 2D culture on both traditional and polyacrylamide hydrogel surfaces in modeling these characteristics and advances in both naturally derived and synthetic scaffolds to facilitate more complex and controllable 3D cancer cell culture. Studies using naturally derived matrix materials like Matrigel and collagen have produced significant findings related to tumor morphogenesis and matrix invasion in a 3D environment and the mechanotransductive signaling that mediates key tumor-matrix interaction. However, lack of precise experimental control over important matrix factors in these matrices have increasingly led investigators to synthetic and semi-synthetic scaffolds that offer the engineering of specific ECM cues and the potential for more advanced experimental manipulations. Synthetic scaffolds composed of poly(ethylene glycol) (PEG), for example, facilitate highly biocompatible 3D culture, modular bioactive features like cell-mediated matrix degradation and complete independent control over matrix bioactivity and mechanics. Future work in PEG or similar reductionist synthetic matrix systems should enable the study of increasingly complex and dynamic tumor-ECM relationships in the hopes that accurate modeling of these relationships may reveal new cancer therapeutics targeting tumor progression and metastasis. © 2013 Published by Elsevier Ltd.

The concentration of extracellular superoxide dismutase in plasma is maintained by LRP-mediated endocytosis

DEFF Research Database (Denmark)

Petersen, Steen V; Thøgersen, Ida B; Valnickova, Zuzana

2010-01-01

In this study, we show that human extracellular superoxide dismutase (EC-SOD) binds to low-density lipoprotein receptor-related protein (LRP). This interaction is most likely responsible for the removal of EC-SOD from the blood circulation via LRP expressed in liver tissue. The receptor recognition...

Extracellular matrix-derived hydrogels for dental stem cell delivery.

Science.gov (United States)

Viswanath, Aiswarya; Vanacker, Julie; Germain, Loïc; Leprince, Julian G; Diogenes, Anibal; Shakesheff, Kevin M; White, Lisa J; des Rieux, Anne

2017-01-01

Decellularized mammalian extracellular matrices (ECM) have been widely accepted as an ideal substrate for repair and remodelling of numerous tissues in clinical and pre-clinical studies. Recent studies have demonstrated the ability of ECM scaffolds derived from site-specific homologous tissues to direct cell differentiation. The present study investigated the suitability of hydrogels derived from different source tissues: bone, spinal cord and dentine, as suitable carriers to deliver human apical papilla derived mesenchymal stem cells (SCAP) for spinal cord regeneration. Bone, spinal cord, and dentine ECM hydrogels exhibited distinct structural, mechanical, and biological characteristics. All three hydrogels supported SCAP viability and proliferation. However, only spinal cord and bone derived hydrogels promoted the expression of neural lineage markers. The specific environment of ECM scaffolds significantly affected the differentiation of SCAP to a neural lineage, with stronger responses observed with spinal cord ECM hydrogels, suggesting that site-specific tissues are more likely to facilitate optimal stem cell behavior for constructive spinal cord regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 319-328, 2017. © 2016 Wiley Periodicals, Inc.

Design and fabrication of porous biodegradable scaffolds: a strategy for tissue engineering.

Science.gov (United States)

Raeisdasteh Hokmabad, Vahideh; Davaran, Soodabeh; Ramazani, Ali; Salehi, Roya

2017-11-01

Current strategies of tissue engineering are focused on the reconstruction and regeneration of damaged or deformed tissues by grafting of cells with scaffolds and biomolecules. Recently, much interest is given to scaffolds which are based on mimic the extracellular matrix that have induced the formation of new tissues. To return functionality of the organ, the presence of a scaffold is essential as a matrix for cell colonization, migration, growth, differentiation and extracellular matrix deposition, until the tissues are totally restored or regenerated. A wide variety of approaches has been developed either in scaffold materials and production procedures or cell sources and cultivation techniques to regenerate the tissues/organs in tissue engineering applications. This study has been conducted to present an overview of the different scaffold fabrication techniques such as solvent casting and particulate leaching, electrospinning, emulsion freeze-drying, thermally induced phase separation, melt molding and rapid prototyping with their properties, limitations, theoretical principles and their prospective in tailoring appropriate micro-nanostructures for tissue regeneration applications. This review also includes discussion on recent works done in the field of tissue engineering.

The danger signal extracellular ATP is an inducer of Fusobacterium nucleatum biofilm dispersal

Directory of Open Access Journals (Sweden)

Qinfeng Ding

2016-11-01

Full Text Available Plaque biofilm is the primary etiological agent of periodontal disease. Biofilm formation progresses through multiple developmental stages beginning with bacterial attachment to a surface, followed by development of microcolonies and finally detachment and dispersal from a mature biofilm as free planktonic bacteria. Tissue damage arising from inflammatory response to biofilm is one of the hallmark features of periodontal disease. A consequence of tissue damage is the release of ATP from within the cell into the extracellular space. Extracellular ATP (eATP is an example of a danger associated molecular pattern (DAMP employed by mammalian cells to elicit inflammatory and damage healing responses. Although the roles of eATP as a signaling molecule in multi-cellular organisms have been relatively well studied, exogenous ATP also influences bacteria biofilm formation. Since plaque biofilms are continuously exposed to various stresses including exposure to the host damage factors eATP, we hypothesized that eATP, in addition to eliciting inflammation could potentially influence the biofilm lifecycle of periodontal associated bacteria. We found that eATP rather than nutritional factors or oxidative stress induced dispersal of Fusobacterium nucleatum, an organism associated with periodontal disease. eATP induced biofilm dispersal through chelating metal ions present in biofilm. Dispersed F. nucleatum biofilm, regardless of natural or induced dispersal by exogenous ATP, were significantly more adhesive and invasive compared to planktonic or biofilm counterparts, and correspondingly activated significantly more pro-inflammatory cytokine production in infected periodontal fibroblasts. Dispersed F. nucleatum also exhibited significantly higher expression of fadA, a virulence factor implicated in adhesion and invasion, compared to planktonic or biofilm bacteria. This study revealed for the first time that periodontal bacterium is capable of co-opting eATP, a

Extracellular matrix adaptation of tendon and skeletal muscle to exercise

DEFF Research Database (Denmark)

Kjaer, Michael; Magnusson, Peter; Krogsgaard, Michael

2006-01-01

The extracellular matrix (ECM) of connective tissues enables linking to other tissues, and plays a key role in force transmission and tissue structure maintenance in tendons, ligaments, bone and muscle. ECM turnover is influenced by physical activity, and both collagen synthesis and metalloprotease......-beta and IL-6 is enhanced following exercise. For tendons, metabolic activity (e.g. detected by positron emission tomography scanning), circulatory responses (e.g. as measured by near-infrared spectroscopy and dye dilution) and collagen turnover are markedly increased after exercise. Tendon blood flow...... is regulated by cyclooxygenase-2 (COX-2)-mediated pathways, and glucose uptake is regulated by specific pathways in tendons that differ from those in skeletal muscle. Chronic loading in the form of physical training leads both to increased collagen turnover as well as to some degree of net collagen synthesis...

Investigating the role of the extracellular matrix on differentiation of human mesenchymal stem cells and MC3T3 cells

NARCIS (Netherlands)

Fernandes, H.A.M.; Dechering, Koen; Someren, Eugene; van Blitterswijk, Clemens; de Boer, Jan

2008-01-01

Human mesenchymal stem cells (hMSCs) are a promising cell source for bone tissue engineering, but due to their limited number and donor variation, other cell types are used to answer relevant questions in bone tissue engineering. Since the extracellular matrix (ECM) is a complex entity with

Is There Volume Transmission Along Extracellular Fluid Pathways Corresponding to the Acupuncture Meridians?

Directory of Open Access Journals (Sweden)

Weibo Zhang

2017-02-01

Full Text Available Volume transmission is a new major communication signaling via extracellular fluid (interstitial fluid pathways. It was proposed by the current authors that such pathways can explain the meridian phenomena and acupuncture effects. To investigate whether meridian-like structures exist in fish body and operate via volume transmission in extracellular fluid pathways, we injected alcian blue (AB under anesthesia into Gephyrocharax melanocheir, which has a translucent body. The migration of AB could be seen directly and was recorded by a digital camera. The fish was then embedded and cut transversely to observe the position of tracks in three dimensions. Eight longitudinal threadlike blue tracks were recognized on the fish. The positions of these threadlike tracks were similar to meridians on the human body. Transverse sections showed that these tracks distributed to different layers of distinct subcutaneous loose connective tissues and intermuscular septa. Lymphatic vessels were sometimes associated with the extracellular blue tracks where the migration of AB occurred. Extracellular fluid pathways were found on fish through their transport of AB. These pathways operating via volume transmission appeared to be similar in positions and functions to the acupuncture meridians in Chinese medicine.

"Tipping" extracellular matrix remodeling towards regression of liver fibrosis

DEFF Research Database (Denmark)

Magdaleno, Fernando; Schierwagen, Robert; Uschner, Frank E

2018-01-01

Fibrosis development was initially conceived as an incessant progressive condition. Nowadays, it has become evident that fibrotic tissue undergoes a continuous two-way process: fibrogenesis and fibrinolysis, characterizing the remodeling of extracellular matrix (ECM). However, in established...... fibrosis, this two-way process is tipped towards fibrogenesis and this leads to a self-perpetuating accumulation of ECM, a distinct metabolic unit, together with other cells and processes promoting fibrosis deposition. Several mechanisms promote fibrosis regression, such as degradation of ECM, infiltration...

Erwinia carotovora extracellular proteases : characterization and role in soft rot

OpenAIRE

Kyöstiö, Sirkka R. M.

1990-01-01

Erwinia carotovora subsp. carotovora (Ecc) strain EC14, a Gram-negative bacterium, causes soft rot on several crops, including potato. Maceration of potato tuber tissue is caused by secreted pectolytic enzymes. Other cell-degrading enzymes may also have roles in pathogenesis, including cellulases, phospholipases, and protease(s). The objectives of this research were to (1) characterize Ecc extracellular protease (Prt) and (2) elucidate its role in potato soft rot. A gene enc...

Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration.

Science.gov (United States)

Pati, Falguni; Song, Tae-Ha; Rijal, Girdhari; Jang, Jinah; Kim, Sung Won; Cho, Dong-Woo

2015-01-01

3D printing technique is the most sophisticated technique to produce scaffolds with tailorable physical properties. But, these scaffolds often suffer from limited biological functionality as they are typically made from synthetic materials. Cell-laid mineralized ECM was shown to be potential for improving the cellular responses and drive osteogenesis of stem cells. Here, we intend to improve the biological functionality of 3D-printed synthetic scaffolds by ornamenting them with cell-laid mineralized extracellular matrix (ECM) that mimics a bony microenvironment. We developed bone graft substitutes by using 3D printed scaffolds made from a composite of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP) and mineralized ECM laid by human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs). A rotary flask bioreactor was used to culture hTMSCs on the scaffolds to foster formation of mineralized ECM. A freeze/thaw cycle in hypotonic buffer was used to efficiently decellularize (97% DNA reduction) the ECM-ornamented scaffolds while preserving its main organic and inorganic components. The ECM-ornamented 3D printed scaffolds supported osteoblastic differentiation of newly-seeded hTMSCs by upregulating four typical osteoblastic genes (4-fold higher RUNX2; 3-fold higher ALP; 4-fold higher osteocalcin; and 4-fold higher osteopontin) and increasing calcium deposition compared to bare 3D printed scaffolds. In vivo, in ectopic and orthotopic models in rats, ECM-ornamented scaffolds induced greater bone formation than that of bare scaffolds. These results suggest a valuable method to produce ECM-ornamented 3D printed scaffolds as off-the-shelf bone graft substitutes that combine tunable physical properties with physiological presentation of biological signals. Copyright © 2014 Elsevier Ltd. All rights reserved.

Syndecans as receptors and organizers of the extracellular matrix.

Science.gov (United States)

Xian, Xiaojie; Gopal, Sandeep; Couchman, John R

2010-01-01

Syndecans are type I transmembrane proteins having a core protein modified with glycosaminoglycan chains, most commonly heparan sulphate. They are an ancient group of molecules, present in invertebrates and vertebrates. Among the plethora of molecules that can interact with heparan sulphate, the collagens and glycoproteins of the extracellular matrix are prominent. Frequently, they do so in conjunction with other receptors, most notably the integrins. For this reason, they are often referred to as "co-receptors". However, just as with integrins, syndecans can interact with actin-associated proteins and signalling molecules, such as protein kinases. Some aspects of syndecan signalling are understood but much remains to be learned. The functions of syndecans in regulating cell adhesion and extracellular matrix assembly are described here. Evidence from null mice suggests that syndecans have roles in postnatal tissue repair, inflammation and tumour progression. Developmental deficits in lower vertebrates in which syndecans are eliminated are also informative and suggest that, in mammals, redundancy is a key issue.

Biomimetics of the extracellular matrix: an integrated three-dimensional fiber-hydrogel composite for cartilage tissue engineering

NARCIS (Netherlands)

Coburn, J.; Gibson, M.; Bandalini, P.A.; Laird, C.; Mao, H.Q.; Moroni, Lorenzo; Seliktar, D.; Elisseeff, J.H.

2011-01-01

The native extracellular matrix (ECM) consists of an integrated fibrous protein network and proteoglycan-based ground (hydrogel) substance. We designed a novel electrospinning technique to engineer a three dimensional fiber-hydrogel composite that mimics the native ECM structure, is injectable, and

Extracellular Nucleotide Hydrolysis in Dermal and Limbal Mesenchymal Stem Cells: A Source of Adenosine Production.

Science.gov (United States)

Naasani, Liliana I Sous; Rodrigues, Cristiano; de Campos, Rafael Paschoal; Beckenkamp, Liziane Raquel; Iser, Isabele C; Bertoni, Ana Paula Santin; Wink, Márcia R

2017-08-01

Human Limbal (L-MSCs) and Dermal Mesenchymal Stem Cell (D-MSCs) possess many properties that increase their therapeutic potential in ophthalmology and dermatology. It is known that purinergic signaling plays a role in many aspects of mesenchymal stem cells physiology. They release and respond to purinergic ligands, altering proliferation, migration, differentiation, and apoptosis. Therefore, more information on these processes would be crucial for establishing future clinical applications using their differentiation potential, but without undesirable side effects. This study evaluated and compared the expression of ecto-nucleotidases, the enzymatic activity of degradation of extracellular nucleotides and the metabolism of extracellular ATP in D-MSCs and L-MSCs, isolated from discard tissues of human skin and sclerocorneal rims. The D-MSCs and L-MSCs showed a differentiation potential into osteogenic, adipogenic, and chondrogenic lineages and the expression of markers CD105 + , CD44 + , CD14 - , CD34 - , CD45 - , as expected. Both cells hydrolyzed low levels of extracellular ATP and high levels of AMP, leading to adenosine accumulation that can regulate inflammation and tissue repair. These cells expressed mRNA for ENTPD1, 2, 3, 5 and 6, and CD73 that corresponded to the observed enzymatic activities. Thus, considering the degradation of ATP and adenosine production, limbal MSCs are very similar to dermal MSCs, indicating that from the aspect of extracellular nucleotide metabolism L-MSCs are very similar to the characterized D-MSCs. J. Cell. Biochem. 118: 2430-2442, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Tissue type plasminogen activator regulates myeloid-cell dependent neoangiogenesis during tissue regeneration

DEFF Research Database (Denmark)

Ohki, Makiko; Ohki, Yuichi; Ishihara, Makoto

2010-01-01

tissue regeneration is not well understood. Bone marrow (BM)-derived myeloid cells facilitate angiogenesis during tissue regeneration. Here, we report that a serpin-resistant form of tPA by activating the extracellular proteases matrix metalloproteinase-9 and plasmin expands the myeloid cell pool......-A. Remarkably, transplantation of BM-derived tPA-mobilized CD11b(+) cells and VEGFR-1(+) cells, but not carrier-mobilized cells or CD11b(-) cells, accelerates neovascularization and ischemic tissue regeneration. Inhibition of VEGF signaling suppresses tPA-induced neovascularization in a model of hind limb...... and mobilizes CD45(+)CD11b(+) proangiogenic, myeloid cells, a process dependent on vascular endothelial growth factor-A (VEGF-A) and Kit ligand signaling. tPA improves the incorporation of CD11b(+) cells into ischemic tissues and increases expression of neoangiogenesis-related genes, including VEGF...

Phytosynthesis of intracellular and extracellular gold nanoparticles by living peanut plant (Arachis hypogaea L.).

Science.gov (United States)

Raju, Dugyala; Mehta, Urmil J; Ahmad, Absar

2012-01-01

Inorganic nanomaterials of different chemical compositions are conventionally synthesized under harsh environments such as extremes of temperature, pressure, and pH. Moreover, these methods are eco-unfriendly and cumbersome, yield bigger particles, and agglomerate because of not being capped by capping agents. In contrast, biological synthesis of inorganic nanomaterials occurs under ambient conditions, namely room temperature, atmospheric pressure, and physiological pH. These methods are reliable, eco-friendly, and cheap. In this paper, we report for the first time the extracellular and intracellular synthesis of gold nanoparticles (GNPs) using living peanut seedlings. The formed GNPs were highly stable in solution and inside the plant tissue. Transmission electron microscopy revealed that extracellular GNPs distributions were in the form of monodispersed nanoparticles. The nanoparticles ranged from 4 to 6 nm in size. The intercellular nanoparticles were of oval shape and size ranged from 5 to 50 nm. Both extracellular and intracellular nanoparticles were further characterized by standard techniques. The formed GNPs inside the plant tissue were estimated by inductively coupled plasma spectrometry. This opens up an exciting possibility of a plant-based nanoparticle synthesis strategy, wherein the nanoparticles may be entrapped in the biomass in the form of a film or produced in the solution, both of which have interesting applications. © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Suppression of ICE and Apoptosis in Mammary Epithelial Cells by Extracellular Matrix

Energy Technology Data Exchange (ETDEWEB)

Boudreau, Nancy; Sympson, C. J.; Werb, Zena; Bissell, Mina J.

1994-12-01

Apoptosis (programmed cell death) plays a major role in development and tissue regeneration. Basement membrane extracellular matrix (ECM), but not fibronectin or collagen, was shown to suppress apoptosis of mammary epithelial cells in tissue culture and in vivo. Apoptosis was induced by antibodies to beta 1 integrins or by overexpression of stromelysin-1, which degrades ECM. Expression of interleukin-1 beta converting enzyme (ICE) correlated with the loss of ECM, and inhibitors of ICE activity prevented apoptosis. These results suggest that ECM regulates apoptosis in mammary epithelial cells through an integrin-dependent negative regulation of ICE expression.

Increased Interleukin-32 Levels in Obesity Promote Adipose Tissue Inflammation and Extracellular Matrix Remodeling: Effect of Weight Loss.

Science.gov (United States)

Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Valentí, Víctor; Moncada, Rafael; Landecho, Manuel F; Silva, Camilo; Salvador, Javier; Frühbeck, Gema

2016-12-01

Interleukin (IL)-32 is a recently described cytokine involved in the regulation of inflammation. We aimed to explore whether IL-32 could function as an inflammatory and angiogenic factor in human obesity and obesity-associated type 2 diabetes. Samples obtained from 90 subjects were used in the study. Obese patients exhibited higher expression levels of IL-32 in visceral adipose tissue (AT) as well as in subcutaneous AT and peripheral blood mononuclear cells. IL32 was mainly expressed by stromovascular fraction cells, and its expression was significantly enhanced by inflammatory stimuli and hypoxia, whereas no changes were found after the incubation with anti-inflammatory cytokines. The addition of exogenous IL-32 induced the expression of inflammation and extracellular matrix-related genes in human adipocyte cultures, and IL32-silenced adipocytes showed a downregulation of inflammatory genes. Furthermore, adipocyte-conditioned media obtained from obese patients increased IL32 gene expression in human monocyte cultures, whereas the adipocyte-conditioned media from lean volunteers had no effect on IL32 mRNA levels. These findings provide evidence, for the first time, about the inflammatory and remodeling properties of IL-32 in AT, implicating this cytokine in obesity-associated comorbidities. © 2016 by the American Diabetes Association.

Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines

Science.gov (United States)

Tosar, Juan Pablo; Gámbaro, Fabiana; Sanguinetti, Julia; Bonilla, Braulio; Witwer, Kenneth W.; Cayota, Alfonso

2015-01-01

Intercellular communication can be mediated by extracellular small regulatory RNAs (sRNAs). Circulating sRNAs are being intensively studied for their promising use as minimally invasive disease biomarkers. To date, most attention is centered on exosomes and microRNAs as the vectors and the secreted species, respectively. However, this field would benefit from an increased understanding of the plethora of sRNAs secreted by different cell types in different extracellular fractions. It is still not clear if specific sRNAs are selected for secretion, or if sRNA secretion is mostly passive. We sequenced the intracellular sRNA content (19–60 nt) of breast epithelial cell lines (MCF-7 and MCF-10A) and compared it with extracellular fractions enriched in microvesicles, exosomes and ribonucleoprotein complexes. Our results are consistent with a non-selective secretion model for most microRNAs, although a few showed secretion patterns consistent with preferential secretion. On the contrary, 5′ tRNA halves and 5′ RNA Y4-derived fragments of 31–33 were greatly and significantly enriched in the extracellular space (even in non-mammary cell lines), where tRNA halves were detected as part of ∼45 kDa ribonucleoprotein complexes. Overall, we show that different sRNA families have characteristic secretion patterns and open the question of the role of these sRNAs in the extracellular space. PMID:25940616

Tissue Engineering Using Transfected Growth-Factor Genes

Science.gov (United States)

Madry, Henning; Langer, Robert S.; Freed, Lisa E.; Trippel, Stephen; Vunjak-Novakovic, Gordana

2005-01-01

A method of growing bioengineered tissues includes, as a major component, the use of mammalian cells that have been transfected with genes for secretion of regulator and growth-factor substances. In a typical application, one either seeds the cells onto an artificial matrix made of a synthetic or natural biocompatible material, or else one cultures the cells until they secrete a desired amount of an extracellular matrix. If such a bioengineered tissue construct is to be used for surgical replacement of injured tissue, then the cells should preferably be the patient s own cells or, if not, at least cells matched to the patient s cells according to a human-leucocyteantigen (HLA) test. The bioengineered tissue construct is typically implanted in the patient's injured natural tissue, wherein the growth-factor genes enhance metabolic functions that promote the in vitro development of functional tissue constructs and their integration with native tissues. If the matrix is biodegradable, then one of the results of metabolism could be absorption of the matrix and replacement of the matrix with tissue formed at least partly by the transfected cells. The method was developed for articular chondrocytes but can (at least in principle) be extended to a variety of cell types and biocompatible matrix materials, including ones that have been exploited in prior tissue-engineering methods. Examples of cell types include chondrocytes, hepatocytes, islet cells, nerve cells, muscle cells, other organ cells, bone- and cartilage-forming cells, epithelial and endothelial cells, connective- tissue stem cells, mesodermal stem cells, and cells of the liver and the pancreas. Cells can be obtained from cell-line cultures, biopsies, and tissue banks. Genes, molecules, or nucleic acids that secrete factors that influence the growth of cells, the production of extracellular matrix material, and other cell functions can be inserted in cells by any of a variety of standard transfection techniques.

Extracellular gadolinium-based contrast media: An overview

International Nuclear Information System (INIS)

Bellin, Marie-France; Van Der Molen, Aart J.

2008-01-01

Increasing use is made of extracellular MRI contrast agents that alter the image contrast following intravenous administration; they predominantly shorten the T1 relaxation time of tissues. The degree and location of these changes provide substantial diagnostic information. However gadolinium-based contrast agents (Gd-CA) are not inert drugs. They may cause acute non-renal adverse reactions (e.g. anaphylactoid reactions), acute renal adverse reactions (e.g. contrast induced nephropathy), delayed adverse reactions (nephrogenic systemic fibrosis) and problems at the site of injection (e.g. local necrosis). This review describes the current status of Gd-CA, their mechanism of action, chemical structure, pharmacokinetics, dosage, elimination, nephrotoxicity and adverse events

Extracellular gadolinium-based contrast media: An overview

Energy Technology Data Exchange (ETDEWEB)

Bellin, Marie-France [University Paris-Sud 11, Department of Radiology, University Hospital Paul-Brousse, AP-HP, 12, Avenue Paul Vaillant-Couturier, 94804 Villejuif Cedex (France)], E-mail: marie-france.bellin@pbr.aphp.fr; Van Der Molen, Aart J. [University Paris-Sud 11, Department of Radiology, University Hospital Paul-Brousse, AP-HP, 12, Avenue Paul Vaillant-Couturier, 94804 Villejuif Cedex (France)

2008-05-15

Increasing use is made of extracellular MRI contrast agents that alter the image contrast following intravenous administration; they predominantly shorten the T1 relaxation time of tissues. The degree and location of these changes provide substantial diagnostic information. However gadolinium-based contrast agents (Gd-CA) are not inert drugs. They may cause acute non-renal adverse reactions (e.g. anaphylactoid reactions), acute renal adverse reactions (e.g. contrast induced nephropathy), delayed adverse reactions (nephrogenic systemic fibrosis) and problems at the site of injection (e.g. local necrosis). This review describes the current status of Gd-CA, their mechanism of action, chemical structure, pharmacokinetics, dosage, elimination, nephrotoxicity and adverse events.

Are specific gene expressions of extracellular matrix and nucleus pulposus affected by primary cell cultures prepared from intact or degenerative intervertebral disc tissues?

Science.gov (United States)

Karaarslan, Numan; Yilmaz, Ibrahim; Ozbek, Hanefi; Sirin Yasar, Duygu; Kaplan, Necati; Akyuva, Yener; Gonultas, Aylin; Ates, Ozkan

2018-01-22

In this scientific research project, the researchers aimed to determine the gene expression patterns of nucleus pulposus (NP) in cell cultures obtained from degenerated or intact tissues. Whereas 12 of the cases were diagnosed with lumbar disc hernia and had undergone lumbar microdiscectomy, 12 cases had undergone traumatic intervertebral discectomy and corpectomy, along with discectomy after spinal trauma. NP-specific markers and gene expressions of the reagents of the extracellular matrix in the experimental setup were tested at the 0th, 24th, and 48th hours by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Visual evaluations were simultaneously made in all samples using invert and fluorescence microscopy. Vitality and proliferation analyses were evaluated by UV spectrophotometer. As a method of statistical evaluation, Spearman was used for categorical variants, and the Pearson correlation was used for variants with numerical and plain distribution. No association was found either between the tissue type and times (r=0.000; p=1.000) or between the region that the tissue was obtained from and hypoxia transcription factor-1 alpha (HIF-1α) gene expression (r=0.098; p=0.245). There was no correlation between cell proliferation and chondroadherin (CHAD) expression or between type II collagen (COL2A1) and CHAD gene expressions. It was found that CHAD and HIF-1α gene expressions and HIF-1α and COL2A1 gene expressions affected cell proliferation. Cell culture setups are of paramount importance because they may influence the pattern of changes in the gene expressions of the cells used in these setups.

EcmPred: Prediction of extracellular matrix proteins based on random forest with maximum relevance minimum redundancy feature selection

KAUST Repository

Kandaswamy, Krishna Kumar Umar; Ganesan, Pugalenthi; Kalies, Kai Uwe; Hartmann, Enno; Martinetz, Thomas M.

2013-01-01

The extracellular matrix (ECM) is a major component of tissues of multicellular organisms. It consists of secreted macromolecules, mainly polysaccharides and glycoproteins. Malfunctions of ECM proteins lead to severe disorders such as marfan

A dual role of the extracellular domain of Drosophila Crumbs for morphogenesis of the embryonic neuroectoderm

Directory of Open Access Journals (Sweden)

Shradha Das

2018-01-01

Full Text Available Epithelia are highly polarised tissues and several highly conserved polarity protein complexes serve to establish and maintain polarity. The transmembrane protein Crumbs (Crb, the central component of the Crb protein complex, is required, among others, for the maintenance of polarity in most epithelia in the Drosophila embryo. However, different epithelia exhibit different phenotypic severity upon loss of crb. Using a transgenomic approach allowed us to more accurately define the role of crb in different epithelia. In particular, we provide evidence that the loss of epithelial tissue integrity in the ventral epidermis of crb mutant embryos is due to impaired actomyosin activity and an excess number of neuroblasts. We demonstrate that the intracellular domain of Crb could only partially rescue this phenotype, while it is able to completely restore tissue integrity in other epithelia. Based on these results we suggest a dual role of the extracellular domain of Crb in the ventral neuroectoderm. First, it is required for apical enrichment of the Crb protein, which in turn regulates actomyosin activity and thereby ensures tissue integrity; and second, the extracellular domain of Crb stabilises the Notch receptor and thereby ensures proper Notch signalling and specification of the correct number of neuroblasts.

Adiponectin Is Involved in Connective Tissue Growth Factor-Induced Proliferation, Migration and Overproduction of the Extracellular Matrix in Keloid Fibroblasts.

Science.gov (United States)

Luo, Limin; Li, Jun; Liu, Han; Jian, Xiaoqing; Zou, Qianlei; Zhao, Qing; Le, Qu; Chen, Hongdou; Gao, Xinghua; He, Chundi

2017-05-12

Adiponectin, an adipocyte-derived hormone, exerts pleiotropic biological effects on metabolism, inflammation, vascular homeostasis, apoptosis and immunity. Recently, adiponectin has been suggested to attenuate the progression of human dermal fibrosis. Connective tissue growth factor (CTGF) is induced in keloids and is thought to be participated in the formation of keloid fibrosis. However, the roles played by adiponectin in keloids remain unclear. In this study, we explored the effects of adiponectin on CTGF-induced cell proliferation, migration and the deposition of extracellular matrix (ECM) and their associated intracellular signalling pathways in keloid fibroblasts (KFs). We also explored possible mechanisms of keloid pathogenesis. Primary fibroblast cultures were established from foreskin biopsies and skin biopsies from patients with keloids. The expression of adiponectin and adiponectin receptors (adipoRs) was evaluated by reverse transcription-PCR (RT-PCR), quantitative real-time RT-PCR, immunofluorescence staining, and immunohistochemical analysis. Next, KFs and normal dermal fibroblasts (NFs) were treated with CTGF in the presence or absence of adiponectin. A cell counting kit-8 (CCK-8) and the Transwell assay were used to examine cell proliferation and migration. The level of the collagen I, fibronectin (FN) and α-smooth muscle actin (α-SMA) mRNAs and proteins were determined by quantitative real-time RT-PCR and western blotting. The effects of RNA interference (RNAi) targeting the adipoR genes were detected. Phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase-protein kinase (PI3K-Akt) were examined by western blotting to further investigate the signalling pathways. Furthermore, inhibitors of signal transduction pathways were investigated. The expression levels of adiponectin and adipoRs were significantly decreased in keloids compared with those

Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury.

Science.gov (United States)

Bosmann, Markus; Grailer, Jamison J; Ruemmler, Robert; Russkamp, Norman F; Zetoune, Firas S; Sarma, J Vidya; Standiford, Theodore J; Ward, Peter A

2013-12-01

We investigated how complement activation promotes tissue injury and organ dysfunction during acute inflammation. Three models of acute lung injury (ALI) induced by LPS, IgG immune complexes, or C5a were used in C57BL/6 mice, all models requiring availability of both C5a receptors (C5aR and C5L2) for full development of ALI. Ligation of C5aR and C5L2 with C5a triggered the appearance of histones (H3 and H4) in bronchoalveolar lavage fluid (BALF). BALF from humans with ALI contained H4 histone. Histones were absent in control BALF from healthy volunteers. In mice with ALI, in vivo neutralization of H4 with IgG antibody reduced the intensity of ALI. Neutrophil depletion in mice with ALI markedly reduced H4 presence in BALF and was highly protective. The direct lung damaging effects of extracellular histones were demonstrated by airway administration of histones into mice and rats (Sprague-Dawley), which resulted in ALI that was C5a receptor-independent, and associated with intense inflammation, PMN accumulation, damage/destruction of alveolar epithelial cells, together with release into lung of cytokines/chemokines. High-resolution magnetic resonance imaging demonstrated lung damage, edema and consolidation in histone-injured lungs. These studies confirm the destructive C5a-dependent effects in lung linked to appearance of extracellular histones.

Circuit Models and Experimental Noise Measurements of Micropipette Amplifiers for Extracellular Neural Recordings from Live Animals

Directory of Open Access Journals (Sweden)

Chang Hao Chen

2014-01-01

Full Text Available Glass micropipettes are widely used to record neural activity from single neurons or clusters of neurons extracellularly in live animals. However, to date, there has been no comprehensive study of noise in extracellular recordings with glass micropipettes. The purpose of this work was to assess various noise sources that affect extracellular recordings and to create model systems in which novel micropipette neural amplifier designs can be tested. An equivalent circuit of the glass micropipette and the noise model of this circuit, which accurately describe the various noise sources involved in extracellular recordings, have been developed. Measurement schemes using dead brain tissue as well as extracellular recordings from neurons in the inferior colliculus, an auditory brain nucleus of an anesthetized gerbil, were used to characterize noise performance and amplification efficacy of the proposed micropipette neural amplifier. According to our model, the major noise sources which influence the signal to noise ratio are the intrinsic noise of the neural amplifier and the thermal noise from distributed pipette resistance. These two types of noise were calculated and measured and were shown to be the dominating sources of background noise for in vivo experiments.

Micromechanical anisotropy and heterogeneity of the meniscus extracellular matrix.

Science.gov (United States)

Li, Qing; Qu, Feini; Han, Biao; Wang, Chao; Li, Hao; Mauck, Robert L; Han, Lin

2017-05-01

To understand how the complex biomechanical functions of the meniscus are endowed by the nanostructure of its extracellular matrix (ECM), we studied the anisotropy and heterogeneity in the micromechanical properties of the meniscus ECM. We used atomic force microscopy (AFM) to quantify the time-dependent mechanical properties of juvenile bovine meniscus at deformation length scales corresponding to the diameters of collagen fibrils. At this scale, anisotropy in the elastic modulus of the circumferential fibers, the major ECM structural unit, can be attributed to differences in fibril deformation modes: uncrimping when normal to the fiber axis, and laterally constrained compression when parallel to the fiber axis. Heterogeneity among different structural units is mainly associated with their variations in microscale fiber orientation, while heterogeneity across anatomical zones is due to alterations in collagen fibril diameter and alignment at the nanoscale. Unlike the elastic modulus, the time-dependent properties are more homogeneous and isotropic throughout the ECM. These results enable a detailed understanding of the meniscus structure-mechanics at the nanoscale, and can serve as a benchmark for understanding meniscus biomechanical functions, documenting disease progression and designing tissue repair strategies. Meniscal damage is a common cause of joint injury, which can lead to the development of post-traumatic osteoarthritis among young adults. Restoration of meniscus function requires repairing its highly heterogeneous and complex extracellular matrix. Employing AFM, this study quantifies the anisotropic and heterogeneous features of the meniscus ECM structure and mechanics. The micromechanical properties are interpreted within the context of the collagen fibril nanostructure and its variation with tissue anatomical locations. These results provide a fundamental structure-mechanics knowledge benchmark, against which, repair and regeneration strategies can

Exercise during long term exposure to space: Value of exercise during space exploration

Science.gov (United States)

1990-01-01

There appear to be two general physiological reasons why exercise will be beneficial to space travelers who will experience a weightless and isolated environment for many months or a few years: (1) to alleviate or prevent tissue atrophy (principally bone and muscle), to maintain cardiovascular function, and to prevent deleterious changes in extracellular and cellular fluid volumes and plasma constituents, especially electrolytes; and (2) to maintain whole organism functional physical and physiological status with special reference to neuromuscular coordination (physical skill) and physical fitness (muscle strength and power, flexibility, and aerobic endurance). The latter reason also relates well to the ability of the crew members to resist both general and local fatigue and thus ensure consistent physical performance. Various forms of exercise, performed regularly, could help alleviate boredom and assist the travelers in coping with stress, anxiety, and depression. The type, frequency, duration and intensity of exercise and ways of ensuring that crew members engage in it are discussed.

HYDROGEL-BASED NANOCOMPOSITES OF THERAPEUTIC PROTEINS FOR TISSUE REPAIR.

Science.gov (United States)

Zhu, Suwei; Segura, Tatiana

2014-05-01

The ability to design artificial extracellular matrices as cell instructive scaffolds has opened the door to technologies capable of studying cell fates in vitro and to guide tissue repair in vivo . One main component of the design of artificial extracellular matrices is the incorporation of protein-based biochemical cues to guide cell phenotypes and multicellular organizations. However, promoting the long-term bioactivity, controlling the bioavailability and understanding how the physical presentations of these proteins impacts cellular fates are among the challenges of the field. Nanotechnolgy has advanced to meet the challenges of protein therapeutics. For example, the approaches to incorporating proteins into tissue repairing scaffolds have ranged from bulk encapsulations to smart nanodepots that protect proteins from degradations and allow opportunities for controlled release.

Extracellular mass transport considerations for space flight research concerning suspended and adherent in vitro cell cultures.

Science.gov (United States)

Klaus, David M; Benoit, Michael R; Nelson, Emily S; Hammond, Timmothy G

2004-03-01

Conducting biological research in space requires consideration be given to isolating appropriate control parameters. For in vitro cell cultures, numerous environmental factors can adversely affect data interpretation. A biological response attributed to microgravity can, in theory, be explicitly correlated to a specific lack of weight or gravity-driven motion occurring to, within or around a cell. Weight can be broken down to include the formation of hydrostatic gradients, structural load (stress) or physical deformation (strain). Gravitationally induced motion within or near individual cells in a fluid includes sedimentation (or buoyancy) of the cell and associated shear forces, displacement of cytoskeleton or organelles, and factors associated with intra- or extracellular mass transport. Finally, and of particular importance for cell culture experiments, the collective effects of gravity must be considered for the overall system consisting of the cells, their environment and the device in which they are contained. This does not, however, rule out other confounding variables such as launch acceleration, on orbit vibration, transient acceleration impulses or radiation, which can be isolated using onboard centrifuges or vibration isolation techniques. A framework is offered for characterizing specific cause-and-effect relationships for gravity-dependent responses as a function of the above parameters.

Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells

OpenAIRE

Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

2015-01-01

Introduction Collagen is a widely used naturally occurring biomaterial for scaffolding, whereas mesenchymal stem cells (MSCs) represent a promising cell source in tissue engineering and regenerative medicine. It is generally known that cells are able to remodel their environment by simultaneous degradation of the scaffolds and deposition of newly synthesized extracellular matrix. Nevertheless, the interactions between MSCs and collagen biomaterials are poorly known, and the strategies enhanci...

A Novel Molecular Diagnostic of Glioblastomas: Detection of an Extracellular Fragment of Protein Tyrosine Phosphatase μ

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Susan M. Burden-Gulley

2010-04-01

Full Text Available We recently found that normal human brain and low-grade astrocytomas express the receptor protein tyrosine phosphatase mu (PTPμ and that the more invasive astrocytomas, glioblastoma multiforme (GBM, downregulate full-length PTPμ expression. Loss of PTPμ expression in GBMs is due to proteolytic cleavage that generates an intracellular and potentially a cleaved and released extracellular fragment of PTPμ. Here, we identify that a cleaved extracellular fragment containing the domains required for PTPμ-mediated adhesion remains associated with GBM tumor tissue. We hypothesized that detection of this fragment would make an excellent diagnostic tool for the localization of tumor tissue within the brain. To this end, we generated a series of fluorescently tagged peptide probes that bind the PTPμ fragment. The peptide probes specifically recognize GBM cells in tissue sections of surgically resected human tumors. To test whether the peptide probes are able to detect GBM tumors in vivo, the PTPμ peptide probes were tested in both mouse flank and intracranial xenograft human glioblastoma tumor model systems. The glial tumors were molecularly labeled with the PTPμ peptide probes within minutes of tail vein injection using the Maestro FLEX In Vivo Imaging System. The label was stable for at least 3 hours. Together, these results indicate that peptide recognition of the PTPμ extracellular fragment provides a novel molecular diagnostic tool for detection of human glioblastomas. Such a tool has clear translational applications and may lead to improved surgical resections and prognosis for patients with this devastating disease.

Engineering of blood vessel patterns by angio-morphogens [angiotropins]: non-mitogenic copper-ribonucleoprotein cytokins [CuRNP ribokines] with their metalloregulated constituents of RAGE-binding S100-EF-hand proteins and extracellular RNA bioaptamers in vascular remodeling of tissue and angiogenesis in vitro

Energy Technology Data Exchange (ETDEWEB)

Wissler, J.H. [ARCONS Applied Research, Bad Nauheim (Germany)

2001-12-01

Tissue vascularization is requisite to successful cell-based therapies, biomaterial design and implant integration. Thus, known problems in ossointegration of avascular implants in connection with the generation of bone tissue reflect arrays of general problems of socio-economic relevance existing in reparative medicine still waiting for to be solved. For this purpose, morphogenesis and remodeling of endothelial angio-architectures in tissue and in vitro by isolated non-mitogenic angio-morphogens [angiotropins] are considered in terms of their structure, function and action mechanisms. Extracellular angiotropins are secreted by activated leukocytes/monocytes/macrophages. They are a family of cytokines with morphogen bioactivity selectively directed to endothelial cells. Their structure was deciphered as metalloregulated copper-ribonucleoproteins [CuRNP ribokines]. They are built up of angiotropin-related S100-EF-hand protein [ARP] and highly modified and edited 5'end-phosphorylated RNA [ARNA], complexed together by copper ions. Oxidant-sensitive ARNA and their precursors represent novel types in a RNA world: They are the first isolated and sequenced forms of extracellular RNA [eRNA], may act as cytokine and bioaptamer, contain isoguanosine [crotonoside] as modified nucleoside and show up copper as RNA-structuring transition metal ion. By metalloregulated bioaptamer functions, ARNA impart novel biofunctions to RAGE-binding S100-EF-hand proteins. Angiotropin morphogens were shown suitable for neointiation and remodeling of blood vessel patterns in different, adult, embryonal and artificial tissues. These neovascular patterns manifest regulated hemodynamics for preventing tissue necrosis, supporting tissue functions and promoting wound healing. As evaluated in skin and muscle vascularization, the neovascular patterns are integrated into homeostatic control mechanisms of tissue. Thus, the morphogens show up beneficial perspectives and are suggested useful tools

Circulating Extracellular Histones Are Clinically Relevant Mediators of Multiple Organ Injury.

Science.gov (United States)

Kawai, Chihiro; Kotani, Hirokazu; Miyao, Masashi; Ishida, Tokiko; Jemail, Leila; Abiru, Hitoshi; Tamaki, Keiji

2016-04-01

Extracellular histones are a damage-associated molecular pattern (DAMP) involved in the pathogenesis of various diseases. The mechanisms of histone-mediated injury in certain organs have been extensively studied, but an understanding of the pathophysiological role of histone-mediated injury in multiple organ injury remains elusive. To elucidate this role, we systemically subjected C57BL/6 mice to various doses of histones and performed a chronological evaluation of the morphological and functional changes in the lungs, liver, and kidneys. Notably, histone administration ultimately led to death after a dose-dependent aggravation of multiple organ injury. In chronological studies, pulmonary and hepatic injuries occurred within 15 minutes, whereas renal injuries presented at a later phase, suggesting that susceptibility to extracellular histones varies among organs. Histones bound to pulmonary and hepatic endothelial cells immediately after administration, leading to endothelial damage, which could be ameliorated by pretreatment with heparin. Furthermore, release of another DAMP, high-mobility group protein box 1, followed the histone-induced tissue damage, and an antibody against the molecule ameliorated hepatic and renal failure in a late phase. These findings indicate that extracellular histones induce multiple organ injury in two progressive stages-direct injury to endothelial cells and the subsequent release of other DAMPs-and that combination therapies against extracellular histones and high-mobility group protein box 1 may be a promising strategy for treating multiple organ injury. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

A structure-based extracellular matrix expansion mechanism of fibrous tissue growth.

Science.gov (United States)

Kalson, Nicholas S; Lu, Yinhui; Taylor, Susan H; Starborg, Tobias; Holmes, David F; Kadler, Karl E

2015-05-20

Embryonic growth occurs predominately by an increase in cell number; little is known about growth mechanisms later in development when fibrous tissues account for the bulk of adult vertebrate mass. We present a model for fibrous tissue growth based on 3D-electron microscopy of mouse tendon. We show that the number of collagen fibrils increases during embryonic development and then remains constant during postnatal growth. Embryonic growth was explained predominately by increases in fibril number and length. Postnatal growth arose predominately from increases in fibril length and diameter. A helical crimp structure was established in embryogenesis, and persisted postnatally. The data support a model where the shape and size of tendon is determined by the number and position of embryonic fibroblasts. The collagen fibrils that these cells synthesise provide a template for postnatal growth by structure-based matrix expansion. The model has important implications for growth of other fibrous tissues and fibrosis.

Extracellular protease mRNAs are predominantly expressed in the stromal areas of microdissected mouse breast carcinomas

DEFF Research Database (Denmark)

Pedersen, Tanja Xenia; Pennington, Caroline J; Almholt, Kasper

2005-01-01

Solid tumors synthesize a number of extracellular matrix-degrading proteases that are important for tumor progression. Based on qualitative in situ hybridization studies in human cancer tissue, a range of components involved in proteolysis appear to be expressed by stromal cells rather than cancer...

Extracellular ATP in the Exocrine Pancreas – ATP Release, Signalling and Metabolism

DEFF Research Database (Denmark)

Kowal, Justyna Magdalena

release. So far, the contribution of duct cells in purinergic signalling has never been studied. This work presents that both acinar and duct cells are sources of extracellular ATP in the exocrine pancreas. Here we show that duct cells release ATP in response to several physiological......ATP plays an important role as an autocrine/paracrine signalling molecule, being released from a number of tissues, in response to physiological and pathophysiological stimuli. Released ATP induces Ca2+ - and/or cAMP - dependent cellular responses via activation of ubiquitously expressed P2X and P2......, particularly during Ca2+ stress conditions. In conclusion, these studies demonstrate a complex regulation of purinergic signalling in exocrine pancreas. A crucial role for duct cells in mediating extracellular nucleotides homeostasis, involving ATP release, subsequent hydrolysis and conversion via...

The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

OpenAIRE

L?sser, Cecilia; Th?ry, Clotilde; Buz?s, Edit I.; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; L?tvall, Jan

2016-01-01

The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field co...

Protein and Glycoprotein Patterns Related to Morphogenesis in Mammillaria gracillis Pfeiff. Tissue Culture

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Biljana Balen

2002-01-01

Full Text Available As plants with Crassulacean Acid Metabolism (CAM, cacti are highly affected by artificial environmental conditions in tissue culture. Plants of Mammillaria gracillis Pfeiff. (Cactaceae propagated in vitro produced callus spontaneously. This habituated callus regenerated normal and hyperhydric shoots without the addition of growth regulators. In order to compare habituated callus with the tumorous one, cactus cells were transformed with two strains of Agrobacterium tumefaciens: the wild strain B6S3 (tumour line TW and the rooty mutant GV3101 (tumour line TR. Gene expression in cactus plants, habituated callus, regenerated shoots and two tumour lines was analysed at the level of cellular and extracellular protein and glycoprotein profiles. Proteins were separated by SDS-polyacrylamide gel electrophoresis and 2-D PAGE electrophoresis and silver stained. Concavalin A-peroxidase staining detected glycoproteins with D-manose in their glycan component on protein blots. Developmentally specific protein patterns of Mammillaria gracillis tissue lines were detected. The 2-D PAGE electrophoresis revealed some tissue specific protein groups. The cellular glycoprotein of 42 kDa detected by ConA was highly expressed in undifferentiated tissues (habituated callus, TW and TR tumours and in hyperhydric regenerants. Tumours produced extracellular proteins of 33, 23 and 22 kDa. The N glycosylation of cellular and extracellular proteins was related to specific developmental stage of cactus tissue.

Evaluation of small intestine grafts decellularization methods for corneal tissue engineering.

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Ana Celeste Oliveira

Full Text Available Advances in the development of cornea substitutes by tissue engineering techniques have focused on the use of decellularized tissue scaffolds. In this work, we evaluated different chemical and physical decellularization methods on small intestine tissues to determine the most appropriate decellularization protocols for corneal applications. Our results revealed that the most efficient decellularization agents were the SDS and triton X-100 detergents, which were able to efficiently remove most cell nuclei and residual DNA. Histological and histochemical analyses revealed that collagen fibers were preserved upon decellularization with triton X-100, NaCl and sonication, whereas reticular fibers were properly preserved by decellularization with UV exposure. Extracellular matrix glycoproteins were preserved after decellularization with SDS, triton X-100 and sonication, whereas proteoglycans were not affected by any of the decellularization protocols. Tissue transparency was significantly higher than control non-decellularized tissues for all protocols, although the best light transmittance results were found in tissues decellularized with SDS and triton X-100. In conclusion, our results suggest that decellularized intestinal grafts could be used as biological scaffolds for cornea tissue engineering. Decellularization with triton X-100 was able to efficiently remove all cells from the tissues while preserving tissue structure and most fibrillar and non-fibrillar extracellular matrix components, suggesting that this specific decellularization agent could be safely used for efficient decellularization of SI tissues for cornea TE applications.

Space Radiation Effects on Human Cells: Modeling DNA Breakage, DNA Damage Foci Distribution, Chromosomal Aberrations and Tissue Effects

Science.gov (United States)

Ponomarev, A. L.; Huff, J. L.; Cucinotta, F. A.

2011-01-01

Future long-tem space travel will face challenges from radiation concerns as the space environment poses health risk to humans in space from radiations with high biological efficiency and adverse post-flight long-term effects. Solar particles events may dramatically affect the crew performance, while Galactic Cosmic Rays will induce a chronic exposure to high-linear-energy-transfer (LET) particles. These types of radiation, not present on the ground level, can increase the probability of a fatal cancer later in astronaut life. No feasible shielding is possible from radiation in space, especially for the heavy ion component, as suggested solutions will require a dramatic increase in the mass of the mission. Our research group focuses on fundamental research and strategic analysis leading to better shielding design and to better understanding of the biological mechanisms of radiation damage. We present our recent effort to model DNA damage and tissue damage using computational models based on the physics of heavy ion radiation, DNA structure and DNA damage and repair in human cells. Our particular area of expertise include the clustered DNA damage from high-LET radiation, the visualization of DSBs (DNA double strand breaks) via DNA damage foci, image analysis and the statistics of the foci for different experimental situations, chromosomal aberration formation through DSB misrepair, the kinetics of DSB repair leading to a model-derived spectrum of chromosomal aberrations, and, finally, the simulation of human tissue and the pattern of apoptotic cell damage. This compendium of theoretical and experimental data sheds light on the complex nature of radiation interacting with human DNA, cells and tissues, which can lead to mutagenesis and carcinogenesis later in human life after the space mission.

Use of tissue equivalent proportional counters to characterize radiation quality on the space shuttle

International Nuclear Information System (INIS)

Braby, L.A.; Conroy, T.J.; Elegy, D.C.; Brackenbush, L.W.

1992-04-01

Tissue equivalent proportional counters (TEPC) are essentially cavity ionization chambers operating at low pressure and with gas gain. A small, battery powered, TEPC spectrometer, which records lineal energy spectra at one minute intervals, has been used on several space shuttle missions. The data it has collected clearly show the South Atlantic anomaly and indicate a mean quality factor somewhat higher than expected. An improved type of instrument has been developed with sufficient memory to record spectra at 10 second intervals, and with increased resolution for low LET events. This type of instrument will be used on most future space shuttle flights and in some international experiments

Biodegradable Polymer-Based Scaffolds for Bone Tissue Engineering

CERN Document Server

Sultana, Naznin

2013-01-01

This book addresses the principles, methods and applications of biodegradable polymer based scaffolds for bone tissue engineering. The general principle of bone tissue engineering is reviewed and the traditional and novel scaffolding materials, their properties and scaffold fabrication techniques are explored. By acting as temporary synthetic extracellular matrices for cell accommodation, proliferation, and differentiation, scaffolds play a pivotal role in tissue engineering. This book does not only provide the comprehensive summary of the current trends in scaffolding design but also presents the new trends and directions for scaffold development for the ever expanding tissue engineering applications.

The Role of Extracellular Matrix Quality in Pulmonary Fibrosis

DEFF Research Database (Denmark)

Kristensen, Jacob Hull; Karsdal, Morten Asser; Genovese, Federica

2014-01-01

This review discusses the role of extracellular matrix (ECM) quality in the pathogenesis of pulmonary fibrosis (PF). In PF, the highly ordered structure of collagens and elastin within the ECM of the lung is severely disrupted and lacks its original tissue quality. Discussions about the ECM have...... focused on the role of protein quantity in relation to the progression of PF, while the importance of lung ECM quality, defined by the levels of ECM protein modifications and by the protein distribution in lung tissue, has not been properly addressed. The quality and function of proteins may be altered...... by different post-translational modifications (PTMs), such as crosslinking, proteolytic cleavage, citrullination, misfolding and glycosylation. This paper is the first to review key data from the literature related to the lung ECM at the molecular level, relate these to changes observed at a macroscopic level...

A Tissue Retrieval and Postharvest Processing Regimen for Rodent Reproductive Tissues Compatible with Long-Term Storage on the International Space Station and Postflight Biospecimen Sharing Program

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Vijayalaxmi Gupta

2015-01-01

Full Text Available Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at −80°C that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASA’s Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

Acute exposure to space flight results in evidence of reduced lymph Transport, tissue fluid Shifts, and immune alterations in the rat gastrointestinal system

Science.gov (United States)

Cromer, W. E.; Zawieja, D. C.

2018-05-01

Space flight causes a number of alterations in physiological systems, changes in the immunological status of subjects, and altered interactions of the host to environmental stimuli. We studied the effect of space flight on the lymphatic system of the gastrointestinal tract which is responsible for lipid transport and immune surveillance which includes the host interaction with the gut microbiome. We found that there were signs of tissue damage present in the space flown animals that was lacking in ground controls (epithelial damage, crypt morphological changes, etc.). Additionally, morphology of the lymphatic vessels in the tissue suggested a collapsed state at time of harvest and there was a profound change in the retention of lipid in the villi of the ileum. Contrary to our assumptions there was a reduction in tissue fluid volume likely associated with other fluid shifts described. The reduction of tissue fluid volume in the colon and ileum is a likely contributing factor to the state of the lymphatic vessels and lipid transport issues observed. There were also associated changes in the number of MHC-II+ immune cells in the colon tissue, which along with reduced lymphatic competence would favor immune dysfunction in the tissue. These findings help expand our understanding of the effects of space flight on various organ systems. It also points out potential issues that have not been closely examined and have to potential for the need of countermeasure development.

Impact of TGF-β inhibition during acute exercise on Achilles tendon extracellular matrix

DEFF Research Database (Denmark)

Potter, Ross M; Huynh, Richard T; Volper, Brent D

2017-01-01

The purpose of this study was to evaluate the role of TGF-β1in regulating tendon extracellular matrix after acute exercise. Wistar rats exercised (n = 15) on a treadmill for four consecutive days (60 min/day) or maintained normal cage activity. After each exercise bout, the peritendinous space of...

A gold nanoparticle coated porcine cholecyst-derived bioscaffold for cardiac tissue engineering.

Science.gov (United States)

Nair, Reshma S; Ameer, Jimna Mohamed; Alison, Malcolm R; Anilkumar, Thapasimuthu V

2017-09-01

Extracellular matrices of xenogeneic origin have been extensively used for biomedical applications, despite the possibility of heterogeneity in structure. Surface modification of biologically derived biomaterials using nanoparticles is an emerging strategy for improving topographical homogeneity when employing these scaffolds for sophisticated tissue engineering applications. Recently, as a tissue engineering scaffold, cholecyst derived extracellular matrix (C-ECM) has been shown to have several advantages over extracellular matrices derived from other organs such as jejunum and urinary bladder. This study explored the possibility of adding gold nanoparticles, which have a large surface area to volume ratio on C-ECM for achieving homogeneity in surface architecture, a requirement for cardiac tissue engineering. In the current study, gold nanoparticles (AuNPs) were synthesized and functionalised for conjugating with a porcine cholecystic extracellular matrix scaffold. The conjugation of nanoparticles to C-ECM was achieved by 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide/N-hydroxysuccinimide chemistry and further characterized by Fourier transform infrared spectroscopy, environmental scanning electron microscopy, energy dispersive X-ray spectroscopy and thermogravimetric analysis. The physical properties of the modified scaffold were similar to the original C-ECM. Biological properties were evaluated by using H9c2 cells, a cardiomyoblast cell line commonly used for cellular and molecular studies of cardiac cells. The modified scaffold was found to be a suitable substrate for the growth and proliferation of the cardiomyoblasts. Further, the non-cytotoxic nature of the modified scaffold was established by direct contact cytotoxicity testing and live/dead staining. Thus, the modified C-ECM appears to be a potential biomaterial for cardiac tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

Specific extracellular matrix remodeling signature of colon hepatic metastases.

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Maguy Del Rio

Full Text Available To identify genes implicated in metastatic colonization of the liver in colorectal cancer, we collected pairs of primary tumors and hepatic metastases before chemotherapy in 13 patients. We compared mRNA expression in the pairs of patients to identify genes deregulated during metastatic evolution. We then validated the identified genes using data obtained by different groups. The 33-gene signature was able to classify 87% of hepatic metastases, 98% of primary tumors, 97% of normal colon mucosa, and 95% of normal liver tissues in six datasets obtained using five different microarray platforms. The identified genes are specific to colon cancer and hepatic metastases since other metastatic locations and hepatic metastases originating from breast cancer were not classified by the signature. Gene Ontology term analysis showed that 50% of the genes are implicated in extracellular matrix remodeling, and more precisely in cell adhesion, extracellular matrix organization and angiogenesis. Because of the high efficiency of the signature to classify colon hepatic metastases, the identified genes represent promising targets to develop new therapies that will specifically affect hepatic metastasis microenvironment.

Intervertebral Disc Tissue Engineering with Natural Extracellular Matrix-Derived Biphasic Composite Scaffolds.

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Baoshan Xu

Full Text Available Tissue engineering has provided an alternative therapeutic possibility for degenerative disc diseases. However, we lack an ideal scaffold for IVD tissue engineering. The goal of this study is to fabricate a novel biomimetic biphasic scaffold for IVD tissue engineering and evaluate the feasibility of developing tissue-engineered IVD in vitro and in vivo. In present study we developed a novel integrated biphasic IVD scaffold using a simple freeze-drying and cross-linking technique of pig bone matrix gelatin (BMG for the outer annulus fibrosus (AF phase and pig acellular cartilage ECM (ACECM for the inner nucleus pulposus (NP phase. Histology and SEM results indicated no residual cells remaining in the scaffold that featured an interconnected porous microstructure (pore size of AF and NP phase 401.4 ± 13.1 μm and 231.6 ± 57.2 μm, respectively. PKH26-labeled AF and NP cells were seeded into the scaffold and cultured in vitro. SEM confirmed that seeded cells could anchor onto the scaffold. Live/dead staining showed that live cells (green fluorescence were distributed in the scaffold, with no dead cells (red fluorescence being found. The cell-scaffold constructs were implanted subcutaneously into nude mice and cultured for 6 weeks in vivo. IVD-like tissue formed in nude mice as confirmed by histology. Cells in hybrid constructs originated from PKH26-labeled cells, as confirmed by in vivo fluorescence imaging system. In conclusion, the study demonstrates the feasibility of developing a tissue-engineered IVD in vivo with a BMG- and ACECM-derived integrated AF-NP biphasic scaffold. As well, PKH26 fluorescent labeling with in vivo fluorescent imaging can be used to track cells and analyse cell--scaffold constructs in vivo.

Extracellular Matrix Molecules Facilitating Vascular Biointegration

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Martin K.C. Ng

2012-08-01

Full Text Available All vascular implants, including stents, heart valves and graft materials exhibit suboptimal biocompatibility that significantly reduces their clinical efficacy. A range of biomolecules in the subendothelial space have been shown to play critical roles in local regulation of thrombosis, endothelial growth and smooth muscle cell proliferation, making these attractive candidates for modulation of vascular device biointegration. However, classically used biomaterial coatings, such as fibronectin and laminin, modulate only one of these components; enhancing endothelial cell attachment, but also activating platelets and triggering thrombosis. This review examines a subset of extracellular matrix molecules that have demonstrated multi-faceted vascular compatibility and accordingly are promising candidates to improve the biointegration of vascular biomaterials.

Nanoscale tissue engineering: spatial control over cell-materials interactions

Science.gov (United States)

Wheeldon, Ian; Farhadi, Arash; Bick, Alexander G.; Jabbari, Esmaiel; Khademhosseini, Ali

2011-01-01

Cells interact with the surrounding environment by making tens to hundreds of thousands of nanoscale interactions with extracellular signals and features. The goal of nanoscale tissue engineering is to harness the interactions through nanoscale biomaterials engineering in order to study and direct cellular behaviors. Here, we review the nanoscale tissue engineering technologies for both two- and three-dimensional studies (2- and 3D), and provide a holistic overview of the field. Techniques that can control the average spacing and clustering of cell adhesion ligands are well established and have been highly successful in describing cell adhesion and migration in 2D. Extension of these engineering tools to 3D biomaterials has created many new hydrogel and nanofiber scaffolds technologies that are being used to design in vitro experiments with more physiologically relevant conditions. Researchers are beginning to study complex cell functions in 3D, however, there is a need for biomaterials systems that provide fine control over the nanoscale presentation of bioactive ligands in 3D. Additionally, there is a need for 2- and 3D techniques that can control the nanoscale presentation of multiple bioactive ligands and the temporal changes in cellular microenvironment. PMID:21451238

Nanoscale tissue engineering: spatial control over cell-materials interactions

International Nuclear Information System (INIS)

Wheeldon, Ian; Farhadi, Arash; Bick, Alexander G; Khademhosseini, Ali; Jabbari, Esmaiel

2011-01-01

Cells interact with the surrounding environment by making tens to hundreds of thousands of nanoscale interactions with extracellular signals and features. The goal of nanoscale tissue engineering is to harness these interactions through nanoscale biomaterials engineering in order to study and direct cellular behavior. Here, we review two- and three-dimensional (2- and 3D) nanoscale tissue engineering technologies, and provide a holistic overview of the field. Techniques that can control the average spacing and clustering of cell adhesion ligands are well established and have been highly successful in describing cell adhesion and migration in 2D. Extension of these engineering tools to 3D biomaterials has created many new hydrogel and nanofiber scaffold technologies that are being used to design in vitro experiments with more physiologically relevant conditions. Researchers are beginning to study complex cell functions in 3D. However, there is a need for biomaterials systems that provide fine control over the nanoscale presentation of bioactive ligands in 3D. Additionally, there is a need for 2- and 3D techniques that can control the nanoscale presentation of multiple bioactive ligands and that can control the temporal changes in the cellular microenvironment. (topical review)

Extracellular ATP reduces HIV-1 transfer from immature dendritic cells to CD4+ T lymphocytes

Directory of Open Access Journals (Sweden)

Barat Corinne

2008-03-01

Full Text Available Abstract Background Dendritic cells (DCs are considered as key mediators of the early events in human immunodeficiency virus type 1 (HIV-1 infection at mucosal sites. Previous studies have shown that surface-bound virions and/or internalized viruses found in endocytic vacuoles of DCs are efficiently transferred to CD4+ T cells. Extracellular adenosine triphosphate (ATP either secreted or released from necrotic cells induces a distorted maturation of DCs, transiently increases their endocytic capacity and affects their migratory capacity. Knowing that high extracellular ATP concentrations are present in situations of tissue injury and inflammation, we investigated the effect of ATP on HIV-1 transmission from DCs to CD4+ T lymphocytes. Results In this study, we show that extracellular ATP reduces HIV-1 transfer from immature monocyte-derived DCs (iDCs to autologous CD4+ T cells. This observed decrease in viral replication was related to a lower proportion of infected CD4+ T cells following transfer, and was seen with both X4- and R5-tropic isolates of HIV-1. Extracellular ATP had no effect on direct CD4+ T cell infection as well as on productive HIV-1 infection of iDCs. These observations indicate that extracellular ATP affects HIV-1 infection of CD4+ T cells in trans with no effect on de novo virus production by iDCs. Additional experiments suggest that extracellular ATP might modulate the trafficking pathway of internalized virions within iDCs leading to an increased lysosomal degradation, which could be partly responsible for the decreased HIV-1 transmission. Conclusion These results suggest that extracellular ATP can act as a factor controlling HIV-1 propagation.

Integrating valve-inspired design features into poly(ethylene glycol) hydrogel scaffolds for heart valve tissue engineering.

Science.gov (United States)

Zhang, Xing; Xu, Bin; Puperi, Daniel S; Yonezawa, Aline L; Wu, Yan; Tseng, Hubert; Cuchiara, Maude L; West, Jennifer L; Grande-Allen, K Jane

2015-03-01

The development of advanced scaffolds that recapitulate the anisotropic mechanical behavior and biological functions of the extracellular matrix in leaflets would be transformative for heart valve tissue engineering. In this study, anisotropic mechanical properties were established in poly(ethylene glycol) (PEG) hydrogels by crosslinking stripes of 3.4 kDa PEG diacrylate (PEGDA) within 20 kDa PEGDA base hydrogels using a photolithographic patterning method. Varying the stripe width and spacing resulted in a tensile elastic modulus parallel to the stripes that was 4.1-6.8 times greater than that in the perpendicular direction, comparable to the degree of anisotropy between the circumferential and radial orientations in native valve leaflets. Biomimetic PEG-peptide hydrogels were prepared by tethering the cell-adhesive peptide RGDS and incorporating the collagenase-degradable peptide PQ (GGGPQG↓IWGQGK) into the polymer network. The specific amounts of RGDS and PEG-PQ within the resulting hydrogels influenced the elongation, de novo extracellular matrix deposition and hydrogel degradation behavior of encapsulated valvular interstitial cells (VICs). In addition, the morphology and activation of VICs grown atop PEG hydrogels could be modulated by controlling the concentration or micro-patterning profile of PEG-RGDS. These results are promising for the fabrication of PEG-based hydrogels using anatomically and biologically inspired scaffold design features for heart valve tissue engineering. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

3D-Printed ABS and PLA Scaffolds for Cartilage and Nucleus Pulposus Tissue Regeneration

OpenAIRE

Rosenzweig, Derek H.; Carelli, Eric; Steffen, Thomas; Jarzem, Peter; Haglund, Lisbet

2015-01-01

Painful degeneration of soft tissues accounts for high socioeconomic costs. Tissue engineering aims to provide biomimetics recapitulating native tissues. Biocompatible thermoplastics for 3D printing can generate high-resolution structures resembling tissue extracellular matrix. Large-pore 3D-printed acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) scaffolds were compared for cell ingrowth, viability, and tissue generation. Primary articular chondrocytes and nucleus pulposus (N...

Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture

Science.gov (United States)

Abbah, Sunny A.; Thomas, Dilip; Browne, Shane; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

2016-02-01

Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-β1). In tendon tissue repair, increased activity of TGF-β1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-β1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-β1 and a range of pro-fibrotic extracellular matrix genes.

A two-phase model of plantar tissue: a step toward prediction of diabetic foot ulceration.

Science.gov (United States)

Sciumè, G; Boso, D P; Gray, W G; Cobelli, C; Schrefler, B A

2014-11-01

A new computational model, based on the thermodynamically constrained averaging theory, has been recently proposed to predict tumor initiation and proliferation. A similar mathematical approach is proposed here as an aid in diabetic ulcer prevention. The common aspects at the continuum level are the macroscopic balance equations governing the flow of the fluid phase, diffusion of chemical species, tissue mechanics, and some of the constitutive equations. The soft plantar tissue is modeled as a two-phase system: a solid phase consisting of the tissue cells and their extracellular matrix, and a fluid one (interstitial fluid and dissolved chemical species). The solid phase may become necrotic depending on the stress level and on the oxygen availability in the tissue. Actually, in diabetic patients, peripheral vascular disease impacts tissue necrosis; this is considered in the model via the introduction of an effective diffusion coefficient that governs transport of nutrients within the microvasculature. The governing equations of the mathematical model are discretized in space by the finite element method and in time domain using the θ-Wilson Method. While the full mathematical model is developed in this paper, the example is limited to the simulation of several gait cycles of a healthy foot. Copyright © 2014 John Wiley & Sons, Ltd.

* Tissue-Specific Extracellular Matrix Enhances Skeletal Muscle Precursor Cell Expansion and Differentiation for Potential Application in Cell Therapy.

Science.gov (United States)

Zhang, Deying; Zhang, Yong; Zhang, Yuanyuan; Yi, Hualin; Wang, Zhan; Wu, Rongpei; He, Dawei; Wei, Guanghui; Wei, Shicheng; Hu, Yun; Deng, Junhong; Criswell, Tracy; Yoo, James; Zhou, Yu; Atala, Anthony

2017-08-01

Skeletal muscle precursor cells (MPCs) are considered a key candidate for cell therapy in the treatment of skeletal muscle dysfunction due to injury, disease, or age. However, expansion of a sufficient number of functional skeletal muscle cells in vitro from a small tissue biopsy has been challenging due to changes in phenotypic expression of these cells under traditional culture conditions. Thus, the aim of the study was to develop a better culture system for the expansion and myo-differentiation of MPCs that could further be used for therapy. For this purpose, we developed an ideal method of tissue decellularization and compared the ability of different matrices to support MPC growth and differentiation. Porcine-derived skeletal muscle and liver and kidney extracellular matrix (ECM) were generated by decellularization methods consisting of distilled water, 0.2 mg/mL DNase, or 5% fetal bovine serum. Acellular matrices were further homogenized, dissolved, and combined with a hyaluronic acid-based hydrogel decorated with heparin (ECM-HA-HP). The cell proliferation and myogenic differentiation capacity of human MPCs were assessed when grown on gel alone, ECM, or each ECM-HA-HP substrate. Human MPC proliferation was significantly enhanced when cultured on the ECM-HA-HP substrates compared to the other substrates tested, with the greatest proliferation on the muscle ECM-HA-HP (mECM-HA-HP) substrate. The number of differentiated myotubes was significantly increased on the mECM-HA-HP substrate compared to the other gel-ECM substrates, as well as the numbers of MPCs expressing specific myogenic cell markers (i.e., myosin, desmin, myoD, and myf5). In conclusion, skeletal mECM-HA-HP as a culture substrate provided an optimal culture microenvironment potentially due to its similarity to the in vivo environment. These data suggest a potential use of skeletal muscle-derived ECM gel for the expansion and differentiation of human MPCs for cell-based therapy for skeletal muscle

Reduced extracellular space in the brain of tenascin-R- and HNK-1-sulphotransferase deficient mice

Czech Academy of Sciences Publication Activity Database

Syková, Eva; Voříšek, Ivan; Mazel, Tomáš; Antonova, Tatiana; Schachner, M.

2005-01-01

Roč. 22, č. 8 (2005), s. 1873-1880 ISSN 0953-816X R&D Projects: GA ČR(CZ) GA309/04/0753; GA MŠk(CZ) 1M0538 Institutional research plan: CEZ:AV0Z50390512 Keywords : diffusion * extracellular matrix Subject RIV: FH - Neurology Impact factor: 3.949, year: 2005

Novel serological neo-epitope markers of extracellular matrix proteins for the detection of portal hypertension

DEFF Research Database (Denmark)

Leeming, Diana Julie; Karsdal, M A; Byrjalsen, I

2013-01-01

The hepatic venous pressure gradient (HVPG) is an invasive, but important diagnostic and prognostic marker in cirrhosis with portal hypertension (PHT). During cirrhosis, remodelling of fibrotic tissue by matrix metalloproteinases (MMPs) is a permanent process generating small fragments of degrade...... extracellular matrix (ECM) proteins known as neoepitopes, which are then released into the circulation....

Recombinant Gelatin Microspheres : Novel Formulations for Tissue Repair?

NARCIS (Netherlands)

Tuin, Annemarie; Kluijtmans, Sebastiaan G.; Bouwstra, Jan B.; Harmsen, Martin C.; Van Luyn, Marja J. A.

Microspheres (MSs) can function as multifunctional scaffolds in different approaches of tissue repair (TR), as a filler, a slow-release depot for growth factors, or a delivery vehicle for cells. Natural cell adhesion-supporting extracellular matrix components like gelatin are good materials for

Tissue Factor-Expressing Tumor-Derived Extracellular Vesicles Activate Quiescent Endothelial Cells via Protease-Activated Receptor-1

Directory of Open Access Journals (Sweden)

Sara P. Y. Che

2017-11-01

Full Text Available Tissue factor (TF-expressing tumor-derived extracellular vesicles (EVs can promote metastasis and pre-metastatic niche formation, but the mechanisms by which this occurs remain largely unknown. We hypothesized that generation of activated factor X (FXa by TF expressed on tumor-derived EV could activate protease-activated receptors (PARs on non-activated endothelial cells to induce a pro-adhesive and pro-inflammatory phenotype. We obtained EV from TF-expressing breast (MDA-MB-231 and pancreatic (BxPC3 and Capan-1 tumor cell lines. We measured expression of E-selectin and secretion of interleukin-8 (IL-8 in human umbilical vein endothelial cells after exposure to EV and various immunologic and chemical inhibitors of TF, FXa, PAR-1, and PAR-2. After 6 h of exposure to tumor-derived EV (pretreated with factor VIIa and FX in vitro, endothelial cells upregulated E-selectin expression and secreted IL-8. These changes were decreased with an anti-TF antibody, FXa inhibitors (FPRCK and EGRCK, and PAR-1 antagonist (E5555, demonstrating that FXa generated by TF-expressing tumor-derived EV was signaling through endothelial PAR-1. Due to weak constitutive PAR-2 expression, these endothelial responses were not induced by a PAR-2 agonist peptide (SLIGKV and were not inhibited by a PAR-2 antagonist (FSLLRY after exposure to tumor-derived EV. In conclusion, we found that TF-expressing cancer-derived EVs activate quiescent endothelial cells, upregulating E-selectin and inducing IL-8 secretion through generation of FXa and cleavage of PAR-1. Conversion of resting endothelial cells to an activated phenotype by TF-expressing cancer-derived EV could promote cancer metastases.

Large area, label-free imaging of extracellular matrix using telecentricity

Science.gov (United States)

Visbal Onufrak, Michelle A.; Konger, Raymond L.; Kim, Young L.

2017-02-01

Subtle alterations in stromal tissue structures and organizations within the extracellular matrix (ECM) have been observed in several types of tissue abnormalities, including early skin cancer and wounds. Current microscopic imaging methods often lack the ability to accurately determine the extent of malignancy over a large area, due to their limited field of view. In this research we focus on the development of simple mesoscopic (i.e. between microscopic and macroscopic) biomedical imaging device for non-invasive assessment of ECM alterations over a large, heterogeneous area. In our technology development, a telecentric lens, commonly used in machine vision systems but rarely used in biomedical imaging, serves as a key platform to visualize alterations in tissue microenvironments in a label-free manner over a clinically relevant area. In general, telecentric imaging represents a simple, alternative method for reducing unwanted scattering or diffuse light caused by the highly anisotropic scattering properties of biological tissue. In particular, under telecentric imaging the light intensity backscattered from biological tissue is mainly sensitive to the scattering anisotropy factor, possibly associated with the ECM. We demonstrate the inherent advantages of combining telecentric lens systems with hyperspectral imaging for providing optical information of tissue scattering in biological tissue of murine models, as well as light absorption of hemoglobin in blood vessel tissue phantoms. Thus, we envision that telecentric imaging could potentially serve for simple site-specific, tissue-based assessment of stromal alterations over a clinically relevant field of view in a label-free manner, for studying diseases associated with disruption of homeostasis in ECM.

3D Bioprinting Technologies for Hard Tissue and Organ Engineering

OpenAIRE

Wang, Xiaohong; Ao, Qiang; Tian, Xiaohong; Fan, Jun; Wei, Yujun; Hou, Weijian; Tong, Hao; Bai, Shuling

2016-01-01

Hard tissues and organs, including the bones, teeth and cartilage, are the most extensively exploited and rapidly developed areas in regenerative medicine field. One prominent character of hard tissues and organs is that their extracellular matrices mineralize to withstand weight and pressure. Over the last two decades, a wide variety of 3D printing technologies have been adapted to hard tissue and organ engineering. These 3D printing technologies have been defined as 3D bioprinting. Especial...

Activation of retinal glial (Müller cells by extracellular ATP induces pronounced increases in extracellular H+ flux.

Directory of Open Access Journals (Sweden)

Boriana K Tchernookova

Full Text Available Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Müller (glial cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPγs at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Müller cells; both the calcium rise and the extracellular H+ fluxes were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin and when the PLC-IP3 signaling pathway was disrupted with 2-APB and U73122. The anion transport inhibitor DIDS also markedly reduced the ATP-induced increase in H+ flux while SITS had no effect. ATP-induced H+ fluxes were also observed from Müller cells isolated from human, rat, monkey, skate and lamprey retinae, suggesting a highly evolutionarily conserved mechanism of potential general importance. Extracellular ATP also induced significant increases in extracellular H+ flux at the level of both the outer and inner plexiform layers in retinal slices of tiger salamander which was significantly reduced by suramin and PPADS. We suggest that the novel H+ flux mediated by ATP-activation of Müller cells and of other glia as well may be a key mechanism modulating neuronal signaling in the vertebrate retina and throughout the brain.

Circulating levels of matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with incisional hernia

DEFF Research Database (Denmark)

Henriksen, Nadia A; Sørensen, Lars T; Jorgensen, Lars N

2013-01-01

Incisional hernia formation is a common complication to laparotomy and possibly associated with alterations in connective tissue metabolism. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are closely involved in the metabolism of the extracellular matrix. Our...

Guiding tissue regeneration with ultrasound in vitro and in vivo

Science.gov (United States)

Dalecki, Diane; Comeau, Eric S.; Raeman, Carol H.; Child, Sally Z.; Hobbs, Laura; Hocking, Denise C.

2015-05-01

Developing new technologies that enable the repair or replacement of injured or diseased tissues is a major focus of regenerative medicine. This paper will discuss three ultrasound technologies under development in our laboratories to guide tissue regeneration both in vitro and in vivo. A critical obstacle in tissue engineering is the need for rapid and effective tissue vascularization strategies. To address this challenge, we are developing acoustic patterning techniques for microvascular tissue engineering. Acoustic radiation forces associated with ultrasound standing wave fields provide a rapid, non-invasive approach to spatially pattern cells in three dimensions without affecting cell viability. Acoustic patterning of endothelial cells leads to the rapid formation of microvascular networks throughout the volumes of three-dimensional hydrogels, and the morphology of the resultant microvessel networks can be controlled by design of the ultrasound field. A second technology under development uses ultrasound to noninvasively control the microstructure of collagen fibers within engineered tissues. The microstructure of extracellular matrix proteins provides signals that direct cell functions critical to tissue regeneration. Thus, controlling collagen microfiber structure with ultrasound provides a noninvasive approach to regulate the mechanical properties of biomaterials and control cellular responses. The third technology employs therapeutic ultrasound to enhance the healing of chronic wounds. Recent studies demonstrate increased granulation tissue thickness and collagen deposition in murine dermal wounds exposed to pulsed ultrasound. In summary, ultrasound technologies offer noninvasive approaches to control cell behaviors and extracellular matrix organization and thus hold great promise to advance tissue regeneration in vitro and in vivo.

Bicarbonate sensing in mouse cortical astrocytes during extracellular acid/base disturbances

Science.gov (United States)

Naoshin, Zinnia; Defren, Sabrina; Schmaelzle, Jana; Weber, Tobias; Schneider, Hans‐Peter

2017-01-01

Key points The present study suggests that the electrogenic sodium–bicarbonate cotransporter, NBCe1, supported by carbonic anhydrase II, CAII, provides an efficient mechanism of bicarbonate sensing in cortical astrocytes. This mechanism is proposed to play a major role in setting the pHi responses to extracellular acid/base challenges in astrocytes.A decrease in extracellular [HCO3 −] during isocapnic acidosis and isohydric hypocapnia, or an increase in intracellular [HCO3 −] during hypercapnic acidosis, was effectively sensed by NBCe1, which carried bicarbonate out of the cells under these conditions, and caused an acidification and sodium fall in WT astrocytes, but not in NBCe1‐knockout astrocytes.Isocapnic acidosis, hypercapnic acidosis and isohydric hypocapnia evoked inward currents in NBCe1‐ and CAII‐expressing Xenopus laevis oocytes, but not in native oocytes, suggesting that NBCe1 operates in the outwardly directed mode under these conditions consistent with our findings in astrocytes.We propose that bicarbonate sensing of astrocytes may have functional significance during extracellular acid/base disturbances in the brain, as it not only alters intracellular pH/[HCO3 −]‐dependent functions of astrocytes, but also modulates the extracellular pH/[HCO3 −] in brain tissue. Abstract Extracellular acid/base status of the mammalian brain undergoes dynamic changes during many physiological and pathological events. Although intracellular pH (pHi) of astrocytes responds to extracellular acid/base changes, the mechanisms mediating these changes have remained unresolved. We have previously shown that the electrogenic sodium–bicarbonate cotransporter, NBCe1, is a high‐affinity bicarbonate carrier in cortical astrocytes. In the present study, we investigated whether NBCe1 plays a role in bicarbonate sensing in astrocytes, and in determining the pHi responses to extracellular acid/base challenges. We measured changes in intracellular H+ and Na+ in

Analyzing the effects of mechanical and osmotic loading on glycosaminoglycan synthesis rate in cartilaginous tissues.

Science.gov (United States)

Gao, Xin; Zhu, Qiaoqiao; Gu, Weiyong

2015-02-26

The glycosaminoglycan (GAG) plays an important role in cartilaginous tissues to support and transmit mechanical loads. Many extracellular biophysical stimuli could affect GAG synthesis by cells. It has been hypothesized that the change of cell volume is a primary mechanism for cells to perceive the stimuli. Experimental studies have shown that the maximum synthesis rate of GAG is achieved at an optimal cell volume, larger or smaller than this level the GAG synthesis rate decreases. Based on the hypothesis and experimental findings in the literature, we proposed a mathematical model to quantitatively describe the cell volume dependent GAG synthesis rate in the cartilaginous tissues. Using this model, we investigated the effects of osmotic loading and mechanical loading on GAG synthesis rate. It is found our proposed mathematical model is able to well describe the change of GAG synthesis rate in isolated cells or in cartilage with variations of the osmotic loading or mechanical loading. This model is important for evaluating the GAG synthesis activity within cartilaginous tissues as well as understanding the role of mechanical loading in tissue growth or degeneration. It is also important for designing a bioreactor system with proper extracellular environment or mechanical loading for growing tissue at the maximum synthesis rate of the extracellular matrix. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modeling microelectrode biosensors: free-flow calibration can substantially underestimate tissue concentrations.

Science.gov (United States)

Newton, Adam J H; Wall, Mark J; Richardson, Magnus J E

2017-03-01

Microelectrode amperometric biosensors are widely used to measure concentrations of analytes in solution and tissue including acetylcholine, adenosine, glucose, and glutamate. A great deal of experimental and modeling effort has been directed at quantifying the response of the biosensors themselves; however, the influence that the macroscopic tissue environment has on biosensor response has not been subjected to the same level of scrutiny. Here we identify an important issue in the way microelectrode biosensors are calibrated that is likely to have led to underestimations of analyte tissue concentrations. Concentration in tissue is typically determined by comparing the biosensor signal to that measured in free-flow calibration conditions. In a free-flow environment the concentration of the analyte at the outer surface of the biosensor can be considered constant. However, in tissue the analyte reaches the biosensor surface by diffusion through the extracellular space. Because the enzymes in the biosensor break down the analyte, a density gradient is set up resulting in a significantly lower concentration of analyte near the biosensor surface. This effect is compounded by the diminished volume fraction (porosity) and reduction in the diffusion coefficient due to obstructions (tortuosity) in tissue. We demonstrate this effect through modeling and experimentally verify our predictions in diffusive environments. NEW & NOTEWORTHY Microelectrode biosensors are typically calibrated in a free-flow environment where the concentrations at the biosensor surface are constant. However, when in tissue, the analyte reaches the biosensor via diffusion and so analyte breakdown by the biosensor results in a concentration gradient and consequently a lower concentration around the biosensor. This effect means that naive free-flow calibration will underestimate tissue concentration. We develop mathematical models to better quantify the discrepancy between the calibration and tissue

Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis

Science.gov (United States)

McKleroy, William; Lee, Ting-Hein

2013-01-01

Pulmonary fibrosis is a vexing clinical problem with no proven therapeutic options. In the normal lung there is continuous collagen synthesis and collagen degradation, and these two processes are precisely balanced to maintain normal tissue architecture. With lung injury there is an increase in the rate of both collagen production and collagen degradation. The increase in collagen degradation is critical in preventing the formation of permanent scar tissue each time the lung is exposed to injury. In pulmonary fibrosis, collagen degradation does not keep pace with collagen production, resulting in extracellular accumulation of fibrillar collagen. Collagen degradation occurs through both extracellular and intracellular pathways. The extracellular pathway involves cleavage of collagen fibrils by proteolytic enzyme including the metalloproteinases. The less-well-described intracellular pathway involves binding and uptake of collagen fragments by fibroblasts and macrophages for lysosomal degradation. The relationship between these two pathways and their relevance to the development of fibrosis is complex. Fibrosis in the lung, liver, and skin has been associated with an impaired degradative environment. Much of the current scientific effort in fibrosis is focused on understanding the pathways that regulate increased collagen production. However, recent reports suggest an important role for collagen turnover and degradation in regulating the severity of tissue fibrosis. The objective of this review is to evaluate the roles of the extracellular and intracellular collagen degradation pathways in the development of fibrosis and to examine whether pulmonary fibrosis can be viewed as a disease of impaired matrix degradation rather than a disease of increased matrix production. PMID:23564511

Secretory proteins of the pulmonary extracellular lining

International Nuclear Information System (INIS)

Gupta, R.P.; Patton, S.E.; Eddy, M.; Smits, H.L.; Jetten, A.M.; Nettesheim, P.; Hook, G.E.R.

1986-01-01

The objective of this investigation was to identify proteins in the pulmonary extracellular lining (EL) that are secreted by cells of the pulmonary epithelium. Pulmonary lavage effluents from the lungs of rabbits were centrifuged to remove all cells and particulate materials. Serum proteins were removed by repeatedly passing concentrated lavage effluent fluid through an affinity column containing IgG fraction of goat anti-rabbit (whole serum) antiserum bound to Sepharose-4B. Nonserum proteins accounted for 21.3 +/- 10.3% of the total soluble proteins in pulmonary lavage effluents. Serum free lavage effluents (SFL) contained 25 identifiable proteins as determined by using SDS-PAGE under reducing conditions. Of these proteins approximately 73% was accounted for by a single protein with MW of 66 kd. The secretory nature of the proteins present in SFL was investigated by studying the incorporation of 35 S-methionine into proteins released by lung slices and trachea followed by SDS-PAGE and autoradiography. Many, but not all proteins present in SFL were identified as proteins secreted by pulmonary tissues. The major secretory proteins appeared to have MWs of 59, 53, 48, 43, 24, 14, and 6 kd under reducing conditions. These data demonstrate the presence of several proteins in the pulmonary extracellular lining that appear to be secreted by the pulmonary epithelium

Bayesian state space models for dynamic genetic network construction across multiple tissues.

Science.gov (United States)

Liang, Yulan; Kelemen, Arpad

2016-08-01

Construction of gene-gene interaction networks and potential pathways is a challenging and important problem in genomic research for complex diseases while estimating the dynamic changes of the temporal correlations and non-stationarity are the keys in this process. In this paper, we develop dynamic state space models with hierarchical Bayesian settings to tackle this challenge for inferring the dynamic profiles and genetic networks associated with disease treatments. We treat both the stochastic transition matrix and the observation matrix time-variant and include temporal correlation structures in the covariance matrix estimations in the multivariate Bayesian state space models. The unevenly spaced short time courses with unseen time points are treated as hidden state variables. Hierarchical Bayesian approaches with various prior and hyper-prior models with Monte Carlo Markov Chain and Gibbs sampling algorithms are used to estimate the model parameters and the hidden state variables. We apply the proposed Hierarchical Bayesian state space models to multiple tissues (liver, skeletal muscle, and kidney) Affymetrix time course data sets following corticosteroid (CS) drug administration. Both simulation and real data analysis results show that the genomic changes over time and gene-gene interaction in response to CS treatment can be well captured by the proposed models. The proposed dynamic Hierarchical Bayesian state space modeling approaches could be expanded and applied to other large scale genomic data, such as next generation sequence (NGS) combined with real time and time varying electronic health record (EHR) for more comprehensive and robust systematic and network based analysis in order to transform big biomedical data into predictions and diagnostics for precision medicine and personalized healthcare with better decision making and patient outcomes.

Growing tissues in real and simulated microgravity: new methods for tissue engineering.

Science.gov (United States)

Grimm, Daniela; Wehland, Markus; Pietsch, Jessica; Aleshcheva, Ganna; Wise, Petra; van Loon, Jack; Ulbrich, Claudia; Magnusson, Nils E; Infanger, Manfred; Bauer, Johann

2014-12-01

Tissue engineering in simulated (s-) and real microgravity (r-μg) is currently a topic in Space medicine contributing to biomedical sciences and their applications on Earth. The principal aim of this review is to highlight the advances and accomplishments in the field of tissue engineering that could be achieved by culturing cells in Space or by devices created to simulate microgravity on Earth. Understanding the biology of three-dimensional (3D) multicellular structures is very important for a more complete appreciation of in vivo tissue function and advancing in vitro tissue engineering efforts. Various cells exposed to r-μg in Space or to s-μg created by a random positioning machine, a 2D-clinostat, or a rotating wall vessel bioreactor grew in the form of 3D tissues. Hence, these methods represent a new strategy for tissue engineering of a variety of tissues, such as regenerated cartilage, artificial vessel constructs, and other organ tissues as well as multicellular cancer spheroids. These aggregates are used to study molecular mechanisms involved in angiogenesis, cancer development, and biology and for pharmacological testing of, for example, chemotherapeutic drugs or inhibitors of neoangiogenesis. Moreover, they are useful for studying multicellular responses in toxicology and radiation biology, or for performing coculture experiments. The future will show whether these tissue-engineered constructs can be used for medical transplantations. Unveiling the mechanisms of microgravity-dependent molecular and cellular changes is an up-to-date requirement for improving Space medicine and developing new treatment strategies that can be translated to in vivo models while reducing the use of laboratory animals.

Temporal Profiles Dissociate Regional Extracellular Ethanol versus Dopamine Concentrations

Science.gov (United States)

2015-01-01

In vivo monitoring of dopamine via microdialysis has demonstrated that acute, systemic ethanol increases extracellular dopamine in regions innervated by dopaminergic neurons originating in the ventral tegmental area and substantia nigra. Simultaneous measurement of dialysate dopamine and ethanol allows comparison of the time courses of their extracellular concentrations. Early studies demonstrated dissociations between the time courses of brain ethanol concentrations and dopaminergic responses in the nucleus accumbens (NAc) elicited by acute ethanol administration. Both brain ethanol and extracellular dopamine levels peak during the first 5 min following systemic ethanol administration, but the dopamine response returns to baseline while brain ethanol concentrations remain elevated. Post hoc analyses examined ratios of the dopamine response (represented as a percent above baseline) to tissue concentrations of ethanol at different time points within the first 25–30 min in the prefrontal cortex, NAc core and shell, and dorsomedial striatum following a single intravenous infusion of ethanol (1 g/kg). The temporal patterns of these “response ratios” differed across brain regions, possibly due to regional differences in the mechanisms underlying the decline of the dopamine signal associated with acute intravenous ethanol administration and/or to the differential effects of acute ethanol on the properties of subpopulations of midbrain dopamine neurons. This Review draws on neurochemical, physiological, and molecular studies to summarize the effects of acute ethanol administration on dopamine activity in the prefrontal cortex and striatal regions, to explore the potential reasons for the regional differences observed in the decline of ethanol-induced dopamine signals, and to suggest directions for future research. PMID:25537116

Extracellular collagenases and the endocytic receptor, urokinase plasminogen activator receptor-associated protein/Endo180, cooperate in fibroblast-mediated collagen degradation

DEFF Research Database (Denmark)

Madsen, Daniel H; Engelholm, Lars H; Ingvarsen, Signe

2007-01-01

in these events. A recently discovered turnover route with importance for tumor growth involves intracellular collagen degradation and is governed by the collagen receptor, urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180). The interplay between this mechanism and extracellular...... collagenolysis is not known. In this report, we demonstrate the existence of a new, composite collagen breakdown pathway. Thus, fibroblast-mediated collagen degradation proceeds preferentially as a sequential mechanism in which extracellular collagenolysis is followed by uPARAP/Endo180-mediated endocytosis......The collagens of the extracellular matrix are the most abundant structural proteins in the mammalian body. In tissue remodeling and in the invasive growth of malignant tumors, collagens constitute an important barrier, and consequently, the turnover of collagen is a rate-limiting process...

No evidence for role of extracellular choline-acetyltransferase in generation of gamma oscillations in rat hippocampal slices in vitro.

Science.gov (United States)

Hollnagel, J O; ul Haq, R; Behrens, C J; Maslarova, A; Mody, I; Heinemann, U

2015-01-22

Acetylcholine (ACh) is well known to induce persistent γ-oscillations in the hippocampus when applied together with physostigmine, an inhibitor of the ACh degrading enzyme acetylcholinesterase (AChE). Here we report that physostigmine alone can also dose-dependently induce γ-oscillations in rat hippocampal slices. We hypothesized that this effect was due to the presence of choline in the extracellular space and that this choline is taken up into cholinergic fibers where it is converted to ACh by the enzyme choline-acetyltransferase (ChAT). Release of ACh from cholinergic fibers in turn may then induce γ-oscillations. We therefore tested the effects of the choline uptake inhibitor hemicholinium-3 (HC-3) on persistent γ-oscillations either induced by physostigmine alone or by co-application of ACh and physostigmine. We found that HC-3 itself did not induce γ-oscillations and also did not prevent physostigmine-induced γ-oscillation while washout of physostigmine and ACh-induced γ-oscillations was accelerated. It was recently reported that ChAT might also be present in the extracellular space (Vijayaraghavan et al., 2013). Here we show that the effect of physostigmine was prevented by the ChAT inhibitor (2-benzoylethyl)-trimethylammonium iodide (BETA) which could indicate extracellular synthesis of ACh. However, when we tested for effects of extracellularly applied acetyl-CoA, a substrate of ChAT for synthesis of ACh, physostigmine-induced γ-oscillations were attenuated. Together, these findings do not support the idea that ACh can be synthesized by an extracellularly located ChAT. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Wetting and dewetting of extracellular matrix and glycocalix models

International Nuclear Information System (INIS)

Tanaka, Motomu; Rehfeldt, Florian; Schneider, Matthias F; Mathe, Gerald; Albersdoerfer, Antero; Neumaier, Klaus R; Purrucker, Oliver; Sackmann, Erich

2005-01-01

In this paper, we study wetting and dewetting of hydrated biopolymer layers mediating cell-cell and cell-tissue contacts, called the extracellular matrix and cell surface glycocalix, by the combination of various physical techniques. Here, the sum of the net effects of the various interfacial forces, which is referred to as the disjoining pressure, is used as a semi-quantitative measure to describe the thermodynamics of hydrated interlayers. The disjoining pressure can be measured by applying external forces to maintain the equilibrium distance between two parallel surfaces (in biology, two neighbouring plasma membranes). Using artificial models of the extracellular matrix and glycocalix, we describe stable cell-cell contacts in terms of the wetting (or spreading) of complex fluids on polymer surfaces. In fact, the adjustment of the wetting interaction via thin hydrating layers enables us to transform three-dimensional cell membranes into quasi-two-dimensional films on macroscopically large surfaces. Fine-tuning of local wetting conditions at the interface further allows for the selective wetting of native cell membranes on microstructured polysaccharide films, which has a large potential for individual detection of biological functions in confined geometries

Involvement of extracellular matrix constituents in breast cancer

Energy Technology Data Exchange (ETDEWEB)

Lochter, Andre; Bissell, Mina J

1995-06-01

It has recently been established that the extracellular matrix is required for normal functional differentiation of mammary epithelia not only in culture, but also in vivo. The mechanisms by which extracellular matrix affects differentiation, as well as the nature of extracellular matrix constituents which have major impacts on mammary gland function, have only now begun to be dissected. The intricate variety of extracellular matrix-mediated events and the remarkable degree of plasticity of extracellular matrix structure and composition at virtually all times during ontogeny, make such studies difficult. Similarly, during carcinogenesis, the extracellular matrix undergoes gross alterations, the consequences of which are not yet precisely understood. Nevertheless, an increasing amount of data suggests that the extracellular matrix and extracellular matrix-receptors might participate in the control of most, if not all, of the successive stages of breast tumors, from appearance to progression and metastasis.

Eosinophilic Otitis Media: the Aftermath of Eosinophil Extracellular Trap Cell Death.

Science.gov (United States)

Ueki, Shigeharu; Ohta, Nobuo; Takeda, Masahide; Konno, Yasunori; Hirokawa, Makoto

2017-05-01

Eosinophilic otitis media (EOM) is a refractory disease characterized by the accumulation of eosinophils in middle ear effusion and mucosa. We summarize current knowledge regarding the clinical characteristics and management of EOM. Although eosinophil activation in inflamed foci is involved in the pathogenesis of EOM, little is known about the fate of the eosinophils and aftermath of their cell death. We discuss the possibility that eosinophils undergo non-apoptotic cell death that worsens tissue damage and increases effusion viscosity. Unlike chronic otitis media, EOM is strongly associated with an allergic background. Corticosteroids are currently the only effective pharmacological treatment, and surgical intervention is often required. Mucosal eosinophils infiltrate extensively into the middle ear cavity where they are stimulated by locally produced activators including interleukin-5 and eotaxin. The eosinophils undergo cytolysis in the effusion, which represents a major fate of activated eosinophils in vivo. Recent data revealed cytolysis could be renamed as extracellular trap cell death (ETosis). ETosis represents suicidal cell death involving total cell degranulation and development of sticky chromatin structures (extracellular traps (ETs)). The characteristics of eosinophil- and neutrophil-derived ET polymers might contribute to the difference in viscosity of secretions between EOM and common chronic otitis media. The extracellular products remaining after eosinophil ETosis are an important aspect of EOM pathology. The concept of ETosis also has novel implications for potential therapeutic modalities in various eosinophilic disorders.

Development of biomaterial scaffold for nerve tissue engineering: Biomaterial mediated neural regeneration

Science.gov (United States)

2009-01-01

Neural tissue repair and regeneration strategies have received a great deal of attention because it directly affects the quality of the patient's life. There are many scientific challenges to regenerate nerve while using conventional autologous nerve grafts and from the newly developed therapeutic strategies for the reconstruction of damaged nerves. Recent advancements in nerve regeneration have involved the application of tissue engineering principles and this has evolved a new perspective to neural therapy. The success of neural tissue engineering is mainly based on the regulation of cell behavior and tissue progression through the development of a synthetic scaffold that is analogous to the natural extracellular matrix and can support three-dimensional cell cultures. As the natural extracellular matrix provides an ideal environment for topographical, electrical and chemical cues to the adhesion and proliferation of neural cells, there exists a need to develop a synthetic scaffold that would be biocompatible, immunologically inert, conducting, biodegradable, and infection-resistant biomaterial to support neurite outgrowth. This review outlines the rationale for effective neural tissue engineering through the use of suitable biomaterials and scaffolding techniques for fabrication of a construct that would allow the neurons to adhere, proliferate and eventually form nerves. PMID:19939265

A collagen-based scaffold delivering exogenous microrna-29B to modulate extracellular matrix remodeling.

Science.gov (United States)

Monaghan, Michael; Browne, Shane; Schenke-Layland, Katja; Pandit, Abhay

2014-04-01

Directing appropriate extracellular matrix remodeling is a key aim of regenerative medicine strategies. Thus, antifibrotic interfering RNA (RNAi) therapy with exogenous microRNA (miR)-29B was proposed as a method to modulate extracellular matrix remodeling following cutaneous injury. It was hypothesized that delivery of miR-29B from a collagen scaffold will efficiently modulate the extracellular matrix remodeling response and reduce maladaptive remodeling such as aggressive deposition of collagen type I after injury. The release of RNA from the scaffold was assessed and its ability to silence collagen type I and collagen type III expression was evaluated in vitro. When primary fibroblasts were cultured with scaffolds doped with miR-29B, reduced levels of collagen type I and collagen type III mRNA expression were observed for up to 2 weeks of culture. When the scaffolds were applied to full thickness wounds in vivo, reduced wound contraction, improved collagen type III/I ratios and a significantly higher matrix metalloproteinase (MMP)-8: tissue inhibitor of metalloproteinase (TIMP)-1 ratio were detected when the scaffolds were functionalized with miR-29B. Furthermore, these effects were significantly influenced by the dose of miR-29B in the collagen scaffold (0.5 versus 5 μg). This study shows a potential of combining exogenous miRs with collagen scaffolds to improve extracellular matrix remodeling following injury.

Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope

Science.gov (United States)

Wang, Han-Wei; Le, Thuc T.; Cheng, Ji-Xin

2008-04-01

A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH 2-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH 2 bonds. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases.

Evaluation of silk biomaterials in combination with extracellular matrix coatings for bladder tissue engineering with primary and pluripotent cells.

Science.gov (United States)

Franck, Debra; Gil, Eun Seok; Adam, Rosalyn M; Kaplan, David L; Chung, Yeun Goo; Estrada, Carlos R; Mauney, Joshua R

2013-01-01

Silk-based biomaterials in combination with extracellular matrix (ECM) coatings were assessed as templates for cell-seeded bladder tissue engineering approaches. Two structurally diverse groups of silk scaffolds were produced by a gel spinning process and consisted of either smooth, compact multi-laminates (Group 1) or rough, porous lamellar-like sheets (Group 2). Scaffolds alone or coated with collagen types I or IV or fibronectin were assessed independently for their ability to support attachment, proliferation, and differentiation of primary cell lines including human bladder smooth muscle cells (SMC) and urothelial cells as well as pluripotent cell populations, such as murine embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells. AlamarBlue evaluations revealed that fibronectin-coated Group 2 scaffolds promoted the highest degree of primary SMC and urothelial cell attachment in comparison to uncoated Group 2 controls and all Group 1 scaffold variants. Real time RT-PCR and immunohistochemical (IHC) analyses demonstrated that both fibronectin-coated silk groups were permissive for SMC contractile differentiation as determined by significant upregulation of α-actin and SM22α mRNA and protein expression levels following TGFβ1 stimulation. Prominent expression of epithelial differentiation markers, cytokeratins, was observed in urothelial cells cultured on both control and fibronectin-coated groups following IHC analysis. Evaluation of silk matrices for ESC and iPS cell attachment by alamarBlue showed that fibronectin-coated Group 2 scaffolds promoted the highest levels in comparison to all other scaffold formulations. In addition, real time RT-PCR and IHC analyses showed that fibronectin-coated Group 2 scaffolds facilitated ESC and iPS cell differentiation toward both urothelial and smooth muscle lineages in response to all trans retinoic acid as assessed by induction of uroplakin and contractile gene and protein expression. These results

Extracellular DNA metabolism in Haloferax volcanii

Directory of Open Access Journals (Sweden)

Scott eChimileski

2014-02-01

Full Text Available Extracellular DNA is found in all environments and is a dynamic component of the micro-bial ecosystem. Microbial cells produce and interact with extracellular DNA through many endogenous mechanisms. Extracellular DNA is processed and internalized for use as genetic information and as a major source of macronutrients, and plays several key roles within prokaryotic biofilms. Hypersaline sites contain some of the highest extracellular DNA con-centrations measured in nature–a potential rich source of carbon, nitrogen and phosphorus for halophilic microorganisms. We conducted DNA growth studies for the halophilic archaeon Haloferax volcanii DS2 and show that this model Halobacteriales strain is capable of using exogenous double-stranded DNA as a nutrient. Further experiments with varying medium composition, DNA concentration and DNA types revealed that DNA is utilized primarily as a phosphorus source, that growth on DNA is concentration-dependent and that DNA isolated from different sources is metabolized selectively, with a bias against highly divergent methylated DNA sources. Additionally, fluorescence microscopy experiments showed that labeled DNA colocalized with Haloferax volcanii cells. The gene Hvo_1477 was also identified using a comparative genomic approach as a factor likely to be involved in extracellular DNA processing at the cell surface, and deletion of Hvo_1477 created an H. volcanii strain deficient in its ability to grow on extracellular DNA. Widespread distribution of Hvo_1477 homologs in archaea suggests metabolism of extracellular DNA may be of broad ecological and physiological relevance in this domain of life.

Dermal extracellular lipid in birds.

Science.gov (United States)

Stromberg, M W; Hinsman, E J; Hullinger, R L

1990-01-01

A light and electron microscopic study of the skin of domestic chickens, seagulls, and antarctic penguins revealed abundant extracellular dermal lipid and intracellular epidermal lipid. Dermal lipid appeared ultrastructurally as extracellular droplets varying from less than 1 micron to more than 25 microns in diameter. The droplets were often irregularly contoured, sometimes round, and of relatively low electron density. Processes of fibrocytes were often seen in contact with extracellular lipid droplets. Sometimes a portion of such a droplet was missing, and this missing part appeared to have been "digested away" by the cell process. In places where cells or cell processes are in contact with fact droplets, there are sometimes extracellular membranous whorls or fragments which have been associated with the presence of fatty acids. Occasionally (in the comb) free fat particles were seen in intimate contact with extravasated erythrocytes. Fat droplets were seen in the lumen of small dermal blood and lymph vessels. We suggest that the dermal extracellular lipid originates in the adipocyte layer and following hydrolysis the free fatty acids diffuse into the epidermis. Here they become the raw material for forming the abundant neutral lipid contained in many of the epidermal cells of both birds and dolphins. The heretofore unreported presence and apparently normal utilization of abundant extracellular lipid in birds, as well as the presence of relatively large droplets of neutral lipid in dermal vessels, pose questions which require a thorough reappraisal of present concepts of the ways in which fat is distributed and utilized in the body.

The study on mutations of the gene of extracellular domain of human thyrotropin receptor in the patients with thyroid diseases

International Nuclear Information System (INIS)

Zhang Zuncheng; Fang Peihua; Tan Jian; Lu Mei

2002-01-01

Objective: To define the sequence of the gene of extracellular domain of normal human thyrotropin receptor (hTSHR) and to investigate the mutations of the gene in the patients with thyroid diseases. Methods: Total RNAs were extracted from the thyroid tissue of four normal controls, twelve Graves' disease, four Hashimoto's thyroiditis and eleven nodular goiter patients. The extracellular domain of hTSHR genes were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced with CEQ 2000 Genetic Analyzer. Results: The normal controls and the patients with thyroid disease had the same gene sequences of the extracellular domain of hTSHR. No mutation was found, except a silent base exchange in exon 7 (Asn187) at 661 base, in which 20 samples were 'T', 11 samples were 'C', without changes of amino acid of the TSHR. Conclusions: This study has not revealed mutations in the gene of extracellular domain of hTSHR. Other molecular pathogenetic mechanisms may be involved and more research is demanded

Extracellular histones reduce survival and angiogenic responses of late outgrowth progenitor and mature endothelial cells.

Science.gov (United States)

Mena, H A; Carestia, A; Scotti, L; Parborell, F; Schattner, M; Negrotto, S

2016-02-01

ESSENTIALS: Extracellular histones are highly augmented in sites of neovessel formation, such as regeneration tissues. We studied histone effect on survival and angiogenic activity of mature and progenitor endothelial cells. Extracellular histones trigger apoptosis and pyroptosis and reduce angiogenesis in vivo and in vitro. Histone blockade can be useful as a therapeutic strategy to improve angiogenesis and tissue regeneration. Extracellular histones are highly augmented in sites of neovessel formation, like regeneration tissues. Their cytotoxic effect has been studied in endothelial cells, although the mechanism involved and their action on endothelial colony-forming cells (ECFCs) remain unknown. To study the effect of histones on ECFC survival and angiogenic functions and compare it with mature endothelial cells. Nuclear morphology analysis showed that each human recombinant histone triggered both apoptotic-like and necrotic-like cell deaths in both mature and progenitor endothelial cells. While H1 and H2A exerted a weak toxicity, H2B, H3 and H4 were the most powerful. The percentage of apoptosis correlated with the percentage of ECFCs exhibiting caspase-3 activation and was zeroed by the pan-caspase inhibitor Z-VAD-FMK. Necrotic-like cell death was also suppressed by this compound and the caspase-1 inhibitor Ac-YVAD-CMK, indicating that histones triggered ECFC pyroptosis. All histones, at non-cytotoxic concentrations, reduced migration and H2B, H3 and H4 induced cell cycle arrest and impaired tubulogenesis via p38 activation. Neutrophil-derived histones exerted similar effects. In vivo blood vessel formation in the quail chorioallantoic membrane was also reduced by H2B, H3 and H4. Their cytotoxic and antiangiogenic effects were suppressed by unfractioned and low-molecular-weight heparins and the combination of TLR2 and TLR4 blocking antibodies. Histones trigger both apoptosis and pyroptosis of ECFCs and inhibit their angiogenic functions. Their cytotoxic and

Pulmonary endothelial activation caused by extracellular histones contributes to neutrophil activation in acute respiratory distress syndrome.

Science.gov (United States)

Zhang, Yanlin; Guan, Li; Yu, Jie; Zhao, Zanmei; Mao, Lijun; Li, Shuqiang; Zhao, Jinyuan

2016-11-21

During the acute respiratory distress syndrome (ARDS), neutrophils play a central role in the pathogenesis, and their activation requires interaction with the endothelium. Extracellular histones have been recognized as pivotal inflammatory mediators. This study was to investigate the role of pulmonary endothelial activation during the extracellular histone-induced inflammatory response in ARDS. ARDS was induced in male C57BL/6 mice by intravenous injection with lipopolysaccharide (LPS) or exogenous histones. Concurrent with LPS administration, anti-histone H4 antibody (anti-H4) or non-specific IgG was administered to study the role of extracellular histones. The circulating von Willebrand factor (vWF) and soluble thrombomodulin (sTM) were measured with ELISA kits at the preset time points. Myeloperoxidase (MPO) activity in lung tissue was measured with a MPO detection kit. The translocation of P-selectin and neutrophil infiltration were measured by immunohistochemical detection. For in vitro studies, histone H4 in the supernatant of mouse lung vascular endothelial cells (MLVECs) was measured by Western blot. The binding of extracellular histones with endothelial membrane was examined by confocal laser microscopy. Endothelial P-selectin translocation was measured by cell surface ELISA. Adhesion of neutrophils to MLVECs was assessed with a color video digital camera. The results showed that during LPS-induced ARDS extracellular histones caused endothelial and neutrophil activation, as seen by P-selectin translocation, release of vWF, an increase of circulating sTM, lung neutrophil infiltration and increased MPO activity. Extracellular histones directly bound and activated MLVECs in a dose-dependent manner. On the contrary, the direct stimulatory effect of exogenous histones on neutrophils was very limited, as measured by neutrophil adhesion and MPO activity. With the contribution of activated endothelium, extracellular histones could effectively activating

Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

Science.gov (United States)

Eiraku, Mototsugu; Sasai, Yoshiki

2011-12-15

Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

Laser tissue welding in ophthalmic surgery.

Science.gov (United States)

Rossi, Francesca; Matteini, Paolo; Ratto, Fulvio; Menabuoni, Luca; Lenzetti, Ivo; Pini, Roberto

2008-09-01

Laser welding of ocular tissues is an alternative technique or adjunct to conventional suturing in ophthalmic surgery. It is based on the photothermal interaction of laser light with the main components of the extracellular matrix of connective tissues. The advantages of the welding procedure with respect to standard suturing and stapling are reduced operation times, lesser inflammation, faster healing and increased ability to induce tissue regeneration. The procedure we set up is based on the use of an infrared diode laser in association with the topical application of the chromophore Indocyanine Green. Laser light may be delivered either continuously or in pulses, thus identifying two different techniques that have been applied clinically in various types of transplants of the cornea.

In vivo measurement of tumor estradiol and Vascular Endothelial Growth Factor in breast cancer patients

International Nuclear Information System (INIS)

Garvin, Stina; Dabrosin, Charlotta

2008-01-01

Angiogenesis, crucial for tumor progression, is a process regulated in the tissue micro-environment. Vascular endothelial growth factor (VEGF) is a potent stimulatory factor of angiogenesis and a negative prognostic indicator of breast cancer. VEGF is biologically active in the extracellular space and hitherto, there has been a lack of techniques enabling sampling of angiogenic molecules such as VEGF in situ. The majority of breast cancers are estrogen-dependent, and estrogen has been shown to regulate VEGF in normal breast tissue and experimental breast cancer. We investigated if microdialysis may be applicable in human breast cancer for sampling of extracellular VEGF in situ and to explore if there is an association with local estradiol and VEGF levels in normal and cancerous breast tissue. Microdialysis was used to sample VEGF and estradiol in tumors and adjacent normal breast tissue in postmenopausal breast cancer patients. VEGF and estradiol were also measured in plasma, and immunohistochemical staining for VEGF was performed on tumor sections. We show that in vivo levels of extracellular VEGF were significantly higher in breast cancer tumors than in normal adjacent breast tissue. There was a significant positive correlation between estradiol and extracellular VEGF in normal breast tissue. However, no correlation was detected between estradiol and VEGF in tumors or between tumor VEGF and plasma VEGF. We conclude that VEGF and estradiol correlates significantly in normal breast tissue. Microdialysis may be used to provide novel insight in breast tumor biology and the regulation of molecules in the extracellular space of human breast tumors in vivo

Development and Characterization of Tissue Equivalent Proportional Counter for Radiation Monitoring in International Space Station

Directory of Open Access Journals (Sweden)

Uk-Won Nam

2013-06-01

Full Text Available Tissue equivalent proportional counter (TEPC can measure the Linear Energy Transfer (LET spectrum and calculate the equivalent dose for the complicated radiation field in space. In this paper, we developed and characterized a TEPC for radiation monitoring in International Space Station (ISS. The prototype TEPC which can simulate a 2 μm of the site diameter for micro-dosimetry has been tested with a standard alpha source (241Am, 5.5 MeV. Also, the calibration of the TEPC was performed by the 252Cf neutron standard source in Korea Research Institute of Standards and Science (KRISS. The determined calibration factor was kf = 3.59×10-7 mSv/R.

An update on the Application of Nanotechnology in Bone Tissue Engineering.

Science.gov (United States)

Griffin, M F; Kalaskar, D M; Seifalian, A; Butler, P E

2016-01-01

Natural bone is a complex and hierarchical structure. Bone possesses an extracellular matrix that has a precise nano-sized environment to encourage osteoblasts to lay down bone by directing them through physical and chemical cues. For bone tissue regeneration, it is crucial for the scaffolds to mimic the native bone structure. Nanomaterials, with features on the nanoscale have shown the ability to provide the appropriate matrix environment to guide cell adhesion, migration and differentiation. This review summarises the new developments in bone tissue engineering using nanobiomaterials. The design and selection of fabrication methods and biomaterial types for bone tissue engineering will be reviewed. The interactions of cells with different nanostructured scaffolds will be discussed including nanocomposites, nanofibres and nanoparticles. Several composite nanomaterials have been able to mimic the architecture of natural bone. Bioceramics biomaterials have shown to be very useful biomaterials for bone tissue engineering as they have osteoconductive and osteoinductive properties. Nanofibrous scaffolds have the ability to provide the appropriate matrix environment as they can mimic the extracellular matrix structure of bone. Nanoparticles have been used to deliver bioactive molecules and label and track stem cells. Future studies to improve the application of nanomaterials for bone tissue engineering are needed.

Safety and Efficacy of Intrapleural Tissue Plasminogen Activator and DNase during Extended Use in Complicated Pleural Space Infections

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Jason R. McClune

2016-01-01

Full Text Available The use of intrapleural therapy with tissue plasminogen activator and DNase improves outcomes in patients with complicated pleural space infections. However, little data exists for the use of combination intrapleural therapy after the initial dosing period of six doses. We sought to describe the safety profile and outcomes of intrapleural therapy beyond this standard dosing. A retrospective review of patients receiving intrapleural therapy with tissue plasminogen activator and DNase was performed at two institutions. We identified 101 patients from January 2013 to August 2015 receiving intrapleural therapy for complicated pleural space infection. The extended use of intrapleural tissue plasminogen activator and DNase therapy beyond six doses was utilized in 20% (20/101 of patients. The mean number of doses in those undergoing extended dosing was 9.8 (range of 7–16. Within the population studied there appears to be no statistically significant increased risk of complications, need for surgical referral, or outcome differences when comparing those receiving standard or extended dosing intrapleural therapy. Future prospective study of intrapleural therapy as an alternative option for patients who fail initial pleural drainage and are unable to tolerate/accept a surgical intervention appears a potential area of study.

An additive manufacturing-based PCL-alginate-chondrocyte bioprinted scaffold for cartilage tissue engineering.

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Kundu, Joydip; Shim, Jin-Hyung; Jang, Jinah; Kim, Sung-Won; Cho, Dong-Woo

2015-11-01

Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self-assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three-dimensional (3D) cell-printed scaffolds using layer-by-layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell-encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell-based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL-alginate gel constructs. PCL-alginate gels containing transforming growth factor-β (TGFβ) showed higher ECM formation. The 3D cell-printed scaffolds of PCL-alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL-alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell-printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology. Copyright © 2013 John Wiley & Sons, Ltd.

The potential for targeting extracellular LOX proteins in human malignancy

DEFF Research Database (Denmark)

Mayorca Guiliani, Alejandro Enrique; Erler, Janine T

2013-01-01

The extracellular matrix (ECM) is the physical scaffold where cells are organized into tissues and organs. The ECM may be modified during cancer to allow and promote proliferation, invasion, and metastasis. The family of lysyl oxidase (LOX) enzymes cross-links collagens and elastin and, therefore......, is a central player in ECM deposition and maturation. Extensive research has revealed how the LOX proteins participate in every stage of cancer progression, and two family members, LOX and LOX-like 2, have been linked to metastasis, the final stage of cancer responsible for over 90% of cancer patient deaths...

The influence of an in vitro generated bone-like extracellular matrix on osteoblastic gene expression of marrow stromal cells.

NARCIS (Netherlands)

Pham, Q.P.; Kasper, F.K.; Baggett, L.S.; Raphael, R.M.; Jansen, J.A.; Mikos, A.G.

2008-01-01

The function and development of cells rely heavily on the signaling interactions with the surrounding extracellular matrix (ECM). Therefore, a tissue engineering scaffold should mimic native ECM to recreate the in vivo environment. Previously, we have shown that an in vitro generated ECM secreted by

Extracellular polymeric substances mediate bioleaching/biocorrosion via interfacial processes involving iron(III) ions and acidophilic bacteria.

Science.gov (United States)

Sand, Wolfgang; Gehrke, Tilman

2006-01-01

Extracellular polymeric substances seem to play a pivotal role in biocorrosion of metals and bioleaching, biocorrosion of metal sulfides for the winning of precious metals as well as acid rock drainage. For better control of both processes, the structure and function of extracellular polymeric substances of corrosion-causing or leaching bacteria are of crucial importance. Our research focused on the extremophilic bacteria Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, because of the "simplicity" and knowledge about the interactions of these bacteria with their substrate/substratum and their environment. For this purpose, the composition of the corresponding extracellular polymeric substances and their functions were analyzed. The extracellular polymeric substances of both species consist mainly of neutral sugars and lipids. The functions of the exopolymers seem to be: (i) to mediate attachment to a (metal) sulfide surface, and (ii) to concentrate iron(III) ions by complexation through uronic acids or other residues at the mineral surface, thus, allowing an oxidative attack on the sulfide. Consequently, dissolution of the metal sulfide is enhanced, which may result in an acceleration of 20- to 100-fold of the bioleaching process over chemical leaching. Experiments were performed to elucidate the importance of the iron(III) ions complexed by extracellular polymeric substances for strain-specific differences in oxidative activity for pyrite. Strains of A. ferrooxidans with a high amount of iron(III) ions in their extracellular polymeric substances possess greater oxidation activity than those with fewer iron(III) ions. These data provide insight into the function of and consequently the advantages that extracellular polymeric substances provide to bacteria. The role of extracellular polymeric substances for attachment under the conditions of a space station and resulting effects like biofouling, biocorrosion, malodorous gases, etc. will be discussed.

Biomaterials in myocardial tissue engineering

Science.gov (United States)

Reis, Lewis A.; Chiu, Loraine L. Y.; Feric, Nicole; Fu, Lara; Radisic, Milica

2016-01-01

Cardiovascular disease is the leading cause of death in the developed world, and as such there is a pressing need for treatment options. Cardiac tissue engineering emerged from the need to develop alternate sources and methods of replacing tissue damaged by cardiovascular diseases, as the ultimate treatment option for many who suffer from end-stage heart failure is a heart transplant. In this review we focus on biomaterial approaches to augment injured or impaired myocardium with specific emphasis on: the design criteria for these biomaterials; the types of scaffolds—composed of natural or synthetic biomaterials, or decellularized extracellular matrix—that have been used to develop cardiac patches and tissue models; methods to vascularize scaffolds and engineered tissue, and finally injectable biomaterials (hydrogels)designed for endogenous repair, exogenous repair or as bulking agents to maintain ventricular geometry post-infarct. The challenges facing the field and obstacles that must be overcome to develop truly clinically viable cardiac therapies are also discussed. PMID:25066525

Injectable biomaterials for adipose tissue engineering

International Nuclear Information System (INIS)

Young, D A; Christman, K L

2012-01-01

Adipose tissue engineering has recently gained significant attention from materials scientists as a result of the exponential growth of soft tissue filler procedures being performed within the clinic. While several injectable materials are currently being marketed for filling subcutaneous voids, they often face limited longevity due to rapid resorption. Their inability to encourage natural adipose formation or ingrowth necessitates repeated injections for a prolonged effect and thus classifies them as temporary fillers. As a result, a significant need for injectable materials that not only act as fillers but also promote in vivo adipogenesis is beginning to be realized. This paper will discuss the advantages and disadvantages of commercially available soft tissue fillers. It will then summarize the current state of research using injectable synthetic materials, biopolymers and extracellular matrix-derived materials for adipose tissue engineering. Furthermore, the successful attributes observed across each of these materials will be outlined along with a discussion of the current difficulties and future directions for adipose tissue engineering. (paper)

Cell based bone tissue engineering in jaw defects

NARCIS (Netherlands)

Meijer, Gert J.; de Bruijn, Joost Dick; Koole, Ron; van Blitterswijk, Clemens

2008-01-01

In 6 patients the potency of bone tissue engineering to reconstruct jaw defects was tested. After a bone marrow aspirate was taken, stem cells were cultured, expanded and grown for 7 days on a bone substitute in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix.

Synthetic biology meets tissue engineering.

Science.gov (United States)

Davies, Jamie A; Cachat, Elise

2016-06-15

Classical tissue engineering is aimed mainly at producing anatomically and physiologically realistic replacements for normal human tissues. It is done either by encouraging cellular colonization of manufactured matrices or cellular recolonization of decellularized natural extracellular matrices from donor organs, or by allowing cells to self-organize into organs as they do during fetal life. For repair of normal bodies, this will be adequate but there are reasons for making unusual, non-evolved tissues (repair of unusual bodies, interface to electromechanical prostheses, incorporating living cells into life-support machines). Synthetic biology is aimed mainly at engineering cells so that they can perform custom functions: applying synthetic biological approaches to tissue engineering may be one way of engineering custom structures. In this article, we outline the 'embryological cycle' of patterning, differentiation and morphogenesis and review progress that has been made in constructing synthetic biological systems to reproduce these processes in new ways. The state-of-the-art remains a long way from making truly synthetic tissues, but there are now at least foundations for future work. © 2016 Authors; published by Portland Press Limited.

INCREASE OF GLYCOSAMINOGLYCANS AND METALLOPROTEINASES 2 AND 9 IN LIVER EXTRACELLULAR MATRIX ON EARLY STAGES OF EXTRAHEPATIC CHOLESTASIS

Directory of Open Access Journals (Sweden)

Pedro Luiz Rodrigues GUEDES

2014-12-01

Full Text Available Context Cholestasis produces hepatocellular injury, leukocyte infiltration, ductular cells proliferation and fibrosis of liver parenchyma by extracellular matrix replacement. Objective Analyze bile duct ligation effect upon glycosaminoglycans content and matrix metalloproteinase (MMPs activities. Methods Animals (6-8 weeks; n = 40 were euthanized 2, 7 or 14 days after bile duct ligation or Sham-surgery. Disease evolution was analyzed by body and liver weight, seric direct bilirubin, globulins, gamma glutamyl transpeptidase (GGT, alkaline phosphatase (Alk-P, alanine and aspartate aminotransferases (ALT and AST, tissue myeloperoxidase and MMP-9, pro MMP-2 and MMP-2 activities, histopathology and glycosaminoglycans content. Results Cholestasis caused cellular damage with elevation of globulins, GGT, Alk-P, ALT, AST. There was neutrophil infiltration observed by the increasing of myeloperoxidase activity on 7 (P = 0.0064 and 14 (P = 0.0002 groups which leads to the magnification of tissue injuries. Bile duct ligation increased pro-MMP-2 (P = 0.0667, MMP-2 (P = 0.0003 and MMP-9 (P<0.0001 activities on 14 days indicating matrix remodeling and establishment of inflammatory process. Bile duct ligation animals showed an increasing on dermatan sulfate and/or heparan sulfate content reflecting extracellular matrix production and growing mitosis due to parenchyma depletion. Conclusions Cholestasis led to many changes on rats’ liver parenchyma, as so as on its extracellular matrix, with major alterations on MMPs activities and glycosaminoglycans content.

Extracellular diffusion parameters in the rat somatosensory cortex during recovery from transient global ischemia/hypoxia

Czech Academy of Sciences Publication Activity Database

Zoremba, N.; Homola, Aleš; Šlais, Karel; Voříšek, Ivan; Rossaint, R.; Lehmenkühler, A.; Syková, Eva

2008-01-01

Roč. 28, č. 10 (2008), s. 1665-1673 ISSN 0271-678X R&D Projects: GA MŠk(CZ) LC554; GA ČR GA305/06/1316 Institutional research plan: CEZ:AV0Z50390512 Keywords : Diffusion * Edema * Extracellular space Subject RIV: FH - Neurology Impact factor: 5.741, year: 2008

The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

Science.gov (United States)

Johnson, Chad J; Cabezas-Olcoz, Jonathan; Kernien, John F; Wang, Steven X; Beebe, David J; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E

2016-09-01

Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.

Natural extracellular nanovesicles and photodynamic molecules: is there a future for drug delivery?

Science.gov (United States)

Kusuzaki, Katsuyuki; Matsubara, Takao; Murata, Hiroaki; Logozzi, Mariantonia; Iessi, Elisabetta; Di Raimo, Rossella; Carta, Fabrizio; Supuran, Claudiu T; Fais, Stefano

2017-12-01

Photodynamic molecules represent an alternative approach for cancer therapy for their property (i) to be photo-reactive; (ii) to be not-toxic for target cells in absence of light; (iii) to accumulate specifically into tumour tissues; (iv) to be activable by a light beam only at the tumour site and (v) to exert cytotoxic activity against tumour cells. However, to date their clinical use is limited by the side effects elicited by systemic administration. Extracellular vesicles are endogenous nanosized-carriers that have been recently introduced as a natural delivery system for therapeutic molecules. We have recently shown the ability of human exosomes to deliver photodynamic molecules. Therefore, this review focussed on extracellular vesicles as a novel strategy for the delivery of photodynamic molecules at cancer sites. This completely new approach may enhance the delivery and decrease the toxicity of photodynamic molecules, therefore, represent the future for photodynamic therapy for cancer treatment.

Extracellular histones play an inflammatory role in acid aspiration-induced acute respiratory distress syndrome.

Science.gov (United States)

Zhang, Yanlin; Wen, Zongmei; Guan, Li; Jiang, Ping; Gu, Tao; Zhao, Jinyuan; Lv, Xin; Wen, Tao

2015-01-01

Systemic inflammation is a key feature in acid aspiration-induced acute respiratory distress syndrome (ARDS), but the factors that trigger inflammation are unclear. The authors hypothesize that extracellular histones, a newly identified inflammatory mediator, play important roles in the pathogenesis of ARDS. The authors used a hydrochloric acid aspiration-induced ARDS model to investigate whether extracellular histones are pathogenic and whether targeting histones are protective. Exogenous histones and antihistone antibody were administered to mice. Heparin can bind to histones, so the authors studied whether heparin could protect from ARDS using cell and mouse models. Furthermore, the authors analyzed whether extracellular histones are clinically involved in ARDS patients caused by gastric aspiration. Extracellular histones in bronchoalveolar lavage fluid of acid-treated mice were significantly higher (1.832 ± 0.698) at 3 h after injury than in sham-treated group (0.63 ± 0.153; P = 0.0252, n = 5 per group). Elevated histones may originate from damaged lung cells and neutrophil infiltration. Exogenous histones aggravated lung injury, whereas antihistone antibody markedly attenuated the intensity of ARDS. Notably, heparin provided a similar protective effect against ARDS. Analysis of plasma from ARDS patients (n = 21) showed elevated histones were significantly correlated with the degree of ARDS and were higher in nonsurvivors (2.723 ± 0.2933, n = 7) than in survivors (1.725 ± 0.1787, P = 0.006, n = 14). Extracellular histones may play a contributory role toward ARDS by promoting tissue damage and systemic inflammation and may become a novel marker reflecting disease activity. Targeting histones by neutralizing antibody or heparin shows potent protective effects, suggesting a potentially therapeutic strategy.

Extracellular histones disarrange vasoactive mediators release through a COX-NOS interaction in human endothelial cells.

Science.gov (United States)

Pérez-Cremades, Daniel; Bueno-Betí, Carlos; García-Giménez, José Luis; Ibañez-Cabellos, José Santiago; Hermenegildo, Carlos; Pallardó, Federico V; Novella, Susana

2017-08-01

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone-mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose-dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX-1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX-2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial-dependent mediators through an up-regulation in COX-2-PGIS-PGI2 pathway which involves a COX-2-dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone-mediated pathologies. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

A comprehensive review of cryogels and their roles in tissue engineering applications.

Science.gov (United States)

Hixon, Katherine R; Lu, Tracy; Sell, Scott A

2017-10-15

The extracellular matrix is fundamental in providing an appropriate environment for cell interaction and signaling to occur. Replicating such a matrix is advantageous in the support of tissue ingrowth and regeneration through the field of tissue engineering. While scaffolds can be fabricated in many ways, cryogels have recently become a popular approach due to their macroporous structure and durability. Produced through the crosslinking of gel precursors followed by a subsequent controlled freeze/thaw cycle, the resulting cryogel provides a unique, sponge-like structure. Therefore, cryogels have proven advantageous for many tissue engineering applications including roles in bioreactor systems, cell separation, and scaffolding. Specifically, the matrix has been demonstrated to encourage the production of various molecules, such as antibodies, and has also been used for cryopreservation. Cryogels can pose as a bioreactor for the expansion of cell lines, as well as a vehicle for cell separation. Lastly, this matrix has shown excellent potential as a tissue engineered scaffold, encouraging regrowth at numerous damaged tissue sites in vivo. This review will briefly discuss the fabrication of cryogels, with an emphasis placed on their application in various facets of tissue engineering to provide an overview of this unique scaffold's past and future roles. Cryogels are unique scaffolds produced through the controlled freezing and thawing of a polymer solution. There is an ever-growing body of literature that demonstrates their applicability in the realm of tissue engineering as extracellular matrix analogue scaffolds; with extensive information having been provided regarding the fabrication, porosity, and mechanical integrity of the scaffolds. Additionally, cryogels have been reviewed with respect to their role in bioseparation and as cellular incubators. This all-inclusive view of the roles that cryogels can play is critical to advancing the technology and expanding its

Therapeutic application of extracellular vesicles in acute and chronic renal injury.

Science.gov (United States)

Rovira, Jordi; Diekmann, Fritz; Campistol, Josep M; Ramírez-Bajo, María José

A new cell-to-cell communication system was discovered in the 1990s, which involves the release of vesicles into the extracellular space. These vesicles shuttle bioactive particles, including proteins, mRNA, miRNA, metabolites, etc. This particular communication has been conserved throughout evolution, which explains why most cell types are capable of producing vesicles. Extracellular vesicles (EVs) are involved in the regulation of different physiological processes, as well as in the development and progression of several diseases. EVs have been widely studied over recent years, especially those produced by embryonic and adult stem cells, blood cells, immune system and nervous system cells, as well as tumour cells. EV analysis from bodily fluids has been used as a diagnostic tool for cancer and recently for different renal diseases. However, this review analyses the importance of EVs generated by stem cells, their function and possible clinical application in renal diseases and kidney transplantation. Copyright © 2016. Published by Elsevier España, S.L.U.

The Double-Edged Sword: Conserved Functions of Extracellular Hsp90 in Wound Healing and Cancer

Energy Technology Data Exchange (ETDEWEB)

Hance, Michael W.; Nolan, Krystal D.; Isaacs, Jennifer S., E-mail: isaacsj@musc.edu [Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Hollings Cancer Center, Charleston, SC 29412 (United States)

2014-05-06

Heat shock proteins (Hsps) represent a diverse group of chaperones that play a vital role in the protection of cells against numerous environmental stresses. Although our understanding of chaperone biology has deepened over the last decade, the “atypical” extracellular functions of Hsps have remained somewhat enigmatic and comparatively understudied. The heat shock protein 90 (Hsp90) chaperone is a prototypic model for an Hsp family member exhibiting a duality of intracellular and extracellular functions. Intracellular Hsp90 is best known as a master regulator of protein folding. Cancers are particularly adept at exploiting this function of Hsp90, providing the impetus for the robust clinical development of small molecule Hsp90 inhibitors. However, in addition to its maintenance of protein homeostasis, Hsp90 has also been identified as an extracellular protein. Although early reports ascribed immunoregulatory functions to extracellular Hsp90 (eHsp90), recent studies have illuminated expanded functions for eHsp90 in wound healing and cancer. While the intended physiological role of eHsp90 remains enigmatic, its evolutionarily conserved functions in wound healing are easily co-opted during malignancy, a pathology sharing many properties of wounded tissue. This review will highlight the emerging functions of eHsp90 and shed light on its seemingly dichotomous roles as a benevolent facilitator of wound healing and as a sinister effector of tumor progression.

Streptococcus suis Serotype 2 Biofilms Inhibit the Formation of Neutrophil Extracellular Traps.

Science.gov (United States)

Ma, Fang; Yi, Li; Yu, Ningwei; Wang, Guangyu; Ma, Zhe; Lin, Huixing; Fan, Hongjie

2017-01-01

Invasive infections caused by Streptococcus suis serotype 2 (SS2) has emerged as a clinical problem in recent years. Neutrophil extracellular traps (NETs) are an important mechanism for the trapping and killing of pathogens that are resistant to phagocytosis. Biofilm formation can protect bacteria from being killed by phagocytes. Until now, there have only been a few studies that focused on the interactions between bacterial biofilms and NETs. SS2 in both a biofilm state and a planktonic cell state were incubated with phagocytes and NETs, and bacterial survival was assessed. DNase I and cytochalasin B were used to degrade NET DNA or suppress phagocytosis, respectively. Extracellular DNA was stained with impermeable fluorescent dye to quantify NET formation. Biofilm formation increased up to 6-fold in the presence of neutrophils, and biofilms were identified in murine tissue. Both planktonic and biofilm cells induced neutrophils chemotaxis to the infection site, with neutrophils increasing by 85.1 and 73.8%, respectively. The bacteria in biofilms were not phagocytized. The bactericidal efficacy of NETs on the biofilms and planktonic cells were equal; however, the biofilm extracellular matrix can inhibit NET release. Although biofilms inhibit NETs release, NETs appear to be an important mechanism to eliminate SS2 biofilms. This knowledge advances the understanding of biofilms and may aid in the development of treatments for persistent infections with a biofilm component.

Extracellular Glycoproteins in Embryogenic Culture of Pumpkin (Cucurbita pepo L.

Directory of Open Access Journals (Sweden)

Hana Čipčić Paljetak

2011-01-01

Full Text Available The extracellular proteins in three distinctly induced embryogenic lines of pumpkin (Cucurbita pepo L. cultivated in four MS media modified regarding the nitrogen composition or auxin presence/absence have been analyzed. Extracellular glycoproteins containing α-D-mannose were specifically detected by the lectine concavalin A. During the cultivation of embryogenic tissue in the medium supplemented with reduced nitrogen, the embryos were mostly arrested at preglobular and globular developmental stages, which coincide with the absence of protein secretion. Secreted glycoproteins of 76, 68, 37 and 34 kDa were detected only if any of the three lines were cultivated in the medium that stimulates embryo development, irrespectively of the addition of 2,4-dichlorophenoxyacetic acid or tunicamycin. The glycoprotein of 64 kDa was detected in all lines cultivated in hormone-free MS medium with conventional nitrogen sources and it appears to be associated with embryo maturation. Tunicamycin treatment did not influence embryogenesis, although it specifically affected glycosylation of proteins in the investigated lines. Our results show that besides auxin, the source of nitrate is of great importance for proper protein glycosylation, excretion and developmental transition of pumpkin somatic embryos.

Human meniscal proteoglycan metabolism in long-term tissue culture

NARCIS (Netherlands)

Verbruggen, G.; Verdonk, R.; Veys, E. M.; van Daele, P.; de Smet, P.; van den Abbeele, K.; Claus, B.; Baeten, D.

1996-01-01

For the purpose of human meniscal allografting, menisci have been maintained viable in in vitro culture. The influence of long-term tissue culture on the extracellular matrix metabolism of the meniscus has been studied. Fetal calf serum (FCS) was used as a supplement for the growth factors necessary

Development of biomaterial scaffold for nerve tissue engineering: Biomaterial mediated neural regeneration

Directory of Open Access Journals (Sweden)

Sethuraman Swaminathan

2009-11-01

Full Text Available Abstract Neural tissue repair and regeneration strategies have received a great deal of attention because it directly affects the quality of the patient's life. There are many scientific challenges to regenerate nerve while using conventional autologous nerve grafts and from the newly developed therapeutic strategies for the reconstruction of damaged nerves. Recent advancements in nerve regeneration have involved the application of tissue engineering principles and this has evolved a new perspective to neural therapy. The success of neural tissue engineering is mainly based on the regulation of cell behavior and tissue progression through the development of a synthetic scaffold that is analogous to the natural extracellular matrix and can support three-dimensional cell cultures. As the natural extracellular matrix provides an ideal environment for topographical, electrical and chemical cues to the adhesion and proliferation of neural cells, there exists a need to develop a synthetic scaffold that would be biocompatible, immunologically inert, conducting, biodegradable, and infection-resistant biomaterial to support neurite outgrowth. This review outlines the rationale for effective neural tissue engineering through the use of suitable biomaterials and scaffolding techniques for fabrication of a construct that would allow the neurons to adhere, proliferate and eventually form nerves.

Extracellular Molecules Involved in Cancer Cell Invasion

International Nuclear Information System (INIS)

Stivarou, Theodora; Patsavoudi, Evangelia

2015-01-01

Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion

Identification of an Extracellular Endoglucanase That Is Required for Full Virulence in Xanthomonas citri subsp. citri.

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Tian Xia

Full Text Available Xanthomonas citri subsp. citri causes citrus canker disease, which is characterized by the formation of water-soaked lesions, white or yellow spongy pustules and brown corky canker. In this work, we report the contribution of extracellular endoglucanase to canker development during infection. The ectopic expression of nine putative cellulases in Escherichia coli indicated that two endoglucanases, BglC3 and EngXCA, show carboxymethyl cellulase activity. Both bglC3 and engXCA genes were transcribed in X. citri subsp. citri, however, only BglC3 protein was detected outside the cell in western blot analysis. The deletion of bglC3 gene resulted in complete loss of extracellular carboxymethyl cellulase activity and delayed the onset of canker symptoms in both infiltration- and wound-inoculation assays. When growing in plant tissue, the cell density of bglC3 mutant was lower than that of the wild type. Our data demonstrated that BglC3 is an extracellular endoglucanase required for the full virulence of X. citri subsp. citri.

[Effect of electroacupuncture intervention on expression of extracellular matrix collagen and metabolic enzymes].

Science.gov (United States)

Liao, Jun; Zhang, Le; Ke, Mei-gui; Xu, Teng

2013-12-01

To observe the effect of electroacupuncture (EA) at "Dazhui" (GV 14) on the contents of extracellular matrix (ECM), collagen type II (COL-II), collagen type V (COL-V), matrix metalloproteinase (MMP)-13, tissue inhibitor of metalloproteinase (TIMP)-1 in rats with cervicovertebral disc degeneration so as to explore its mechanism underlying relief of intervertebral disc degeneration. A total of 28 SD rats were randomly divided into sham group (n = 7), model group (n = 7), EA group (n = 7) and medication group (n = 7). The model of cervical intervertebral disc degeneration was established by trans-section of the deep neck splenius, the longest muscles of head, neck costocervicalis, head semi-spinatus muscle, supraspinous ligament and interspinal ligaments of cervical 2-7 segments, etc. to produce imbalance between the dynamic and static force. EA was applied to "Dazhui" (GV 14) for 30 min, once daily for 28 days, with a 2 days' interval between two courses. Animals of the medication group were treated by oral administration of meloxicam tablets (0.75 mg/kg) once daily for 28 days, with a 2 days' interval between two courses. Immunohistochemistry was used to measure the expression of ECM, COL- II, COL-V, MMP-13 and TIMP-1 in the cervicovertebral disc tissue. Compared with the sham group, the expression levels of ECM and COL-II proteins in the cervicovertebral disc tissue were significantly decreased in the model group (P 0.05). EA of "Dazhui" (GV 14) can effectively regulate extracellular matrix system in rats with cervical intervertebral disc degeneration, which is possibly related to its effect in relieving cervical spondylosis.

Measuring principles of frictional coefficients in cartilaginous tissues and its substitutes

NARCIS (Netherlands)

Huyghe, J.M.R.J.; Janssen, C.F.; Donkelaar, van C.C.; Lanir, Y.

2002-01-01

The frictional properties of cartilaginous tissues, such as the hydraulic permeability, the electro-osmotic permeability, the diffusion coefficients of various ions and solutes, and the electrical conductance, are vital data to characterise the extracellular environment in which chondrocytes reside.

HEPATIC TISSUE ENGINEERING (MODERN STATE OF THIS PROBLEM

Directory of Open Access Journals (Sweden)

Y.S. Gulay

2014-01-01

Full Text Available In this article it was discussed the problem of creation implanted hepatic tissue engineering designs as a modern stage of complex investigation for working out bioartifi cial liver support systems. It was determined that for the positive decision of numerous biological and technological problems it is necessary: to use matrices with determined properties, which mimic properties of hepatic extracellular matrix; to use technology for stereotype sowing of these matrices by both parenchymal and non-parenchymal hepatic cells and to improve the technologies for making and assembling of hepatic tissue-engineering designs.

Hyperplasia of elastic tissue in hepatic schistosomal fibrosis

Directory of Open Access Journals (Sweden)

Zilton A. Andrade

1991-12-01

Full Text Available Elastic tissue hyperplasia, revealed by means of histological, immunocytochemical and ultrastructural methods, appeared as a prominent change in surgical liver biopsies taken from 61 patients with schistosomal periportal and septal fibrosis. Such hyperplasia was absent in ecperimental murine schistosomiasis, including mice with "pipe-stem" fibrosis. Displaced connective tissue cells in periportal areas, such as smooth muscle cells, more frequently observed in human material, could be the site of excessive elastin synthesis, and could explain the differences observed in human and experimental materials. Elastic tissue, sometimes represented by its microfibrillar components, also appeared to be more condensed in areas of matrix (collagen degradation, suggesting a participation of this tissue in the remodelling of the extracellular matrix. By its rectratile properties elastic tissue hyperplasia in hepatic schistosomiasis can cause vascular narrowing and thus play a role in the pathogenesis of portal hypeertension.

Placental vascular responses are dependent on surrounding tissue

DEFF Research Database (Denmark)

Brøgger, Torbjørn Halle

-depth understanding of the mechanism regulating blood flow and perfusion is necessary if we are to come up with new ideas for intervention and treatment. Method: From fresh born placentas stem villi arteries were carefully dissected. The artery branches were divided. The surrounding tissue was removed from one end...... and was left untouched in the other end. Then using wire myography they were investigated in terms of contractility and sensitivity to physiological relevant human-like agonists. Results: Sensitivity to PGF2α, Tx-analog, 5-HT and endothelin-1 was significantly lower in arteries with intact surrounding tissue...... compared to arteries stripped of the tissue. The maximal force development was also significantly lower in arteries with surrounding tissue, when they were depolarized high extracellular [K+] or stimulated with PGF2α or endotheline-1. Conclusion: The perivascular tissue significantly alters stem villi...

Placental vascular responses are dependent on surrounding tissue

DEFF Research Database (Denmark)

Brøgger, Torbjørn Halle

. Materials and methods. From fresh born placentas, stem villi arteries were carefully dissected. The artery branches were divided. The surrounding tissue was removed from one end and was left untouched in the other end.Then, using wire myography, they were investigated in terms of contractility...... and sensitivity to physiological relevant human-like agonists. Results. Sensitivity to PGF2α, Tx-analog, 5-HT and endothelin-1 was significantly lower in arteries with intact surrounding tissue compared to arteries stripped of the tissue. The maximal force development was also significantly lower in arteries...... with surrounding tissue when they were depolarized high extracellular [K+] or stimulated with PGF2α or endotheline-1. Conclusion. The perivascular tissue significantly alters stem villi arteries' sensitivity and force development in a suppressive way. This implicates a new aspect of blood flow regulation...

Extracellular matrix control of mammary gland morphogenesis and tumorigenesis: insights from imaging

Energy Technology Data Exchange (ETDEWEB)

Ghajar, Cyrus M; Bissell, Mina J

2008-10-23

The extracellular matrix (ECM), once thought to solely provide physical support to a tissue, is a key component of a cell's microenvironment responsible for directing cell fate and maintaining tissue specificity. It stands to reason, then, that changes in the ECM itself or in how signals from the ECM are presented to or interpreted by cells can disrupt tissue organization; the latter is a necessary step for malignant progression. In this review, we elaborate on this concept using the mammary gland as an example. We describe how the ECM directs mammary gland formation and function, and discuss how a cell's inability to interpret these signals - whether as a result of genetic insults or physicochemical alterations in the ECM - disorganizes the gland and promotes malignancy. By restoring context and forcing cells to properly interpret these native signals, aberrant behavior can be quelled and organization re-established. Traditional imaging approaches have been a key complement to the standard biochemical, molecular, and cell biology approaches used in these studies. Utilizing imaging modalities with enhanced spatial resolution in live tissues may uncover additional means by which the ECM regulates tissue structure, on different length scales, through its pericellular organization (short-scale) and by biasing morphogenic and morphostatic gradients (long-scale).

Extracellular vesicles: Exosomes, microvesicles, and friends

NARCIS (Netherlands)

Raposo, G.; Stoorvogel, W.|info:eu-repo/dai/nl/074352385

2013-01-01

Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for

T Cell Interstitial Migration: Motility Cues from the Inflamed Tissue for Micro- and Macro-Positioning.

Science.gov (United States)

Gaylo, Alison; Schrock, Dillon C; Fernandes, Ninoshka R J; Fowell, Deborah J

2016-01-01

Effector T cells exit the inflamed vasculature into an environment shaped by tissue-specific structural configurations and inflammation-imposed extrinsic modifications. Once within interstitial spaces of non-lymphoid tissues, T cells migrate in an apparent random, non-directional, fashion. Efficient T cell scanning of the tissue environment is essential for successful location of infected target cells or encounter with antigen-presenting cells that activate the T cell's antimicrobial effector functions. The mechanisms of interstitial T cell motility and the environmental cues that may promote or hinder efficient tissue scanning are poorly understood. The extracellular matrix (ECM) appears to play an important scaffolding role in guidance of T cell migration and likely provides a platform for the display of chemotactic factors that may help to direct the positioning of T cells. Here, we discuss how intravital imaging has provided insight into the motility patterns and cellular machinery that facilitates T cell interstitial migration and the critical environmental factors that may optimize the efficiency of effector T cell scanning of the inflamed tissue. Specifically, we highlight the local micro-positioning cues T cells encounter as they migrate within inflamed tissues, from surrounding ECM and signaling molecules, as well as a requirement for appropriate long-range macro-positioning within distinct tissue compartments or at discrete foci of infection or tissue damage. The central nervous system (CNS) responds to injury and infection by extensively remodeling the ECM and with the de novo generation of a fibroblastic reticular network that likely influences T cell motility. We examine how inflammation-induced changes to the CNS landscape may regulate T cell tissue exploration and modulate function.

Assessment of Energy Metabolic Changes in Adipose Tissue-Derived Stem Cells

NARCIS (Netherlands)

Hajmousa, Ghazaleh; Harmsen, Martin C; Di Nardo, Paolo; Dhingra, Sanjiv; Singla, Dinender K.

2017-01-01

Adipose tissue-derived stem cells (ADSC) are promising candidates for therapeutic applications in cardiovascular regenerative medicine. By definition, the phenotype ADSCs, e.g., the ubiquitous secretion of growth factors, cytokines, and extracellular matrix components is not met in vivo, which

Full-length fibronectin drives fibroblast accumulation at the surface of collagen microtissues during cell-induced tissue morphogenesis

NARCIS (Netherlands)

Foolen, J.; Shiu, J.-Y.; Mitsi, M.; Zhang, Y.; Chen, C.; Vogel, Viola

2016-01-01

Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is

Fabrication and characterization of scaffold from cadaver goat-lung tissue for skin tissue engineering applications.

Science.gov (United States)

Gupta, Sweta K; Dinda, Amit K; Potdar, Pravin D; Mishra, Narayan C

2013-10-01

The present study aims to fabricate scaffold from cadaver goat-lung tissue and evaluate it for skin tissue engineering applications. Decellularized goat-lung scaffold was fabricated by removing cells from cadaver goat-lung tissue enzymatically, to have cell-free 3D-architecture of natural extracellular matrix. DNA quantification assay and Hematoxylin and eosin staining confirmed the absence of cellular material in the decellularized lung-tissue. SEM analysis of decellularized scaffold shows the intrinsic porous structure of lung tissue with well-preserved pore-to-pore interconnectivity. FTIR analysis confirmed non-denaturation and well maintainance of collagenous protein structure of decellularized scaffold. MTT assay, SEM analysis and H&E staining of human skin-derived Mesenchymal Stem cell, seeded over the decellularized scaffold, confirms stem cell attachment, viability, biocompatibility and proliferation over the decellularized scaffold. Expression of Keratin18 gene, along with CD105, CD73 and CD44, by human skin-derived Mesenchymal Stem cells over decellularized scaffold signifies that the cells are viable, proliferating and migrating, and have maintained their critical cellular functions in the presence of scaffold. Thus, overall study proves the applicability of the goat-lung tissue derived decellularized scaffold for skin tissue engineering applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Sources of extracellular tau and its signaling.

Science.gov (United States)

Avila, Jesús; Simón, Diana; Díaz-Hernández, Miguel; Pintor, Jesús; Hernández, Félix

2014-01-01

The pathology associated with tau protein, tauopathy, has been recently analyzed in different disorders, leading to the suggestion that intracellular and extracellular tau may itself be the principal agent in the transmission and spreading of tauopathies. Tau pathology is based on an increase in the amount of tau, an increase in phosphorylated tau, and/or an increase in aggregated tau. Indeed, phosphorylated tau protein is the main component of tau aggregates, such as the neurofibrillary tangles present in the brain of Alzheimer's disease patients. It has been suggested that intracellular tau could be toxic to neurons in its phosphorylated and/or aggregated form. However, extracellular tau could also damage neurons and since neuronal death is widespread in Alzheimer's disease, mainly among cholinergic neurons, these cells may represent a possible source of extracellular tau. However, other sources of extracellular tau have been proposed that are independent of cell death. In addition, several ways have been proposed for cells to interact with, transmit, and spread extracellular tau, and to transduce signals mediated by this tau. In this work, we will discuss the role of extracellular tau in the spreading of the tau pathology.

Polymer hydrogels usable for nervous tissue repair

Czech Academy of Sciences Publication Activity Database

Lesný, Petr; DeCroos, Jane; Přádný, Martin; Vacík, Jiří; Michálek, Jiří; Woerly, S.; Syková, Eva

2002-01-01

Roč. 23, - (2002), s. 243-247 ISSN 0891-0618 R&D Projects: GA MŠk LN00A065; GA ČR GV307/96/K226 Institutional research plan: CEZ:AV0Z5039906 Keywords : Extracellular space * Astrocytes Subject RIV: FH - Neurology Impact factor: 2.696, year: 2002

Tumorigenic Potential of Extracellular Matrix Metalloproteinase Inducer

Science.gov (United States)

Zucker, Stanley; Hymowitz, Michelle; Rollo, Ellen E.; Mann, Richard; Conner, Cathleen E.; Cao, Jian; Foda, Hussein D.; Tompkins, David C.; Toole, Bryan P.

2001-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs. PMID:11395366

Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

Science.gov (United States)

DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

2015-05-01

Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Analysis of extracellular RNA by digital PCR

Directory of Open Access Journals (Sweden)

Kenji eTakahashi

2014-06-01

Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

Extracellular Molecules Involved in Cancer Cell Invasion

Directory of Open Access Journals (Sweden)

Theodora Stivarou

2015-01-01

Full Text Available Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion.

Myogenic Progenitor Cells Control Extracellular Matrix Production by Fibroblasts during Skeletal Muscle Hypertrophy.

Science.gov (United States)

Fry, Christopher S; Kirby, Tyler J; Kosmac, Kate; McCarthy, John J; Peterson, Charlotte A

2017-01-05

Satellite cells, the predominant stem cell population in adult skeletal muscle, are activated in response to hypertrophic stimuli and give rise to myogenic progenitor cells (MPCs) within the extracellular matrix (ECM) that surrounds myofibers. This ECM is composed largely of collagens secreted by interstitial fibrogenic cells, which influence satellite cell activity and muscle repair during hypertrophy and aging. Here we show that MPCs interact with interstitial fibrogenic cells to ensure proper ECM deposition and optimal muscle remodeling in response to hypertrophic stimuli. MPC-dependent ECM remodeling during the first week of a growth stimulus is sufficient to ensure long-term myofiber hypertrophy. MPCs secrete exosomes containing miR-206, which represses Rrbp1, a master regulator of collagen biosynthesis, in fibrogenic cells to prevent excessive ECM deposition. These findings provide insights into how skeletal stem and progenitor cells interact with other cell types to actively regulate their extracellular environments for tissue maintenance and adaptation. Copyright © 2017 Elsevier Inc. All rights reserved.

Of extracellular matrix, scaffolds, and signaling: Tissuearchitectureregulates development, homeostasis, and cancer

Energy Technology Data Exchange (ETDEWEB)

Nelson, Celeste M.; Bissell, Mina J.

2006-03-09

The microenvironment surrounding cells influences gene expression, such that a cell's behavior is largely determined by its interactions with the extracellular matrix, neighboring cells, and soluble cues released locally or by distant tissues. We describe the essential role of context and organ structure in directing mammary gland development and differentiated function, and in determining response to oncogenic insults including mutations. We expand on the concept of 'dynamic reciprocity' to present an integrated view of development, cancer, and aging, and posit that genes are like piano keys: while essential, it is the context that makes the music.

Nanotopography-guided tissue engineering and regenerative medicine☆

Science.gov (United States)

Kim, Hong Nam; Jiao, Alex; Hwang, Nathaniel S.; Kim, Min Sung; Kang, Do Hyun; Kim, Deok-Ho; Suh, Kahp-Yang

2017-01-01

Human tissues are intricate ensembles of multiple cell types embedded in complex and well-defined structures of the extracellular matrix (ECM). The organization of ECM is frequently hierarchical from nano to macro, with many proteins forming large scale structures with feature sizes up to several hundred microns. Inspired from these natural designs of ECM, nanotopography-guided approaches have been increasingly investigated for the last several decades. Results demonstrate that the nanotopography itself can activate tissue-specific function in vitro as well as promote tissue regeneration in vivo upon transplantation. In this review, we provide an extensive analysis of recent efforts to mimic functional nanostructures in vitro for improved tissue engineering and regeneration of injured and damaged tissues. We first characterize the role of various nanostructures in human tissues with respect to each tissue-specific function. Then, we describe various fabrication methods in terms of patterning principles and material characteristics. Finally, we summarize the applications of nanotopography to various tissues, which are classified into four types depending on their functions: protective, mechano-sensitive, electro-active, and shear stress-sensitive tissues. Some limitations and future challenges are briefly discussed at the end. PMID:22921841

Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers.

OpenAIRE

Herrick, S. E.; Sloan, P.; McGurk, M.; Freak, L.; McCollum, C. N.; Ferguson, M. W.

1992-01-01

As part of a major clinical trial, sequential biopsies were taken from the margins of venous leg ulcers during their healing. The changing patterns of tissue architecture and extracellular matrix synthesis during healing were documented histologically and immunocytochemically. Initial biopsies were similar in appearance: prominent fibrin cuffs, variable inflammation, hemosiderin, and red blood cell extravasation. So called "fibrin cuffs" were highly organized structures composed of laminin, f...

Invited review: mesenchymal progenitor cells in intramuscular connective tissue development.

Science.gov (United States)

Miao, Z G; Zhang, L P; Fu, X; Yang, Q Y; Zhu, M J; Dodson, M V; Du, M

2016-01-01

The abundance and cross-linking of intramuscular connective tissue contributes to the background toughness of meat, and is thus undesirable. Connective tissue is mainly synthesized by intramuscular fibroblasts. Myocytes, adipocytes and fibroblasts are derived from a common pool of progenitor cells during the early embryonic development. It appears that multipotent mesenchymal stem cells first diverge into either myogenic or non-myogenic lineages; non-myogenic mesenchymal progenitors then develop into the stromal-vascular fraction of skeletal muscle wherein adipocytes, fibroblasts and derived mesenchymal progenitors reside. Because non-myogenic mesenchymal progenitors mainly undergo adipogenic or fibrogenic differentiation during muscle development, strengthening progenitor proliferation enhances the potential for both intramuscular adipogenesis and fibrogenesis, leading to the elevation of both marbling and connective tissue content in the resulting meat product. Furthermore, given the bipotent developmental potential of progenitor cells, enhancing their conversion to adipogenesis reduces fibrogenesis, which likely results in the overall improvement of marbling (more intramuscular adipocytes) and tenderness (less connective tissue) of meat. Fibrogenesis is mainly regulated by the transforming growth factor (TGF) β signaling pathway and its regulatory cascade. In addition, extracellular matrix, a part of the intramuscular connective tissue, provides a niche environment for regulating myogenic differentiation of satellite cells and muscle growth. Despite rapid progress, many questions remain in the role of extracellular matrix on muscle development, and factors determining the early differentiation of myogenic, adipogenic and fibrogenic cells, which warrant further studies.

Calculation of microplanar beam dose profiles in a tissue/lung/tissue phantom

International Nuclear Information System (INIS)

Company, F.Z.; Allen, B.J.

1998-01-01

Recent advances in synchrotron generated x-ray beams with a high fluence rate permit investigation of the application of an array of closely spaced, parallel or converging microplanar beams in radiotherapy. The proposed technique takes advantage of the hypothesized repair mechanism of capillary cells between alternate microbeam zones, which regenerates the lethally irradiated endothelial cells. The lateral and depth doses of 100 keV microplanar beams are investigated for different beam dimensions and spacings in a tissue, lung and tissue/lung/tissue phantom. The EGS4 Monte Carlo code is used to calculate dose profiles at different depths and bundles of beams (up to 20x20cm square cross section). The maximum dose on the beam axis (peak) and the minimum interbeam dose (valley) are compared at different depths, bundles, heights, widths and beam spacings. (author)

[Inhibitory proteins of neuritic regeneration in the extracellular matrix: structure, molecular interactions and their functions. Mechanisms of extracellular balance].

Science.gov (United States)

Vargas, Javier; Uribe-Escamilla, Rebeca; Alfaro-Rodríguez, Alfonso

2013-01-01

After injury of the central nervous system (CNS) in higher vertebrates, neurons neither grow nor reconnect with their targets because their axons or dendrites cannot regenerate within the injured site. In the CNS, the signal from the environment regulating neurite regeneration is not exclusively generated by one molecular group. This signal is generated by the interaction of various types of molecules such as extracellular matrix proteins, soluble factors and surface membrane molecules; all these elements interact with one another generating the matrix's biological state: the extracellular balance. Proteins in the balanced extracellular matrix, support and promote cellular physiological states, including neuritic regeneration. We have reviewed three types of proteins of the extracellular matrix possessing an inhibitory effect and that are determinant of neuritic regeneration failure in the CNS: chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and tenascin. We also review some of the mechanisms involved in the balance of extracellular proteins such as isomerization, epimerization, sulfation and glycosylation as well as the assemblage of the extracellular matrix, the interaction between the matrix and soluble factors and its proteolytic degradation. In the final section, we have presented some examples of the matrix's role in development and in tumor propagation.

Silk fibroin porous scaffolds for nucleus pulposus tissue engineering

International Nuclear Information System (INIS)

Zeng, Chao; Yang, Qiang; Zhu, Meifeng; Du, Lilong; Zhang, Jiamin; Ma, Xinlong; Xu, Baoshan; Wang, Lianyong

2014-01-01

Intervertebral discs (IVDs) are structurally complex tissue that hold the vertebrae together and provide mobility to spine. The nucleus pulposus (NP) degeneration often results in degenerative IVD disease that is one of the most common causes of back and neck pain. Tissue engineered nucleus pulposus offers an alternative approach to regain the function of the degenerative IVD. The aim of this study is to determine the feasibility of porous silk fibroin (SF) scaffolds fabricated by paraffin-sphere-leaching methods with freeze-drying in the application of nucleus pulposus regeneration. The prepared scaffold possessed high porosity of 92.38 ± 5.12% and pore size of 165.00 ± 8.25 μm as well as high pore interconnectivity and appropriate mechanical properties. Rabbit NP cells were seeded and cultured on the SF scaffolds. Scanning electron microscopy, histology, biochemical assays and mechanical tests revealed that the porous scaffolds could provide an appropriate microstructure and environment to support adhesion, proliferation and infiltration of NP cells in vitro as well as the generation of extracellular matrix. The NP cell–scaffold construction could be preliminarily formed after subcutaneously implanted in a nude mice model. In conclusion, The SF porous scaffold offers a potential candidate for tissue engineered NP tissue. - Highlights: • Paraffin microsphere-leaching method is used to fabricate silk fibroin scaffold. • The scaffold has appropriate mechanical property, porosity and pore size • The scaffold supports growth and infiltration of nucleus pulposus cells. • Nucleus pulposus cells can secrete extracellular matrix in the scaffolds. • The scaffold is a potential candidate for tissue engineered nucleus pulposus

Silk fibroin porous scaffolds for nucleus pulposus tissue engineering

Energy Technology Data Exchange (ETDEWEB)

Zeng, Chao; Yang, Qiang [Department of Spine Surgery, Tianjin Hospital, Tianjin 300211 (China); Tianjin Medical University, Tianjin 300070 (China); Zhu, Meifeng [The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071 (China); Du, Lilong [Department of Spine Surgery, Tianjin Hospital, Tianjin 300211 (China); Tianjin Medical University, Tianjin 300070 (China); Zhang, Jiamin [The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ma, Xinlong [Department of Spine Surgery, Tianjin Hospital, Tianjin 300211 (China); Xu, Baoshan, E-mail: xubaoshan99@126.com [Department of Spine Surgery, Tianjin Hospital, Tianjin 300211 (China); Wang, Lianyong, E-mail: wly@nankai.edu.cn [The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071 (China)

2014-04-01

Intervertebral discs (IVDs) are structurally complex tissue that hold the vertebrae together and provide mobility to spine. The nucleus pulposus (NP) degeneration often results in degenerative IVD disease that is one of the most common causes of back and neck pain. Tissue engineered nucleus pulposus offers an alternative approach to regain the function of the degenerative IVD. The aim of this study is to determine the feasibility of porous silk fibroin (SF) scaffolds fabricated by paraffin-sphere-leaching methods with freeze-drying in the application of nucleus pulposus regeneration. The prepared scaffold possessed high porosity of 92.38 ± 5.12% and pore size of 165.00 ± 8.25 μm as well as high pore interconnectivity and appropriate mechanical properties. Rabbit NP cells were seeded and cultured on the SF scaffolds. Scanning electron microscopy, histology, biochemical assays and mechanical tests revealed that the porous scaffolds could provide an appropriate microstructure and environment to support adhesion, proliferation and infiltration of NP cells in vitro as well as the generation of extracellular matrix. The NP cell–scaffold construction could be preliminarily formed after subcutaneously implanted in a nude mice model. In conclusion, The SF porous scaffold offers a potential candidate for tissue engineered NP tissue. - Highlights: • Paraffin microsphere-leaching method is used to fabricate silk fibroin scaffold. • The scaffold has appropriate mechanical property, porosity and pore size • The scaffold supports growth and infiltration of nucleus pulposus cells. • Nucleus pulposus cells can secrete extracellular matrix in the scaffolds. • The scaffold is a potential candidate for tissue engineered nucleus pulposus.

The effects of matrix inhomogeneities on the cellular mechanical environment in tissue-engineered cartilage: an in silico investigation

NARCIS (Netherlands)

Khoshgoftar, M.; Wilson, W.; Ito, K.; Donkelaar, C.C. van

2014-01-01

Mechanical stimulation during cartilage tissue-engineering enhances extracellular matrix (ECM) synthesis and thereby improves the mechanical properties of tissue engineered (TE) cartilage. Generally, these mechanical stimuli are of a fixed magnitude. However, as a result of ECM synthesis and spatial

Extracellular Neutrophil Proteases Are Efficient Regulators of IL-1, IL-33, and IL-36 Cytokine Activity but Poor Effectors of Microbial Killing.

Science.gov (United States)

Clancy, Danielle M; Sullivan, Graeme P; Moran, Hannah B T; Henry, Conor M; Reeves, Emer P; McElvaney, Noel G; Lavelle, Ed C; Martin, Seamus J

2018-03-13

Neutrophil granule proteases are thought to function as anti-microbial effectors, cooperatively hydrolyzing microorganisms within phagosomes, or upon deployment into the extracellular space. However, evidence also suggests that neutrophil proteases play an important role in the coordination and escalation of inflammatory reactions, but how this is achieved has been obscure. IL-1 family cytokines are important initiators of inflammation and are typically released via necrosis but require proteolytic processing for activation. Here, we show that proteases liberated from activated neutrophils can positively or negatively regulate the activity of six IL-1 family cytokines (IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ) with exquisite sensitivity. In contrast, extracellular neutrophil proteases displayed very poor bactericidal activity, exhibiting 100-fold greater potency toward cytokine processing than bacterial killing. Thus, in addition to their classical role as phagocytes, neutrophils play an important immunoregulatory role through deployment of their granule proteases into the extracellular space to process multiple IL-1 family cytokines. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Preventing tissue fibrosis by local biomaterials interfacing of specific cryptic extracellular matrix information

Science.gov (United States)

Horejs, Christine-Maria; St-Pierre, Jean-Philippe; Ojala, Juha R. M.; Steele, Joseph A. M.; da Silva, Patricia Barros; Rynne-Vidal, Angela; Maynard, Stephanie A.; Hansel, Catherine S.; Rodríguez-Fernández, Clara; Mazo, Manuel M.; You, Amanda Y. F.; Wang, Alex J.; von Erlach, Thomas; Tryggvason, Karl; López-Cabrera, Manuel; Stevens, Molly M.

2017-01-01

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin β1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the β1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets. PMID:28593951

Preventing tissue fibrosis by local biomaterials interfacing of specific cryptic extracellular matrix information

Science.gov (United States)

Horejs, Christine-Maria; St-Pierre, Jean-Philippe; Ojala, Juha R. M.; Steele, Joseph A. M.; da Silva, Patricia Barros; Rynne-Vidal, Angela; Maynard, Stephanie A.; Hansel, Catherine S.; Rodríguez-Fernández, Clara; Mazo, Manuel M.; You, Amanda Y. F.; Wang, Alex J.; von Erlach, Thomas; Tryggvason, Karl; López-Cabrera, Manuel; Stevens, Molly M.

2017-06-01

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin β1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the β1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets.

Biomimetic electrospun nanofibers for tissue regeneration

International Nuclear Information System (INIS)

Liao, Susan; Li Bojun; Ma Zuwei; Wei He; Chan Casey; Ramakrishna, Seeram

2006-01-01

Nanofibers exist widely in human tissue with different patterns. Electrospinning nanotechnology has recently gained a new impetus due to the introduction of the concept of biomimetic nanofibers for tissue regeneration. The advanced electrospinning technique is a promising method to fabricate a controllable continuous nanofiber scaffold similar to the natural extracellular matrix. Thus, the biomedical field has become a significant possible application field of electrospun fibers. Although electrospinning has developed rapidly over the past few years, electrospun nanofibers are still at a premature research stage. Further comprehensive and deep studies on electrospun nanofibers are essential for promoting their biomedical applications. Current electrospun fiber materials include natural polymers, synthetic polymers and inorganic substances. This review briefly describes several typically electrospun nanofiber materials or composites that have great potential for tissue regeneration, and describes their fabrication, advantages, drawbacks and future prospects. (topical review)

Transcriptome of extracellular vesicles released by hepatocytes.

Directory of Open Access Journals (Sweden)

Felix Royo

Full Text Available The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

Bromide space, total body water, and sick cell syndrome

International Nuclear Information System (INIS)

Schober, O.; Hundeshagen, H.; Lehr, L.

1982-01-01

Displacements of the bromide space (Br-82-C, as a marker for the extracellular fluid compartment) are caused by an enhanced anatomical space and/or increased permeability of cells to bromide. The ratio Br-82-C: total body water (TBW) was evaluated to be 0.83 +- 0.17 in critically ill patients (n = 38) compared with the normal value of 0.46 +- 0.04 (n = 10). Because of normal TBW in critically ill patients (TBW = 505 +- 68 ml/kg), an increased bromide penetration into cells seems to be responsible for the enlarged ratio Br-82-C: TBW. Taking into consideration measurements in patients with malabsorption (Br-82-C: TBW = 0.56 +- 0.13; n = 13) and carcinoma of the rectum and colon (Br-82-C: TBW = 0.66 +- 0.24; n = 18) we think that the bromide space is a good measurement of the effective extracellular water. (orig.)

Emerging roles of extracellular vesicles in the adaptive response of tumour cells to microenvironmental stress

Directory of Open Access Journals (Sweden)

Paulina Kucharzewska

2013-03-01

Full Text Available Cells are constantly subjected to various types of endogenous and exogenous stressful stimuli, which can cause serious and even permanent damage. The ability of a cell to sense and adapt to environmental alterations is thus vital to maintain tissue homeostasis during development and adult life. Here, we review some of the major phenotypic characteristics of the hostile tumour microenvironment and the emerging roles of extracellular vesicles in these events.

Nanomaterials for Craniofacial and Dental Tissue Engineering.

Science.gov (United States)

Li, G; Zhou, T; Lin, S; Shi, S; Lin, Y

2017-07-01

Tissue engineering shows great potential as a future treatment for the craniofacial and dental defects caused by trauma, tumor, and other diseases. Due to the biomimetic features and excellent physiochemical properties, nanomaterials are of vital importance in promoting cell growth and stimulating tissue regeneration in tissue engineering. For craniofacial and dental tissue engineering, the frequently used nanomaterials include nanoparticles, nanofibers, nanotubes, and nanosheets. Nanofibers are attractive for cell invasion and proliferation because of their resemblance to extracellular matrix and the presence of large pores, and they have been used as scaffolds in bone, cartilage, and tooth regeneration. Nanotubes and nanoparticles improve the mechanical and chemical properties of scaffold, increase cell attachment and migration, and facilitate tissue regeneration. In addition, nanofibers and nanoparticles are also used as a delivery system to carry the bioactive agent in bone and tooth regeneration, have better control of the release speed of agent upon degradation of the matrix, and promote tissue regeneration. Although applications of nanomaterials in tissue engineering remain in their infancy with numerous challenges to face, the current results indicate that nanomaterials have massive potential in craniofacial and dental tissue engineering.

On the role of type IX collagen in the extracellular matrix of cartilage: type IX collagen is localized to intersections of collagen fibrils

OpenAIRE

1986-01-01

The tissue distribution of type II and type IX collagen in 17-d-old chicken embryo was studied by immunofluorescence using polyclonal antibodies against type II collagen and a peptic fragment of type IX collagen (HMW), respectively. Both proteins were found only in cartilage where they were co-distributed. They occurred uniformly throughout the extracellular matrix, i.e., without distinction between pericellular, territorial, and interterritorial matrices. Tissues that undergo endochondral bo...

Extracellular histones induce tissue factor expression in vascular endothelial cells via TLR and activation of NF-κB and AP-1.

Science.gov (United States)

Yang, Xinyu; Li, Lin; Liu, Jin; Lv, Ben; Chen, Fangping

2016-01-01

Extracellular histones have been recognized recently as proinflammatory mediators; they are released from dying cells in response to inflammatory challenge, contributing to endothelial cell dysfunction, thrombin formation, organ failure, and death during sepsis. Clinical studies suggest that the plasma concentration of the histone-DNA complex is correlated with the severity of DIC and is a poor independent prognostic marker in sepsis. In addition, platelet activation stimulates thrombus formation. Whether histones contribute to procoagulant activity in other ways remains elusive. In this study, we confirmed that histones induce tissue factor (TF) expression in a concentration- and time-dependent manner in vascular endothelial cells (ECs) and macrophages. However, histones did not affect TF pathway inhibitor expression. Moreover, blocking the cell surface receptors TLR4 and TLR2 with specific neutralizing antibodies significantly reduced histone-induced TF expression. Furthermore, histones enhanced the nuclear translocation of NF-κB (c-Rel/p65) and AP-1 expression in a time-dependent manner in ECs. Mutating NF-κB and AP-1 significantly reduced histone-induced TF expression. Altogether, our experiments suggest that histone induces TF expression in ECs via cell surface receptors TLR4 and TLR2, simultaneously depending on the activation of the transcription factors NF-κB and AP-1. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular regulation of CCN2 in the intervertebral disc: lessons learned from other connective tissues.

Science.gov (United States)

Tran, Cassie M; Shapiro, Irving M; Risbud, Makarand V

2013-08-08

Connective tissue growth factor (CCN2/CTGF) plays an important role in extracellular matrix synthesis, especially in skeletal tissues such as cartilage, bone, and the intervertebral disc. As a result there is a growing interest in examining the function and regulation of this important molecule in the disc. This review discusses the regulation of CCN2 by TGF-β and hypoxia, two critical determinants that characterize the disc microenvironment, and discusses known functions of CCN2 in the disc. The almost ubiquitous regulation of CCN2 by TGF-β, including that seen in the disc, emphasizes the importance of the TGF-β-CCN2 relationship, especially in terms of extracellular matrix synthesis. Likewise, the unique cross-talk between CCN2 and HIF-1 in the disc highlights the tissue and niche specific mode of regulation. Taken together the current literature supports an anabolic role for CCN2 in the disc and its involvement in the maintenance of tissue homeostasis during both health and disease. Further studies of CCN2 in this tissue may reveal valuable targets for the biological therapy of disc degeneration. © 2013 Elsevier B.V. All rights reserved.

Characterisation of tissue factor-bearing extracellular vesicles with AFM: comparison of air-tapping-mode AFM and liquid Peak Force AFM

Directory of Open Access Journals (Sweden)

Julie Hardij

2013-08-01

Full Text Available Introduction: Extracellular vesicles (EVs are shed from cells and carry markers of the parent cells. Vesicles derived from cancer cells reach the bloodstream and locally influence important physiological processes. It has been previously shown that procoagulant vesicles are circulating in patients’ fluids. These EVs are therefore considered as promising biomarkers for the thrombotic risk. Because of their small size, classical methods such as flow cytometry suffer from limitation for their characterisation. Atomic force microscopy (AFM has been proposed as a promising complementary method for the characterisation of EVs. Objectives: The objectives of this study are: (a to develop and validate AFM with specific antibodies (anti-TF and (b to compare air and liquid modes for EVs’ size and number determination as potential biomarkers of the prothrombotic risk. Methods: AFM multimode nanoscope III was used for air tapping mode (TM. AFM catalyst was used for liquid Peak Force Tapping (PFT mode. Vesicles are generated according to Davila et al.'s protocol. Substrates are coated with various concentrations of antibodies, thanks to ethanolamine and glutaraldehyde. Results: Vesicles were immobilised on antibody-coated surfaces to select tissue factor (TF-positive vesicles. The size range of vesicles observed in liquid PFT mode is 6–10 times higher than in air mode. This corresponds to the data found in the literature. Conclusion: We recommend liquid PFT mode to analyse vesicles on 5 µg/ml antibody-coated substrates.

Evaluation of pancreatic cancer by multiple breath-hold dynamic contrast-enhanced magnetic resonance imaging at 3.0 T

International Nuclear Information System (INIS)

Yao, Xiuzhong; Zeng, Mengsu; Wang, He; Sun, Fei; Rao, Shengxiang; Ji, Yuan

2012-01-01

Objective: To investigate the microcirculation in pancreatic cancer by pharmacokinetic analysis of multiple breath-hold dynamic contrast-enhanced magnetic resonance imaging at 3.0 T. Materials and methods: Multiple breath-hold dynamic contrast-enhanced magnetic resonance imaging was performed in 40 healthy volunteers and 40 patients with pancreatic cancer proven by histopathology using an axial three-dimensions fat-saturated T1-weighted spoiled-gradient echo sequence at 3.0 T. A two compartment model with T1 correction was used to quantify the transfer constant, the rate constant of backflux from the extravascular extracellular space to the plasma and the extravascular extracellular space fractional volume in pancreatic cancer, obstructive pancreatitis distal to the malignant tumor, adjacent pancreatic tissue proximal to the tumor and normal pancreas. All parameters were statistically analyzed. Results: Statistical differences were noticed in both the transfer constant (p = 0.000075) and the rate constant of backflux (p = 0.006) among different tissues. Both the transfer constant and the rate constant of backflux in pancreatic cancer were statistically lower than those in normal pancreas and adjacent pancreatic tissue (p < 0.05). Both the transfer constant and the rate constant of backflux in obstructive pancreatitis were statistically lower than those in normal pancreas and adjacent pancreatic tissue (p < 0.05). The extravascular extracellular space fractional volume in pancreatic cancer was statistically lager than that in normal pancreas (p = 0.002). Conclusion: Multiple breath-hold dynamic contrast-enhanced magnetic resonance imaging offers a useful technique to evaluate the microenvironment in pancreatic cancer at 3.0 T. Compared to normal pancreas, pancreatic cancer has lower transfer constant, rate constant of backflux and larger extravascular extracellular space fractional volume.

Evaluation of pancreatic cancer by multiple breath-hold dynamic contrast-enhanced magnetic resonance imaging at 3.0 T

Energy Technology Data Exchange (ETDEWEB)

Yao, Xiuzhong, E-mail: yao.xiuzhong@zs-hospital.sh.cn [Department of Radiology, Zhongshan Hospital of Fudan University and Department of Medical Image, Shanghai Medical College of Fudan University, No. 138, Fenglin Road, Xuhui District, Shanghai 200032 (China); Zeng, Mengsu, E-mail: zengmengsu@gmail.com [Department of Radiology, Zhongshan Hospital of Fudan University and Department of Medical Image, Shanghai Medical College of Fudan University, No. 138, Fenglin Road, Xuhui District, Shanghai 200032 (China); Wang, He, E-mail: herry258@hotmail.com [Global Applied Science Laboratory of GE Healthcare, No. 1, Huatuo Road, Zhangjiang Hi-tech Park, Pudong District, Shanghai 201203 (China); Sun, Fei, E-mail: fei.sun@med.ge.com [Global Applied Science Laboratory of GE Healthcare, No. 1, Huatuo Road, Zhangjiang Hi-tech Park, Pudong District, Shanghai 201203 (China); Rao, Shengxiang, E-mail: rao.shengxiang@zs-hospital.sh.cn [Department of Radiology, Zhongshan Hospital of Fudan University and Department of Medical Image, Shanghai Medical College of Fudan University, No. 138, Fenglin Road, Xuhui District, Shanghai 200032 (China); Ji, Yuan, E-mail: Ji.yuan@zs-hospital.sh.cn [Department of Pathology, Zhongshan Hospital of Fudan University, No. 138, Fenglin Road, Xuhui District, Shanghai 200032 (China)

2012-08-15

Objective: To investigate the microcirculation in pancreatic cancer by pharmacokinetic analysis of multiple breath-hold dynamic contrast-enhanced magnetic resonance imaging at 3.0 T. Materials and methods: Multiple breath-hold dynamic contrast-enhanced magnetic resonance imaging was performed in 40 healthy volunteers and 40 patients with pancreatic cancer proven by histopathology using an axial three-dimensions fat-saturated T1-weighted spoiled-gradient echo sequence at 3.0 T. A two compartment model with T1 correction was used to quantify the transfer constant, the rate constant of backflux from the extravascular extracellular space to the plasma and the extravascular extracellular space fractional volume in pancreatic cancer, obstructive pancreatitis distal to the malignant tumor, adjacent pancreatic tissue proximal to the tumor and normal pancreas. All parameters were statistically analyzed. Results: Statistical differences were noticed in both the transfer constant (p = 0.000075) and the rate constant of backflux (p = 0.006) among different tissues. Both the transfer constant and the rate constant of backflux in pancreatic cancer were statistically lower than those in normal pancreas and adjacent pancreatic tissue (p < 0.05). Both the transfer constant and the rate constant of backflux in obstructive pancreatitis were statistically lower than those in normal pancreas and adjacent pancreatic tissue (p < 0.05). The extravascular extracellular space fractional volume in pancreatic cancer was statistically lager than that in normal pancreas (p = 0.002). Conclusion: Multiple breath-hold dynamic contrast-enhanced magnetic resonance imaging offers a useful technique to evaluate the microenvironment in pancreatic cancer at 3.0 T. Compared to normal pancreas, pancreatic cancer has lower transfer constant, rate constant of backflux and larger extravascular extracellular space fractional volume.

Extracellular proteins: Novel key components of metal resistance in cyanobacteria?

Directory of Open Access Journals (Sweden)

Joaquin eGiner-Lamia

2016-06-01

Full Text Available Metals are essential for all living organisms and required for fundamental biochemical processes. However, when in excess, metals can turn into highly-toxic agents able to disrupt cell membranes, alter enzymatic activities and damage DNA. Metal concentrations are therefore tightly controlled inside cells, particularly in cyanobacteria. Cyanobacteria are ecologically relevant prokaryotes that perform oxygenic photosynthesis and can be found in many different marine and freshwater ecosystems, including environments contaminated with heavy metals. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been widely studied in cyanobacteria. So far, most studies have focused on how cells are capable of controlling their internal metal pools, with a strong bias towards the analysis of intracellular processes. Ultrastructure, modulation of physiology, dynamic changes in transcription and protein levels have been studied, but what takes place in the extracellular environment when cells are exposed to an unbalanced metal availability remains largely unknown. The interest in studying the subset of proteins present in the extracellular space has only recently begun and the identification and functional analysis of the cyanobacterial exoproteomes are just emerging. Remarkably, metal-related proteins such as the copper-chaperone CopM or the iron-binding protein FutA2 have already been identified outside the cell. With this perspective, we aim to raise the awareness that metal-resistance mechanisms are not yet fully known and hope to motivate future studies assessing the role of extracellular proteins on bacterial metal homeostasis, with a special focus on cyanobacteria.

Cells involved in extracellular matrix remodeling after acute myocardial infarction

International Nuclear Information System (INIS)

Garcia, Larissa Ferraz; Mataveli, Fábio D’Aguiar; Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell; Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva

2015-01-01

Evaluate the effects of VEGF_1_6_5 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF_1_6_5 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF_1_6_5. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF_1_6_5, suggesting greater tissue differentiation. The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF_1_6_5 seems to provide a protective effect in the treatment of acute myocardial infarct

Cells involved in extracellular matrix remodeling after acute myocardial infarction

Energy Technology Data Exchange (ETDEWEB)

Garcia, Larissa Ferraz [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Mataveli, Fábio D’Aguiar [Universidade Federal de São Paulo, São Paulo, SP (Brazil); Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva [Universidade Federal de São Paulo, São Paulo, SP (Brazil)

2015-07-01

Evaluate the effects of VEGF{sub 165} gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF{sub 165} treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF{sub 165}. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF{sub 165}, suggesting greater tissue differentiation. The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF{sub 165} seems to provide a protective effect in the treatment of acute myocardial infarct.

Extracellular adenosine controls NKT-cell-dependent hepatitis induction.

Science.gov (United States)

Subramanian, Meenakshi; Kini, Radhika; Madasu, Manasa; Ohta, Akiko; Nowak, Michael; Exley, Mark; Sitkovsky, Michail; Ohta, Akio

2014-04-01

Extracellular adenosine regulates inflammatory responses via the A2A adenosine receptor (A2AR). A2AR deficiency results in much exaggerated acute hepatitis, indicating nonredundancy of adenosine-A2AR pathway in inhibiting immune activation. To identify a critical target of immunoregulatory effect of extracellular adenosine, we focused on NKT cells, which play an indispensable role in hepatitis. An A2AR agonist abolished NKT-cell-dependent induction of acute hepatitis by concanavalin A (Con A) or α-galactosylceramide in mice, corresponding to downregulation of activation markers and cytokines in NKT cells and of NK-cell co-activation. These results show that A2AR signaling can downregulate NKT-cell activation and suppress NKT-cell-triggered inflammatory responses. Next, we hypothesized that NKT cells might be under physiological control of the adenosine-A2AR pathway. Indeed, both Con A and α-galactosylceramide induced more severe hepatitis in A2AR-deficient mice than in WT controls. Transfer of A2AR-deficient NKT cells into A2AR-expressing recipients resulted in exaggeration of Con A-induced liver damage, suggesting that NKT-cell activation is controlled by endogenous adenosine via A2AR, and this physiological regulatory mechanism of NKT cells is critical in the control of tissue-damaging inflammation. The current study suggests the possibility to manipulate NKT-cell activity in inflammatory disorders through intervention to the adenosine-A2AR pathway. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Extracellular miRNA-21 as a novel biomarker in glioma: evidence from meta-analysis, clinical validation and experimental investigations

Science.gov (United States)

Liu, Tian; Wang, Zhixin; Tai, Minghui; Meng, Fandi; Zhang, Jingyao; Wan, Yong; Mao, Ping; Dong, Xiaoqun; Liu, Chang; Niu, Wenquan; Dong, Shunbin

2016-01-01

Evidence is accumulating highlighting the importance of extracellular miRNA as a novel biomarker for diagnosing various kinds of malignancies. MiR-21 is one of the most studied miRNAs and is over-expressed in cancer tissues. To explore the clinical implications and secretory mechanisms of extracellular miR-21, we firstly meta-analyzed the diagnostic efficiency of extracellular miR-21 in different cancer types. Eighty-one studies based on 59 articles were finally included. In our study, extracellular miR-21 was observed to exhibit an outstanding diagnostic accuracy in detecting brain cancer (area under the summary receiver operating characteristic curve or AUC = 0.94), and this accuracy was more obvious in glioma diagnosis (AUC = 0.95). Our validation study (n = 45) further confirmed the diagnostic and prognostic role of miR-21 in cerebrospinal fluid (CSF) for glioma. These findings inspired us to explore the biological function of miR-21. We next conducted mechanistic investigations to explain the secretory mechanisms of extracellular miR-21 in glioma. TGF-β/Smad3 signaling was identified to participate in mediating the release of miR-21 from glioma cells. Further targeting TGF-β/Smad3 signaling using galunisertib, an inhibitor of the TGF-β type I receptor kinase, can attenuate the secretion of miR-21 from glioma cells. Taken together, CSF-based miR-21 might serve as a potential biomarker for diagnosing brain cancer, especially for patients with glioma. Moreover, extracellular levels of miR-21 were affected by exogenous TGF-β activity and galunisertib treatment. PMID:27166186

Extracellular peptidase hunting for improvement of protein production in plant cells and roots

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Jérôme eLallemand

2015-02-01

Full Text Available Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion, in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA and human serum immunoglobulins G (hIgGs. Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing.

A multi-scale controlled tissue engineering scaffold prepared by 3D printing and NFES technology

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Feifei Yan

2014-03-01

Full Text Available The current focus in the field of life science is the use of tissue engineering scaffolds to repair human organs, which has shown great potential in clinical applications. Extracellular matrix morphology and the performance and internal structure of natural organs are required to meet certain requirements. Therefore, integrating multiple processes can effectively overcome the limitations of the individual processes and can take into account the needs of scaffolds for the material, structure, mechanical properties and many other aspects. This study combined the biological 3D printing technology and the near-field electro-spinning (NFES process to prepare a multi-scale controlled tissue engineering scaffold. While using 3D printing technology to directly prepare the macro-scaffold, the compositing NFES process to build tissue micro-morphology ultimately formed a tissue engineering scaffold which has the specific extracellular matrix structure. This scaffold not only takes into account the material, structure, performance and many other requirements, but also focuses on resolving the controllability problems in macro- and micro-forming which further aim to induce cell directed differentiation, reproduction and, ultimately, the formation of target tissue organs. It has in-depth immeasurable significance to build ideal scaffolds and further promote the application of tissue engineering.

Targeting diseased tissues by pHLIP insertion at low cell surface pH

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Oleg A. Andreev

2014-03-01

Full Text Available The discovery of the pH Low Insertion Peptides (pHLIPs provides an opportunity to develop imaging and drug delivery agents targeting extracellular acidity. Extracellular acidity is associated with many pathological states, such as those in cancer, ischemic stroke, neurotrauma, infection, lacerations and others. The metabolism of cells in injured or diseased tissues often results in the acidification of the extracellular environment, so acidosis might be useful as a general marker for the imaging and treatment of diseased states if an effective targeting method can be developed. The molecular mechanism of a pHLIP peptide is based on pH-dependent membrane-associated folding. pHLIPs, being moderately hydrophobic peptides, have high affinities for cellular membranes at normal pH, but fold and insert across membranes at low pH, allowing them to sense pH at the surfaces of cells in diseased tissues, where it is the lowest. Here we discuss the main principles of pHLIP interactions with membrane lipid bilayers at neutral and low pHs, the possibility of tuning the folding and insertion pH by peptide sequence variation, and potential applications of pHLIPs for imaging, therapy and image-guided interventions.

Targeting diseased tissues by pHLIP insertion at low cell surface pH.

Science.gov (United States)

Andreev, Oleg A; Engelman, Donald M; Reshetnyak, Yana K

2014-01-01

The discovery of the pH Low Insertion Peptides (pHLIPs®) provides an opportunity to develop imaging and drug delivery agents targeting extracellular acidity. Extracellular acidity is associated with many pathological states, such as those in cancer, ischemic stroke, neurotrauma, infection, lacerations, and others. The metabolism of cells in injured or diseased tissues often results in the acidification of the extracellular environment, so acidosis might be useful as a general marker for the imaging and treatment of diseased states if an effective targeting method can be developed. The molecular mechanism of a pHLIP peptide is based on pH-dependent membrane-associated folding. pHLIPs, being moderately hydrophobic peptides, have high affinities for cellular membranes at normal pH, but fold and insert across membranes at low pH, allowing them to sense pH at the surfaces of cells in diseased tissues, where it is the lowest. Here we discuss the main principles of pHLIP interactions with membrane lipid bilayers at neutral and low pHs, the possibility of tuning the folding and insertion pH by peptide sequence variation, and potential applications of pHLIPs for imaging, therapy and image-guided interventions.

Microgravity cultivation of cells and tissues

Science.gov (United States)

Freed, L. E.; Pellis, N.; Searby, N.; de Luis, J.; Preda, C.; Bordonaro, J.; Vunjak-Novakovic, G.

1999-01-01

In vitro studies of cells and tissues in microgravity, either simulated by cultivation conditions on earth or actual, during spaceflight, are expected to help identify mechanisms underlying gravity sensing and transduction in biological organisms. In this paper, we review rotating bioreactor studies of engineered skeletal and cardiovascular tissues carried out in unit gravity, a four month long cartilage tissue engineering study carried out aboard the Mir Space Station, and the ongoing laboratory development and testing of a system for cell and tissue cultivation aboard the International Space Station.

The interplay between tissue growth and scaffold degradation in engineered tissue constructs

KAUST Repository

O’Dea, R. D.

2012-09-18

In vitro tissue engineering is emerging as a potential tool to meet the high demand for replacement tissue, caused by the increased incidence of tissue degeneration and damage. A key challenge in this field is ensuring that the mechanical properties of the engineered tissue are appropriate for the in vivo environment. Achieving this goal will require detailed understanding of the interplay between cell proliferation, extracellular matrix (ECM) deposition and scaffold degradation. In this paper, we use a mathematical model (based upon a multiphase continuum framework) to investigate the interplay between tissue growth and scaffold degradation during tissue construct evolution in vitro. Our model accommodates a cell population and culture medium, modelled as viscous fluids, together with a porous scaffold and ECM deposited by the cells, represented as rigid porous materials. We focus on tissue growth within a perfusion bioreactor system, and investigate how the predicted tissue composition is altered under the influence of (1) differential interactions between cells and the supporting scaffold and their associated ECM, (2) scaffold degradation, and (3) mechanotransduction-regulated cell proliferation and ECM deposition. Numerical simulation of the model equations reveals that scaffold heterogeneity typical of that obtained from μCT scans of tissue engineering scaffolds can lead to significant variation in the flow-induced mechanical stimuli experienced by cells seeded in the scaffold. This leads to strong heterogeneity in the deposition of ECM. Furthermore, preferential adherence of cells to the ECM in favour of the artificial scaffold appears to have no significant influence on the eventual construct composition; adherence of cells to these supporting structures does, however, lead to cell and ECM distributions which mimic and exaggerate the heterogeneity of the underlying scaffold. Such phenomena have important ramifications for the mechanical integrity of

Targeting Extracellular DNA to Deliver IGF-1 to the Injured Heart

Science.gov (United States)

Khan, Raffay S.; Martinez, Mario D.; Sy, Jay C.; Pendergrass, Karl D.; Che, Pao-Lin; Brown, Milton E.; Cabigas, E. Bernadette; Dasari, Madhuri; Murthy, Niren; Davis, Michael E.

2014-03-01

There is a great need for the development of therapeutic strategies that can target biomolecules to damaged myocardium. Necrosis of myocardium during a myocardial infarction (MI) is characterized by extracellular release of DNA, which can serve as a potential target for ischemic tissue. Hoechst, a histological stain that binds to double-stranded DNA can be conjugated to a variety of molecules. Insulin-like growth factor-1 (IGF-1), a small protein/polypeptide with a short circulating-half life is cardioprotective following MI but its clinical use is limited by poor delivery, as intra-myocardial injections have poor retention and chronic systemic presence has adverse side effects. Here, we present a novel delivery vehicle for IGF-1, via its conjugation to Hoechst for targeting infarcted tissue. Using a mouse model of ischemia-reperfusion, we demonstrate that intravenous delivery of Hoechst-IGF-1 results in activation of Akt, a downstream target of IGF-1 and protects from cardiac fibrosis and dysfunction following MI.

Variation in primary and culture-expanded cells derived from connective tissue progenitors in human bone marrow space, bone trabecular surface and adipose tissue.

Science.gov (United States)

Qadan, Maha A; Piuzzi, Nicolas S; Boehm, Cynthia; Bova, Wesley; Moos, Malcolm; Midura, Ronald J; Hascall, Vincent C; Malcuit, Christopher; Muschler, George F

2018-03-01

Connective tissue progenitors (CTPs) embody the heterogeneous stem and progenitor cell populations present in native tissue. CTPs are essential to the formation and remodeling of connective tissue and represent key targets for tissue-engineering and cell-based therapies. To better understand and characterize CTPs, we aimed to compare the (i) concentration and prevalence, (ii) early in vitro biological behavior and (iii) expression of surface-markers and transcription factors among cells derived from marrow space (MS), trabecular surface (TS), and adipose tissues (AT). Cancellous-bone and subcutaneous-adipose tissues were collected from 8 patients. Cells were isolated and cultured. Colony formation was assayed using Colonyze software based on ASTM standards. Cell concentration ([Cell]), CTP concentration ([CTP]) and CTP prevalence (P CTP ) were determined. Attributes of culture-expanded cells were compared based on (i) effective proliferation rate and (ii) expression of surface-markers CD73, CD90, CD105, SSEA-4, SSEA-3, SSEA-1/CD15, Cripto-1, E-Cadherin/CD324, Ep-CAM/CD326, CD146, hyaluronan and transcription factors Oct3/4, Sox-2 and Nanog using flow cytometry. Mean [Cell], [CTP] and P CTP were significantly different between MS and TS samples (P = 0.03, P = 0.008 and P= 0.0003), respectively. AT-derived cells generated the highest mean total cell yield at day 6 of culture-4-fold greater than TS and more than 40-fold greater than MS per million cells plated. TS colonies grew with higher mean density than MS colonies (290 ± 11 versus 150 ± 11 cell per mm 2 ; P = 0.0002). Expression of classical-mesenchymal stromal cell (MSC) markers was consistently recorded (>95%) from all tissue sources, whereas all the other markers were highly variable. The prevalence and biological potential of CTPs are different between patients and tissue sources and lack variation in classical MSC markers. Other markers are more likely to discriminate differences

Use of integrin-linked kinase to extend function of encapsulated pancreatic tissue

International Nuclear Information System (INIS)

Blanchette, James O; Langer, Steven J; Leinwand, Leslie L; Sahai, Suchit; Topiwala, Pritesh S; Anseth, Kristi S

2010-01-01

We have studied the impact of overexpression of an intracellular signaling protein, integrin-linked kinase (ILK), on the survival and function of encapsulated islet tissue used for the treatment of type 1 diabetes. The dimensions of the encapsulated tissue can impact the stresses placed on the tissue and ILK overexpression shows the ability to extend function of dissociated cells as well as intact islets. These results suggest that lost cell-extracellular matrix interactions in cell encapsulation systems can lead to decreased insulin secretion and ILK signaling is a target to overcome this phenomenon. (communication)

Use of integrin-linked kinase to extend function of encapsulated pancreatic tissue

Energy Technology Data Exchange (ETDEWEB)

Blanchette, James O [Department of Chemical Engineering, University of South Carolina, Columbia, SC (United States); Langer, Steven J; Leinwand, Leslie L [Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO (United States); Sahai, Suchit; Topiwala, Pritesh S [Biomedical Engineering Program, University of South Carolina, Columbia, SC (United States); Anseth, Kristi S, E-mail: blanchej@cec.sc.ed [Howard Hughes Medical Institute, Boulder, CO (United States)

2010-12-15

We have studied the impact of overexpression of an intracellular signaling protein, integrin-linked kinase (ILK), on the survival and function of encapsulated islet tissue used for the treatment of type 1 diabetes. The dimensions of the encapsulated tissue can impact the stresses placed on the tissue and ILK overexpression shows the ability to extend function of dissociated cells as well as intact islets. These results suggest that lost cell-extracellular matrix interactions in cell encapsulation systems can lead to decreased insulin secretion and ILK signaling is a target to overcome this phenomenon. (communication)

Lung tissue remodeling in the acute respiratory distress syndrome

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Souza Alba Barros de

2003-01-01

Full Text Available Acute respiratory distress syndrome (ARDS is characterized by diffuse alveolar damage, and evolves progressively with three phases: exsudative, fibroproliferative, and fibrotic. In the exudative phase, there are interstitial and alveolar edemas with hyaline membrane. The fibropro­liferative phase is characterized by exudate organization and fibroelastogenesis. There is proliferation of type II pneumocytes to cover the damaged epithelial surface, followed by differentiation into type I pneumocytes. The fibroproliferative phase starts early, and its severity is related to the patient?s prognosis. The alterations observed in the phenotype of the pulmonary parenchyma cells steer the tissue remodeling towards either progressive fibrosis or the restoration of normal alveolar architecture. The fibrotic phase is characterized by abnormal and excessive deposition of extracellular matrix proteins, mainly collagen. The dynamic control of collagen deposition and degradation is regulated by metalloproteinases and their tissular regulators. The deposition of proteoglycans in the extracellular matrix of ARDS patients needs better study. The regulation of extracellular matrix remodeling, in normal conditions or in several pulmonary diseases, such as ARDS, results from a complex mechanism that integrate the transcription of elements that destroy the matrix protein and produce activation/inhibition of several cellular types of lung tissue. This review article will analyze the ECM organization in ARDS, the different pulmonary parenchyma remodeling mechanisms, and the role of cytokines in the regulation of the different matrix components during the remodeling process.

Experiment K-7-29: Connective Tissue Studies. Part 3; Rodent Tissue Repair: Skeletal Muscle

Science.gov (United States)

Stauber, W.; Fritz, V. K.; Burkovskaya, T. E.; Ilyina-Kakueva, E. I.

1994-01-01

Myofiber injury-repair was studied in the rat gastrocnemius following a crush injury to the lower leg prior to flight in order to understand if the regenerative responses of muscles are altered by the lack of gravitational forces during Cosmos 2044 flight. After 14 days of flight, the gastrocnemius muscle was removed from the 5 injured flight rodents and various Earth-based treatment groups for comparison. The Earth-based animals consisted of three groups of five rats with injured muscles from a simulated, tail-suspended, and vivarium as well as an uninjured basal group. The gastrocnemius muscle from each was evaluated by histochemical and immunohistochemical techniques to document myofiber, vascular, and connective tissue alterations following injury. In general the repair process was somewhat similar in all injured muscle samples with regard to extracellular matrix organization and myofiber regeneration. Small and large myofibers were present with a newly organized extracellular matrix indicative of myogenesis and muscle regeneration. In the tail-suspended animals, a more complete repair was observed with no enlarged area of non-muscle cells or matrix material visible. In contrast, the muscle samples from the flight animals were less well differentiated with more macrophages and blood vessels in the repair region but small myofibers and proteoglycans, nevertheless, were in their usual configuration. Thus, myofiber repair did vary in muscles from the different groups, but for the most part, resulted in functional muscle tissue.

Biomimetic extracellular matrix mediated somatic stem cell differentiation: applications in dental pulp tissue regeneration

Science.gov (United States)

Ravindran, Sriram; George, Anne

2015-01-01

Dental caries is one of the most widely prevalent infectious diseases in the world. It affects more than half of the world's population. The current treatment for necrotic dental pulp tissue arising from dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality making it prone for secondary infections. Over the past decade, several tissue-engineering approaches have attempted regeneration of the dental pulp tissue. Although several studies have highlighted the potential of dental stem cells, none have transitioned into a clinical setting owing to limited availability of dental stem cells and the need for growth factor delivery systems. Our strategy is to utilize the intact ECM of pulp cells to drive lineage specific differentiation of bone marrow derived mesenchymal stem cells. From a clinical perspective, pulp ECM scaffolds can be generated using cell lines and patient specific somatic stem cells can be used for regeneration. Our published results have shown the feasibility of using pulp ECM scaffolds for odontogenic differentiation of non-dental mesenchymal cells. This focused review discusses the issues surrounding dental pulp tissue regeneration and the potential of our strategy to overcome these issues. PMID:25954205

Measurement of the Extracellular pH of Adherently Growing Mammalian Cells with High Spatial Resolution Using a Voltammetric pH Microsensor.

Science.gov (United States)

Munteanu, Raluca-Elena; Stǎnicǎ, Luciana; Gheorghiu, Mihaela; Gáspár, Szilveszter

2018-05-15

There are only a few tools suitable for measuring the extracellular pH of adherently growing mammalian cells with high spatial resolution, and none of them is widely used in laboratories around the world. Cell biologists very often limit themselves to measuring the intracellular pH with commercially available fluorescent probes. Therefore, we built a voltammetric pH microsensor and investigated its suitability for monitoring the extracellular pH of adherently growing mammalian cells. The voltammetric pH microsensor consisted of a 37 μm diameter carbon fiber microelectrode modified with reduced graphene oxide and syringaldazine. While graphene oxide was used to increase the electrochemically active surface area of our sensor, syringaldazine facilitated pH sensing through its pH-dependent electrochemical oxidation and reduction. The good sensitivity (60 ± 2.5 mV/pH unit), reproducibility (coefficient of variation ≤3% for the same pH measured with 5 different microsensors), and stability (pH drift around 0.05 units in 3 h) of the built voltammetric pH sensors were successfully used to investigate the acidification of the extracellular space of both cancer cells and normal cells. The results indicate that the developed pH microsensor and the perfected experimental protocol based on scanning electrochemical microscopy can reveal details of the pH regulation of cells not attainable with pH sensors lacking spatial resolution or which cannot be reproducibly positioned in the extracellular space.

Plasma biomarker discovery in preeclampsia using a novel differential isolation technology for circulating extracellular vesicles.

Science.gov (United States)

Tan, Kok Hian; Tan, Soon Sim; Sze, Siu Kwan; Lee, Wai Kheong Ryan; Ng, Mor Jack; Lim, Sai Kiang

2014-10-01

To circumvent the complex protein milieu of plasma and discover robust predictive biomarkers for preeclampsia (PE), we investigate if phospholipid-binding ligands can reduce the milieu complexity by extracting plasma extracellular vesicles for biomarker discovery. Cholera toxin B chain (CTB) and annexin V (AV) which respectively binds GM1 ganglioside and phosphatidylserine were used to isolate extracellular vesicles from plasma of PE patients and healthy pregnant women. The proteins in the vesicles were identified using enzyme-linked immunosorbent assay, antibody array, and mass spectrometry. CTB and AV were found to bind 2 distinct groups of extracellular vesicles. Antibody array and enzyme-linked immunosorbent assay revealed that PE patients had elevated levels of CD105, interleukin-6, placental growth factor, tissue inhibitor of metallopeptidase 1, and atrial natriuretic peptide in cholera toxin B- but not AV-vesicles, and elevated levels of plasminogen activator inhibitor-1, pro-calcitonin, S100b, tumor growth factor β, vascular endothelial growth factor receptor 1, brain natriuretic peptide, and placental growth factor in both cholera toxin B- and AV-vesicles. CD9 level was elevated in cholera toxin B-vesicles but reduced in AV vesicles of PE patients. Proteome analysis revealed that in cholera toxin B-vesicles, 87 and 222 proteins were present only in PE patients and healthy pregnant women respectively while in AV-vesicles, 104 and 157 proteins were present only in PE and healthy pregnant women, respectively. This study demonstrated for the first time that CTB and AV bind unique extracellular vesicles, and their protein cargo reflects the disease state of the patient. The successful use of these 2 ligands to isolate circulating plasma extracellular vesicles for biomarker discovery in PE represents a novel technology for biomarker discovery that can be applied to other specialties. Copyright © 2014 Elsevier Inc. All rights reserved.

Biotechnological experiments in space flights on board of space stations

Science.gov (United States)

Nechitailo, Galina S.

2012-07-01

Space flight conditions are stressful for any plant and cause structural-functional transition due to mobiliation of adaptivity. In space flight experiments with pea tissue, wheat and arabidopsis we found anatomical-morphological transformations and biochemistry of plants. In following experiments, tissue of stevia (Stevia rebaudiana), potato (Solanum tuberosum), callus culture and culture and bulbs of suffron (Crocus sativus), callus culture of ginseng (Panax ginseng) were investigated. Experiments with stevia carried out in special chambers. The duration of experiment was 8-14 days. Board lamp was used for illumination of the plants. After experiment the plants grew in the same chamber and after 50 days the plants were moved into artificial ionexchange soil. The biochemical analysis of plants was done. The total concentration of glycozides and ratio of stevioside and rebauside were found different in space and ground plants. In following generations of stevia after flight the total concentration of stevioside and rebauside remains higher than in ground plants. Experiments with callus culture of suffron carried out in tubes. Duration of space flight experiment was 8-167 days. Board lamp was used for illumination of the plants. We found picrocitina pigment in the space plants but not in ground plants. Tissue culture of ginseng was grown in special container in thermostate under stable temperature of 22 ± 0,5 C. Duration of space experiment was from 8 to 167 days. Biological activity of space flight culutre was in 5 times higher than the ground culture. This difference was observed after recultivation of space flight samples on Earth during year after flight. Callus tissue of potato was grown in tubes in thermostate under stable temperature of 22 ± 0,5 C. Duration of space experiment was from 8 to 14 days. Concentration of regenerates in flight samples was in 5 times higher than in ground samples. The space flight experiments show, that microgravity and other

Modeling the Human Scarred Heart In Vitro : Toward New Tissue Engineered Models

NARCIS (Netherlands)

Deddens, Janine C.; Sadeghi, Amir Hossein; Hjortnaes, Jesper; van Laake, Linda W.; Buijsrogge, Marc; Doevendans, Pieter A.; Khademhosseini, Ali; Sluijter, Joost P G

2017-01-01

Cardiac remodeling is critical for effective tissue healing, however, excessive production and deposition of extracellular matrix components contribute to scarring and failing of the heart. Despite the fact that novel therapies have emerged, there are still no lifelong solutions for this problem. An

Composite microsphere-functionalized scaffold for the controlled release of small molecules in tissue engineering

Directory of Open Access Journals (Sweden)

Laura Pandolfi

2016-01-01

Full Text Available Current tissue engineering strategies focus on restoring damaged tissue architectures using biologically active scaffolds. The ideal scaffold would mimic the extracellular matrix of any tissue of interest, promoting cell proliferation and de novo extracellular matrix deposition. A plethora of techniques have been evaluated to engineer scaffolds for the controlled and targeted release of bioactive molecules to provide a functional structure for tissue growth and remodeling, as well as enhance recruitment and proliferation of autologous cells within the implant. Recently, novel approaches using small molecules, instead of growth factors, have been exploited to regulate tissue regeneration. The use of small synthetic molecules could be very advantageous because of their stability, tunability, and low cost. Herein, we propose a chitosan–gelatin scaffold functionalized with composite microspheres consisting of mesoporous silicon microparticles and poly(dl-lactic-co-glycolic acid for the controlled release of sphingosine-1-phospate, a small molecule of interest. We characterized the platform with scanning electron microscopy, Fourier transform infrared spectroscopy, and confocal microscopy. Finally, the biocompatibility of this multiscale system was analyzed by culturing human mesenchymal stem cells onto the scaffold. The presented strategy establishes the basis of a versatile scaffold for the controlled release of small molecules and for culturing mesenchymal stem cells for regenerative medicine applications.

Collagen Quantification in Tissue Specimens.

Science.gov (United States)

Coentro, João Quintas; Capella-Monsonís, Héctor; Graceffa, Valeria; Wu, Zhuning; Mullen, Anne Maria; Raghunath, Michael; Zeugolis, Dimitrios I

2017-01-01

Collagen is the major extracellular protein in mammals. Accurate quantification of collagen is essential in the biomaterials (e.g., reproducible collagen scaffold fabrication), drug discovery (e.g., assessment of collagen in pathophysiologies, such as fibrosis), and tissue engineering (e.g., quantification of cell-synthesized collagen) fields. Although measuring hydroxyproline content is the most widely used method to quantify collagen in biological specimens, the process is very laborious. To this end, the Sircol™ Collagen Assay is widely used due to its inherent simplicity and convenience. However, this method leads to overestimation of collagen content due to the interaction of Sirius red with basic amino acids of non-collagenous proteins. Herein, we describe the addition of an ultrafiltration purification step in the process to accurately determine collagen content in tissues.

The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles.

Science.gov (United States)

Lässer, Cecilia; Théry, Clotilde; Buzás, Edit I; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; Lötvall, Jan

2016-01-01

The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, "Basics of Extracellular Vesicles," uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform "Coursera" and is free of charge.

Bacterial binding to extracellular proteins - in vitro adhesion

DEFF Research Database (Denmark)

Schou, C.; Fiehn, N.-E.

1999-01-01

Viridans streptococci, bacterial adherence, extracellular matrix proteins, surface receptors, endocarditis......Viridans streptococci, bacterial adherence, extracellular matrix proteins, surface receptors, endocarditis...

Illustration of extensive extracellular matrix at the epithelial-mesenchymal interface within the renal stem/progenitor cell niche

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Minuth Will W

2012-09-01

Full Text Available Abstract Background Stem/progenitor cells are promising candidates to treat diseased renal parenchyma. However, implanted stem/progenitor cells are exposed to a harmful atmosphere of degenerating parenchyma. To minimize hampering effects after an implantation investigations are in progress to administer these cells within an artificial polyester interstitum supporting survival. Learning from nature the renal stem/progenitor cell niche appears as a valuable model. At this site epithelial stem/progenitor cells within the collecting duct ampulla face mesenchymal stem/progenitor cells. Both cell types do not have close contact but are separated by a wide interstitium. Methods To analyze extracellular matrix in this particular interstitium, special contrasting for transmission electron microscopy was performed. Kidneys of neonatal rabbits were fixed in solutions containing glutaraldehyde (GA or in combination with cupromeronic blue, ruthenium red and tannic acid. Results GA revealed a basal lamina at the ampulla and a bright but inconspicuously looking interstitial space. In contrast, GA containing cupromeronic blue exhibits numerous proteoglycan braces lining from the ampulla towards the interstitial space. GA containing ruthenium red or tannic acid demonstrates clouds of extracellular matrix protruding from the basal lamina of the ampulla to the surface of mesenchymal stem/progenitor cells. Conclusions The actual data show that the interstitium between epithelial and mesenchymal stem/progenitor cells contains much more and up to date unknown extracellular matrix than earlier observed by classical GA fixation.

Physical, Spatial, and Molecular Aspects of Extracellular Matrix of In Vivo Niches and Artificial Scaffolds Relevant to Stem Cells Research

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Maria Akhmanova

2015-01-01

Full Text Available Extracellular matrix can influence stem cell choices, such as self-renewal, quiescence, migration, proliferation, phenotype maintenance, differentiation, or apoptosis. Three aspects of extracellular matrix were extensively studied during the last decade: physical properties, spatial presentation of adhesive epitopes, and molecular complexity. Over 15 different parameters have been shown to influence stem cell choices. Physical aspects include stiffness (or elasticity, viscoelasticity, pore size, porosity, amplitude and frequency of static and dynamic deformations applied to the matrix. Spatial aspects include scaffold dimensionality (2D or 3D and thickness; cell polarity; area, shape, and microscale topography of cell adhesion surface; epitope concentration, epitope clustering characteristics (number of epitopes per cluster, spacing between epitopes within cluster, spacing between separate clusters, cluster patterns, and level of disorder in epitope arrangement, and nanotopography. Biochemical characteristics of natural extracellular matrix molecules regard diversity and structural complexity of matrix molecules, affinity and specificity of epitope interaction with cell receptors, role of non-affinity domains, complexity of supramolecular organization, and co-signaling by growth factors or matrix epitopes. Synergy between several matrix aspects enables stem cells to retain their function in vivo and may be a key to generation of long-term, robust, and effective in vitro stem cell culture systems.

Gene Expression Changes in Femoral Head Necrosis of Human Bone Tissue

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Bernadett Balla

2011-01-01

Full Text Available Osteonecrosis of the femoral head (ONFH is the result of an interruption of the local circulation and the injury of vascular supply of bone. Multiple factors have been implicated in the development of the disease. However the mechanism of ischemia and necrosis in non-traumatic ONFH is not clear. The aim of our investigation was to identify genes that are differently expressed in ONFH vs. non-ONFH human bone and to describe the relationships between these genes using multivariate data analysis. Six bone tissue samples from ONFH male patients and 8 bone tissue samples from non-ONFH men were examined. The expression differences of selected 117 genes were analyzed by TaqMan probe-based quantitative real-time RT-PCR system. The significance test indicated marked differences in the expression of nine genes between ONFH and non-ONFH individuals. These altered genes code for collagen molecules, an extracellular matrix digesting metalloproteinase, a transcription factor, an adhesion molecule, and a growth factor. Canonical variates analysis demonstrated that ONFH and non-ONFH bone tissues can be distinguished by the multiple expression profile analysis of numerous genes controlled via canonical TGFB pathway as well as genes coding for extracellular matrix composing collagen type molecules. The markedly altered gene expression profile observed in the ONFH of human bone tissue may provide further insight into the pathogenetic process of osteonecrotic degeneration of bone.

Electrospun conductive nanofibrous scaffolds for engineering cardiac tissue and 3D bioactuators.

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Wang, Ling; Wu, Yaobin; Hu, Tianli; Guo, Baolin; Ma, Peter X

2017-09-01

Mimicking the nanofibrous structure similar to extracellular matrix and conductivity for electrical propagation of native myocardium would be highly beneficial for cardiac tissue engineering and cardiomyocytes-based bioactuators. Herein, we developed conductive nanofibrous sheets with electrical conductivity and nanofibrous structure composed of poly(l-lactic acid) (PLA) blending with polyaniline (PANI) for cardiac tissue engineering and cardiomyocytes-based 3D bioactuators. Incorporating of varying contents of PANI from 0wt% to 3wt% into the PLA polymer, the electrospun nanofibrous sheets showed enhanced conductivity while maintaining the same fiber diameter. These PLA/PANI conductive nanofibrous sheets exhibited good cell viability and promoting effect on differentiation of H9c2 cardiomyoblasts in terms of maturation index and fusion index. Moreover, PLA/PANI nanofibrous sheets enhanced the cell-cell interaction, maturation and spontaneous beating of primary cardiomyocytes. Furthermore, the cardiomyocytes-laden PLA/PANI conductive nanofibrous sheets can form 3D bioactuators with tubular and folding shapes, and spontaneously beat with much higher frequency and displacement than that on cardiomyocytes-laden PLA nanofibrous sheets. Therefore, these PLA/PANI conductive nanofibrous sheets with conductivity and extracellular matrix like nanostructure demonstrated promising potential in cardiac tissue engineering and cardiomyocytes-based 3D bioactuators. Cardiomyocytes-based bioactuators have been paid more attention due to their spontaneous motion by integrating cardiomyocytes into polymer structures, but developing suitable scaffolds for bioactuators remains challenging. Electrospun nanofibrous scaffolds have been widely used in cardiac tissue engineering because they can mimic the extracellular matrix of myocardium. Developing conductive nanofibrous scaffolds by electrospinning would be beneficial for cardiomyocytes-based bioactuators, but such scaffolds have been

Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA

International Nuclear Information System (INIS)

Spicer, E.K.; Horton, R.; Bloem, L.

1987-01-01

Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. λ Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC)

Simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell cultures and in sub-regions of guinea pig brain

DEFF Research Database (Denmark)

Schou-Pedersen, Anne Marie Voigt; Hansen, Stine Normann; Tveden-Nyborg, Pernille

2016-01-01

In the present paper, we describe a validated chromatographic method for the simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell culture and in sub-regions of the guinea pig brain. Electrochemical...... of intracellular and extracellular amounts of monoamine neurotransmitters and their metabolites in guinea pig frontal cortex and hippocampal primary neuronal cell cultures. Noradrenaline, dopamine and serotonin were found to be in a range from 0.31 to 1.7 pmol per 2 million cells intracellularly, but only...... the biogenic metabolites could be detected extracellularly. Distinct differences in monoamine concentrations were observed when comparing concentrations in guinea pig frontal cortex and cerebellum tissue with higher amounts of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid...

Quantitative modeling of clinical, cellular, and extracellular matrix variables suggest prognostic indicators in cancer: a model in neuroblastoma.

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Tadeo, Irene; Piqueras, Marta; Montaner, David; Villamón, Eva; Berbegall, Ana P; Cañete, Adela; Navarro, Samuel; Noguera, Rosa

2014-02-01

Risk classification and treatment stratification for cancer patients is restricted by our incomplete picture of the complex and unknown interactions between the patient's organism and tumor tissues (transformed cells supported by tumor stroma). Moreover, all clinical factors and laboratory studies used to indicate treatment effectiveness and outcomes are by their nature a simplification of the biological system of cancer, and cannot yet incorporate all possible prognostic indicators. A multiparametric analysis on 184 tumor cylinders was performed. To highlight the benefit of integrating digitized medical imaging into this field, we present the results of computational studies carried out on quantitative measurements, taken from stromal and cancer cells and various extracellular matrix fibers interpenetrated by glycosaminoglycans, and eight current approaches to risk stratification systems in patients with primary and nonprimary neuroblastoma. New tumor tissue indicators from both fields, the cellular and the extracellular elements, emerge as reliable prognostic markers for risk stratification and could be used as molecular targets of specific therapies. The key to dealing with personalized therapy lies in the mathematical modeling. The use of bioinformatics in patient-tumor-microenvironment data management allows a predictive model in neuroblastoma.

Bioreactor-induced mesenchymal progenitor cell differentiation and elastic fiber assembly in engineered vascular tissues.

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Lin, Shigang; Mequanint, Kibret

2017-09-01

In vitro maturation of engineered vascular tissues (EVT) requires the appropriate incorporation of smooth muscle cells (SMC) and extracellular matrix (ECM) components similar to native arteries. To this end, the aim of the current study was to fabricate 4mm inner diameter vascular tissues using mesenchymal progenitor cells seeded into tubular scaffolds. A dual-pump bioreactor operating either in perfusion or pulsatile perfusion mode was used to generate physiological-like stimuli to promote progenitor cell differentiation, extracellular elastin production, and tissue maturation. Our data demonstrated that pulsatile forces and perfusion of 3D tubular constructs from both the lumenal and ablumenal sides with culture media significantly improved tissue assembly, effectively inducing mesenchymal progenitor cell differentiation to SMCs with contemporaneous elastin production. With bioreactor cultivation, progenitor cells differentiated toward smooth muscle lineage characterized by the expression of smooth muscle (SM)-specific markers smooth muscle alpha actin (SM-α-actin) and smooth muscle myosin heavy chain (SM-MHC). More importantly, pulsatile perfusion bioreactor cultivation enhanced the synthesis of tropoelastin and its extracellular cross-linking into elastic fiber compared with static culture controls. Taken together, the current study demonstrated progenitor cell differentiation and vascular tissue assembly, and provides insights into elastin synthesis and assembly to fibers. Incorporation of elastin into engineered vascular tissues represents a critical design goal for both mechanical and biological functions. In the present study, we seeded porous tubular scaffolds with multipotent mesenchymal progenitor cells and cultured in dual-pump pulsatile perfusion bioreactor. Physiological-like stimuli generated by bioreactor not only induced mesenchymal progenitor cell differentiation to vascular smooth muscle lineage but also actively promoted elastin synthesis and

Excessive Extracellular ATP Desensitizes P2Y2 and P2X4 ATP Receptors Provoking Surfactant Impairment Ending in Ventilation-Induced Lung Injury

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Djo Hasan

2018-04-01

Full Text Available Stretching the alveolar epithelial type I (AT I cells controls the intercellular signaling for the exocytosis of surfactant by the AT II cells through the extracellular release of adenosine triphosphate (ATP (purinergic signaling. Extracellular ATP is cleared by extracellular ATPases, maintaining its homeostasis and enabling the lung to adapt the exocytosis of surfactant to the demand. Vigorous deformation of the AT I cells by high mechanical power ventilation causes a massive release of extracellular ATP beyond the clearance capacity of the extracellular ATPases. When extracellular ATP reaches levels >100 μM, the ATP receptors of the AT II cells become desensitized and surfactant impairment is initiated. The resulting alteration in viscoelastic properties and in alveolar opening and collapse time-constants leads to alveolar collapse and the redistribution of inspired air from the alveoli to the alveolar ducts, which become pathologically dilated. The collapsed alveoli connected to these dilated alveolar ducts are subject to a massive strain, exacerbating the ATP release. After reaching concentrations >300 μM extracellular ATP acts as a danger-associated molecular pattern, causing capillary leakage, alveolar space edema, and further deactivation of surfactant by serum proteins. Decreasing the tidal volume to 6 mL/kg or less at this stage cannot prevent further lung injury.

Why regenerative medicine needs an extracellular matrix.

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Prestwich, Glenn D; Healy, Kevin E

2015-01-01

Regenerative medicine is now coming of age. Many attempts at cell therapy have failed to show significant efficacy, and the umbrella term 'stem cell therapy' is perceived in some quarters as hype or just expensive and unnecessary medical tourism. Here we present a short editorial in three parts. First, we examine the importance of using a semisynthetic extracellular matrix (ECM) mimetic, or sECM, to deliver and retain therapeutic cells at the site of administration. Second, we describe one approach in which biophysical and biochemical properties are tailored to each tissue type, which we call "design for optimal functionality." Third, we describe an alternative approach to sECM design and implementation, called "design for simplicity," in which a deconstructed, minimalist sECM is employed and biology is allowed to perform the customization in situ. We opine that an sECM, whether minimal or instructive, is an essential contributor to improve the outcomes of cell-based therapies.

Towards integrating extracellular matrix and immunological pathways.

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Boyd, David F; Thomas, Paul G

2017-10-01

The extracellular matrix (ECM) is a complex and dynamic structure made up of an estimated 300 different proteins. The ECM is also a rich source of cytokines and growth factors in addition to numerous bioactive ECM degradation products that influence cell migration, proliferation, and differentiation. The ECM is constantly being remodeled during homeostasis and in a wide range of pathological contexts. Changes in the ECM modulate immune responses, which in turn regulate repair and regeneration of tissues. Here, we review the many components of the ECM, enzymes involved in ECM remodeling, and the signals that feed into immunological pathways in the context of a dynamic ECM. We highlight studies that have taken an integrative approach to studying immune responses in the context of the ECM and studies that use novel proteomic strategies. Finally, we discuss research challenges relevant to the integration of immune and ECM networks and propose experimental and translational approaches to resolve these issues. Copyright © 2017 Elsevier Ltd. All rights reserved.

Extracellular Matrix-Regulated Gene Expression RequiresCooperation of SWI/SNF and Transcription Factors

Energy Technology Data Exchange (ETDEWEB)

Xu, Ren; Spencer, Virginia A.; Bissell, Mina J.

2006-05-25

Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation (ChIP) assays and mammary-specific genes as models, we show here that extracellular matrix (ECM) molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the {beta}- and ?-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both {beta}- and ?-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. ChIP analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Coimmunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, C/EBP{beta}, and glucocorticoid receptor (GR). Thus, ECM- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors.

Extracellular matrix of cultured glial cells: Selective expression of chondroitin 4-sulfate by type-2 astrocytes and their progenitors

International Nuclear Information System (INIS)

Gallo, V.; Bertolotto, A.

1990-01-01

We have studied the extracellular matrix composition of cultured glial cells by immunocytochemistry with different monoclonal and polyclonal antibodies. Double immunofluorescence experiments and metabolic labeling with [3H]glucosamine performed in different types of cerebellar and cortical cultures showed that bipotential progenitors for type-2 astrocytes and for oligodendrocytes synthesize chondroitin sulfate (CS) and deposit this proteoglycan in their extracellular matrix. The distribution of the various [3H]glucosamine-labeled glycosaminoglycans between the intracellular and the extracellular space was different. CS was present both within the cells and in the culture medium, although in different amounts. Bi-potential progenitors became also O4-positive during their development in vitro. At the stage of O4-positivity they were still stained with antibodies against CS. However, when the progenitor cells were maintained in serum-free medium and differentiated into Gal-C-positive oligodendrocytes, they became CS-negative. In the presence of fetal calf serum in the culture medium, the bipotential progenitors differentiated into GFAP-positive type-2 astrocytes. These cells still expressed CS: their Golgi area and their surface were stained with anti-CS antibodies. Staining with monoclonal antibodies specific for different types of CS (4-sulfate, 6-sulfate, and unsulfated) revealed that both bipotential progenitors and type-2 astrocytes synthesized only chondroitin 4-sulfate. Type-1 astrocytes were negative for both the polyclonal and the monoclonal anti-CS antibodies. Finally, type-2 astrocytes and their progenitors were weakly stained with anti-laminin antibodies and unstained with anti-fibronectin. Type-1 astrocytes were positive for both anti-laminin and anti-fibronectin antibodies and appeared to secrete fibronectin in the extracellular space

Characterization of yeast extracellular vesicles: evidence for the participation of different pathways of cellular traffic in vesicle biogenesis.

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Débora L Oliveira

2010-06-01

Full Text Available Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown.We characterized extracellular vesicle production in wild type (WT and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100-300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex or MVB functionality (vps23, late endosomal trafficking revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells.Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the

In vivo imaging of cerebral hemodynamics and tissue scattering in rat brain using a surgical microscope camera system

Science.gov (United States)

Nishidate, Izumi; Kanie, Takuya; Mustari, Afrina; Kawauchi, Satoko; Sato, Shunichi; Sato, Manabu; Kokubo, Yasuaki

2018-02-01

We investigated a rapid imaging method to monitor the spatial distribution of total hemoglobin concentration (CHbT), the tissue oxygen saturation (StO2), and the scattering power b in the expression of musp=a(lambda)^-b as the scattering parameters in cerebral cortex using a digital red-green-blue camera. In the method, Monte Carlo simulation (MCS) for light transport in brain tissue is used to specify a relation among the RGB-values and the concentration of oxygenated hemoglobin (CHbO), that of deoxygenated hemoglobin (CHbR), and the scattering power b. In the present study, we performed sequential recordings of RGB images of in vivo exposed brain of rats while changing the fraction of inspired oxygen (FiO2), using a surgical microscope camera system. The time courses of CHbO, CHbR, CHbT, and StO2 indicated the well-known physiological responses in cerebral cortex. On the other hand, a fast decrease in the scattering power b was observed immediately after the respiratory arrest, which is similar to the negative deflection of the extracellular DC potential so-called anoxic depolarization. It is said that the DC shift coincident with a rise in extracellular potassium and can evoke cell deformation generated by water movement between intracellular and extracellular compartments, and hence the light scattering by tissue. Therefore, the decrease in the scattering power b after the respiratory arrest is indicative of changes in light scattering by tissue. The results in this study indicate potential of the method to evaluate the pathophysiological conditions and loss of tissue viability in brain tissue.

Rapid clearance of xanthines from airway and pulmonary tissues

International Nuclear Information System (INIS)

Kroell, F.K.; Karlsson, J.A.; Nilsson, E.; Ryrfeldt, A.; Persson, C.G.

1990-01-01

The airway and pulmonary fate of two antiasthma xanthines was examined in a guinea pig perfused lung preparation where the airway mechanics and airway microvascular perfusion are maintained at near normal values. 14C-theophylline or 14C-enprofylline was infused for 10, 30, and 300 s into the pulmonary artery of the guinea pig isolated lung. The radioactivity increased rapidly (within 10 s) in tracheobronchial as well as in lung tissue, confirming that the large airway microcirculation was well supplied also by the perfusion. The effluent concentrations of total 3H and 14C radioactivity at the onset, during, and after intrapulmonary infusion of 14C-labeled xanthines and 3H-sucrose were closely associated, suggesting that the xanthines, like sucrose, largely distributed in extracellular fluid and were not taken up by the tissues. No metabolites of enprofylline or theophylline could be detected in the lung tissue or lung effluent, suggesting that xanthines are not biotransformed by the guinea pig lung. After intratracheal instillation of 14C-theophylline, the peak radioactivity in the lung effluent appeared in the second 15-s fraction after instillation, and after 10 and 60 min, 68.1 +/- 4.7% and 86.9 +/- 8.4%, respectively, of the given dose had appeared in the lung effluent. The present data suggest a mainly extracellular distribution and a rapid clearance of xanthines from the lung and airway tissues. The rapid disappearance of topical theophylline may explain the lack of success of inhalation therapy with this drug

Astrocytic mechanisms explaining neural-activity-induced shrinkage of extraneuronal space

DEFF Research Database (Denmark)

Østby, Ivar; Øyehaug, Leiv; Einevoll, Gaute T

2009-01-01

Neuronal stimulation causes approximately 30% shrinkage of the extracellular space (ECS) between neurons and surrounding astrocytes in grey and white matter under experimental conditions. Despite its possible implications for a proper understanding of basic aspects of potassium clearance and astr......Neuronal stimulation causes approximately 30% shrinkage of the extracellular space (ECS) between neurons and surrounding astrocytes in grey and white matter under experimental conditions. Despite its possible implications for a proper understanding of basic aspects of potassium clearance...... concentrations observed in connection with neuronal stimulation, the actions of the Na(+)/K(+)/Cl(-) (NKCC1) and the Na(+)/HCO(3) (-) (NBC) cotransporters appear to be critical determinants for achieving observed quantitative levels of ECS shrinkage. Considering the current state of knowledge, the model...

Fabrication and characterization of scaffold from cadaver goat-lung tissue for skin tissue engineering applications

Energy Technology Data Exchange (ETDEWEB)

Gupta, Sweta K. [Department of Polymer and Process Engineering, Indian Institute of Technology, Roorkee (India); Dinda, Amit K. [Department of Pathology, All India Institute of Medical Sciences, New Delhi (India); Potdar, Pravin D. [Department of Molecular Medicine, Jaslok Hospital and Research Centre, Mumbai (India); Mishra, Narayan C., E-mail: mishrawise@gmail.com [Department of Polymer and Process Engineering, Indian Institute of Technology, Roorkee (India)

2013-10-15

The present study aims to fabricate scaffold from cadaver goat-lung tissue and evaluate it for skin tissue engineering applications. Decellularized goat-lung scaffold was fabricated by removing cells from cadaver goat-lung tissue enzymatically, to have cell-free 3D-architecture of natural extracellular matrix. DNA quantification assay and Hematoxylin and eosin staining confirmed the absence of cellular material in the decellularized lung-tissue. SEM analysis of decellularized scaffold shows the intrinsic porous structure of lung tissue with well-preserved pore-to-pore interconnectivity. FTIR analysis confirmed non-denaturation and well maintainance of collagenous protein structure of decellularized scaffold. MTT assay, SEM analysis and H and E staining of human skin-derived Mesenchymal Stem cell, seeded over the decellularized scaffold, confirms stem cell attachment, viability, biocompatibility and proliferation over the decellularized scaffold. Expression of Keratin18 gene, along with CD105, CD73 and CD44, by human skin-derived Mesenchymal Stem cells over decellularized scaffold signifies that the cells are viable, proliferating and migrating, and have maintained their critical cellular functions in the presence of scaffold. Thus, overall study proves the applicability of the goat-lung tissue derived decellularized scaffold for skin tissue engineering applications. - Highlights: • We successfully fabricated decellularized scaffold from cadaver goat-lung tissue. • Decellularized goat-lung scaffolds were found to be highly porous. • Skin derived MSC shows high cell viability and proliferation over the scaffold. • Phenotype of MSCs was well maintained over the scaffold. • The scaffold shows potential for applications in skin tissue engineering.

Fabrication and characterization of scaffold from cadaver goat-lung tissue for skin tissue engineering applications

International Nuclear Information System (INIS)

Gupta, Sweta K.; Dinda, Amit K.; Potdar, Pravin D.; Mishra, Narayan C.

2013-01-01

The present study aims to fabricate scaffold from cadaver goat-lung tissue and evaluate it for skin tissue engineering applications. Decellularized goat-lung scaffold was fabricated by removing cells from cadaver goat-lung tissue enzymatically, to have cell-free 3D-architecture of natural extracellular matrix. DNA quantification assay and Hematoxylin and eosin staining confirmed the absence of cellular material in the decellularized lung-tissue. SEM analysis of decellularized scaffold shows the intrinsic porous structure of lung tissue with well-preserved pore-to-pore interconnectivity. FTIR analysis confirmed non-denaturation and well maintainance of collagenous protein structure of decellularized scaffold. MTT assay, SEM analysis and H and E staining of human skin-derived Mesenchymal Stem cell, seeded over the decellularized scaffold, confirms stem cell attachment, viability, biocompatibility and proliferation over the decellularized scaffold. Expression of Keratin18 gene, along with CD105, CD73 and CD44, by human skin-derived Mesenchymal Stem cells over decellularized scaffold signifies that the cells are viable, proliferating and migrating, and have maintained their critical cellular functions in the presence of scaffold. Thus, overall study proves the applicability of the goat-lung tissue derived decellularized scaffold for skin tissue engineering applications. - Highlights: • We successfully fabricated decellularized scaffold from cadaver goat-lung tissue. • Decellularized goat-lung scaffolds were found to be highly porous. • Skin derived MSC shows high cell viability and proliferation over the scaffold. • Phenotype of MSCs was well maintained over the scaffold. • The scaffold shows potential for applications in skin tissue engineering

Generalized connective tissue disease in Crtap-/- mouse.

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Dustin Baldridge

2010-05-01

Full Text Available Mutations in CRTAP (coding for cartilage-associated protein, LEPRE1 (coding for prolyl 3-hydroxylase 1 [P3H1] or PPIB (coding for Cyclophilin B [CYPB] cause recessive forms of osteogenesis imperfecta and loss or decrease of type I collagen prolyl 3-hydroxylation. A comprehensive analysis of the phenotype of the Crtap-/- mice revealed multiple abnormalities of connective tissue, including in the lungs, kidneys, and skin, consistent with systemic dysregulation of collagen homeostasis within the extracellular matrix. Both Crtap-/- lung and kidney glomeruli showed increased cellular proliferation. Histologically, the lungs showed increased alveolar spacing, while the kidneys showed evidence of segmental glomerulosclerosis, with abnormal collagen deposition. The Crtap-/- skin had decreased mechanical integrity. In addition to the expected loss of proline 986 3-hydroxylation in alpha1(I and alpha1(II chains, there was also loss of 3Hyp at proline 986 in alpha2(V chains. In contrast, at two of the known 3Hyp sites in alpha1(IV chains from Crtap-/- kidneys there were normal levels of 3-hydroxylation. On a cellular level, loss of CRTAP in human OI fibroblasts led to a secondary loss of P3H1, and vice versa. These data suggest that both CRTAP and P3H1 are required to maintain a stable complex that 3-hydroxylates canonical proline sites within clade A (types I, II, and V collagen chains. Loss of this activity leads to a multi-systemic connective tissue disease that affects bone, cartilage, lung, kidney, and skin.

Knee Ligament Injury and the Clinical Application of Tissue Engineering Techniques: A Systematic Review.

Science.gov (United States)

Riley, Thomas C; Mafi, Reza; Mafi, Pouya; Khan, Wasim S

2018-02-23