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C2—Nanobiology
Comparative Biochemistry and Physiology, Part A 143 (2006) S123 – S125 www.elsevier.com/locate/cbpa
Society for Experimental Biology Annual Main Meeting 2nd–7th April 2006, University of Kent at Canterbury, UK
C2–NANOBIOLOGY Organised by M. Riehle (University of Glasgow) and R. Handy (University of Plymouth) Sponsored by: Company of Biologists
C2.1 Nanomaterials-sources, classification, and toxicity C. Buzea, I. Pacheco, K. Robbie, (Queen’s University) We identify the main sources of nanoparticles, classify nanomaterials from a morphological perspective, and summarize the current knowledge on the toxicology of nanoparticles, including epidemiological, animal, clinical, and in vitro studies. Life forms on Earth have always been exposed to nanoparticles from natural sources (dust storms, volcanic ash, fires, micro-organisms), and have developed advanced defenses to their toxicity. Industrial development in recent centuries has created many anthropogenic sources of nanoparticulate pollution, particularly from combustion-based engine transportation. While the human immune system has adapted during evolution to protect against nano and micro particle intruders (with the reticuloendothelial system playing a major role in this defence), recent epidemiological studies have shown strong correlation between particulate air pollution levels and mortality rates, respiratory and cardiovascular diseases, and various types of cancers. The adverse health effects of nanoparticles depend on particle chemistry, concentration, size, aspect ratio, crystallinity, and agglomeration state. Animal and human studies show that inhaled nanoparticle are not efficiently removed by macrophage clearance mechanisms in the lung, causing lung damage and distribution to other organs through circulation within the blood and lymphatic networks. The key to understanding nanoparticle toxicity lies in their minute size, smaller than cells and cellular organelles. Nanoparticles penetrate these basic blocks, produce physical damage, inducing harmful inflammatory response. Ongoing study and development of nanoparticulate materials mandates a diligent multidisciplinary exploration of their toxic effects on living organisms. We present a summary of existing knowledge, and the beginning of our study of biotoxicity of manufactured nanostructured particles and surfaces.
C2.2 Nanoparticle toxicity V. Stone, (Napier University) The number and diversity of newly engineered nanoparticles is steadily increasing. Due to the wide variety of applications of nanoparticles, doi:10.1016/j.cbpa.2006.01.049
including food, medicine, clothing, and cosmetics, human exposure is inevitable and so it is important to consider the hazard of these materials. Very little is known regarding the toxicity of new nanoparticles such as fullerenes, nanotubes and quantum dots. However the toxicology of bulk manufactured nanoparticles such as carbon black, TiO2 and polystyrene beads has been investigated, in relation to the mechanisms driving the adverse health effects of ambient air pollution. These studies provide a platform on which an effective strategy for nanoparticle toxicity testing can be established. We have previously reported that carbon (14 nm diameter) or polystyrene (54 nm diameter) nanoparticles generate more inflammation in the rat lung than larger particles, and this inflammation correlates with the surface area of the particle dose. The smaller particles have been demonstrated in a number of assays to generate more reactive oxygen species (ROS) than larger particles leading to oxidative stress in the human A549 lung epithelial cell line and in macrophages. In addition non-lethal doses of these particles induced a significant increase in cytosolic calcium leading to the production of the pro-inflammatory cytokine tumour necrosis factor alpha. In conclusion, the relatively small size of nanoparticles enhances their potency in a number of in vivo and in vitro systems resulting in the production of oxidants and the generation of pro-inflammatory mediators. Funding: The Colt Foundation and BP.
C2.3 Predicting the mechanisms of nano-toxicity from the membrane biology of fishes: Is nano pollution a problem for aquatic life? R. Handy, (University of Plymouth) The idea of nano-pollution has sparked the imagination of the media, and the scientific community is speculating on the possible effects (or not) of nano-technology and nano-pollution. Here, we propose a mechanistic, hypothesis-led approach to rationalising the possible effects on nano-materials on fish and other aquatic animals. This includes a brief review of the microenvironment and surface chemistry of epithelia to indicate how we expect nano-materials to interact with the gills of aquatic animals. A mechanistic approach as to how nano-
S124
Abstracts / Comparative Biochemistry and Physiology, Part A 143 (2006) S123 – S125
materials are absorbed includes aspects of diffusion, and the ability of nano-particles to use ion or water channels on biological membranes. Concerns at the cellular level include the accumulation of nanoparticles in cells, and subsequent inflammation reactions or responses of the immune system. The role of blood circulation in distributing nanoparticles to internal organs (target organ toxicity) is discussed, and mechanisms of clearance from the body. Finally, based on the above, the likely health effects on animals that may be diagnostic of nano pollutant exposure.
roughness or highly defined topography yield enriched osteoblast populations, the addition of nanodisorder (T 50 nm) give the optimal performance in regard to in vitro osteogenesis. Acknowledgements: MJD is a BBSRC David Phillips Fellow, NG is a Royal Society of Edinburgh Fellow, DS is supported by EC grant Nanocues. RT is supported by BBSRC and ROC is supported by BBSRC and EPSRC. We also thank Prof Adam Curtis.
C2.6 Nanoscale investigations of forces produced by cells C2.4 Artificial micro-and nanostructured surfaces in biomedicine
A. Curtis, M. Dalby, N. Gadegaard, M. Riehle, L. Csaderova, (University of Glasgow)
F. Walboomers, C. Bruckmann, W. Loesberg, K. Matsuzaka, J. Parker, J. Jansen, (Radboud University)
Abstract not submitted.
The materials used to produce medical devices are also referred to as Fbiomaterials_. To improve biomaterial performance, research is aiming to improve overall design, mechanical properties, and surface chemical/physicochemical properties. Especially the use of surface topography is a proven powerful tool to influence the behaviour of adherent cells. Already since the beginning of last century biologists study substrate surface topography (i.e. both nonspecific roughnesses as well as dedicated textures). It is well known that applying a micrometer pattern of grooves and ridges on a culturing substrate will induce adherent cells to spread out and migrate in accordance with the pattern. This phenomenon is also known as Fcontact guidance_. In this presentation, a number of current topics in the field of contact guidance will be discussed. First, the patterns used nowadays more and more are designed to mimic the micro and nano designs of nature itself. For instance, it has been shown that fibroblasts derived from the periodontal ligament produce more extracellular matrix, when cultured on substrate surfaces resembling the micrometer-sized pits as present on the dentin. Second, also culture conditions become more and more complex. Recent literature shows examples of researchers studying the interaction between surface topography-driven processes, and mechanical stimuli resembling the movements in a living organism. Third, with the advances in machining techniques, it is evident that the boundaries of contact guidance are challenged, gradually moving form micro- to nanometer-sized patters. Finally, the translation of all such cell culture models to application in medical devices will be discussed.
C2.7 Function of toe pad cell nano-structuring in adhesion in tree frogs
C2.5 Nanotopographical stimulation of mesenchymal stem cells
J. Smith, M. Riehle, J. Barnes, R. Downie, (University of Glasgow) Tree frogs are noted for their ability to climb on smooth vertical surfaces. Consensus is that they are able to do this via wet adhesion, facilitated by disc-like toe pads, which have mucosal glands opening to their ventral surface. Across many families in which arboreal frogs are found, toe pads differ to only a small degree in their gross morphology but the specialised epithelial cells that cover them are remarkably similar. Cells are columnar and divided at their apices, which are hexagonal. This apical surface is further divided into hexagonal sub-units by nano-structural Fpegs_. These are often considered to be residual artefacts. However, use of interference reflection microscopy (IRM), has allowed us to observe how toe pad cells contact the surface and has suggested that these features may be of key importance to adhesive ability. Across much of the toe pad, cells are in much closer contact than would be expected for a wet adhesive system. This is particularly the case for the nano-structural features, where typical contact distances are between 0 – 20 nm. This suggests direct contact between cell and surface at these junctions, which has significant implications for our understanding of the adhesive system. Furthermore, using IRM over a range of orientations shows that as frogs approach the vertical, the numbers of nano-structural pegs in close contact increase, thereby creating greater friction and adhesion. Sylgard models with nano-structured surfaces will be investigated in an attempt to recreate tree frogs’ sticking ability to assess the biomimetic potential of their adhesive system.
M. Dalby, N. Gadegaard, M. O. Riehle, C. Wilkinson, (University of Glasgow); D. Sutherland, (Chalmers University of Technology); S. Affrossman, (University of Strathclyde)
C2.8 How the hinge angle of the integrin headpiece is switched open: a hypothesis of integrin activation
Due to rapidly developing nanofabrication technology, biologists are being able to observe the scale of possible cellular reactions to their nanoenvironment. One area of particular interest is that of nanocues in stem cell differentiation. We have tested osteogenesis of primary human mesenchymal stem cells using microarray and histological analysis. Our results show that whilst traditional approaches of use of random
E. Faucher, V. Vogel, (Swiss Federal Institute of Technology); M. Gao, K. Schulten, (University of Illinois) Experiments have shown that ligand binding to integrins causes the hA/hybrid domain hinge angle in the integrin headpiece to switch from closed to open and this conformational change has been linked to
Abstracts / Comparative Biochemistry and Physiology, Part A 143 (2006) S123 – S125
the switch from low to high ligand binding affinity. However, the ligand-bound avh3 integrin crystal structure, which was formed by soaking RGD-ligand into preformed unliganded integrin crystals, displays the closed hinge angle conformation. Here we demonstrate through molecular dynamics (MD) simulations that when the legless headpiece of this structure is docked to fibronectin’s 10 th type III module (FnIII10) and allowed to equilibrate, the closed hinge angle spontaneously opens. These simulations reveal how a minor structural perturbation at the ligand binding site can be amplified by elastic
S125
distortions propagated across the hA domain to the hA/hybrid domain interface. Changes within the hA domain that are characteristic of the transition from the closed to the open hinge angle conformation are described. We present this dynamical picture, together with currently available experimental data, in the framework of a two-state hA domain model. Finally, we show how force can accelerate the opening of the closed hinge angle in steered molecular dynamics (SMD) simulations and so extend our model to include the potential role of mechanical force in integrin activation.
Society for Experimental Biology Annual Main Meeting 2nd–7th April 2006, University of Kent at Canterbury, UK
C2–NANOBIOLOGY Organised by M. Riehle (University of Glasgow) and R. Handy (University of Plymouth) Sponsored by: Company of Biologists
C2.1 Nanomaterials-sources, classification, and toxicity C. Buzea, I. Pacheco, K. Robbie, (Queen’s University) We identify the main sources of nanoparticles, classify nanomaterials from a morphological perspective, and summarize the current knowledge on the toxicology of nanoparticles, including epidemiological, animal, clinical, and in vitro studies. Life forms on Earth have always been exposed to nanoparticles from natural sources (dust storms, volcanic ash, fires, micro-organisms), and have developed advanced defenses to their toxicity. Industrial development in recent centuries has created many anthropogenic sources of nanoparticulate pollution, particularly from combustion-based engine transportation. While the human immune system has adapted during evolution to protect against nano and micro particle intruders (with the reticuloendothelial system playing a major role in this defence), recent epidemiological studies have shown strong correlation between particulate air pollution levels and mortality rates, respiratory and cardiovascular diseases, and various types of cancers. The adverse health effects of nanoparticles depend on particle chemistry, concentration, size, aspect ratio, crystallinity, and agglomeration state. Animal and human studies show that inhaled nanoparticle are not efficiently removed by macrophage clearance mechanisms in the lung, causing lung damage and distribution to other organs through circulation within the blood and lymphatic networks. The key to understanding nanoparticle toxicity lies in their minute size, smaller than cells and cellular organelles. Nanoparticles penetrate these basic blocks, produce physical damage, inducing harmful inflammatory response. Ongoing study and development of nanoparticulate materials mandates a diligent multidisciplinary exploration of their toxic effects on living organisms. We present a summary of existing knowledge, and the beginning of our study of biotoxicity of manufactured nanostructured particles and surfaces.
C2.2 Nanoparticle toxicity V. Stone, (Napier University) The number and diversity of newly engineered nanoparticles is steadily increasing. Due to the wide variety of applications of nanoparticles, doi:10.1016/j.cbpa.2006.01.049
including food, medicine, clothing, and cosmetics, human exposure is inevitable and so it is important to consider the hazard of these materials. Very little is known regarding the toxicity of new nanoparticles such as fullerenes, nanotubes and quantum dots. However the toxicology of bulk manufactured nanoparticles such as carbon black, TiO2 and polystyrene beads has been investigated, in relation to the mechanisms driving the adverse health effects of ambient air pollution. These studies provide a platform on which an effective strategy for nanoparticle toxicity testing can be established. We have previously reported that carbon (14 nm diameter) or polystyrene (54 nm diameter) nanoparticles generate more inflammation in the rat lung than larger particles, and this inflammation correlates with the surface area of the particle dose. The smaller particles have been demonstrated in a number of assays to generate more reactive oxygen species (ROS) than larger particles leading to oxidative stress in the human A549 lung epithelial cell line and in macrophages. In addition non-lethal doses of these particles induced a significant increase in cytosolic calcium leading to the production of the pro-inflammatory cytokine tumour necrosis factor alpha. In conclusion, the relatively small size of nanoparticles enhances their potency in a number of in vivo and in vitro systems resulting in the production of oxidants and the generation of pro-inflammatory mediators. Funding: The Colt Foundation and BP.
C2.3 Predicting the mechanisms of nano-toxicity from the membrane biology of fishes: Is nano pollution a problem for aquatic life? R. Handy, (University of Plymouth) The idea of nano-pollution has sparked the imagination of the media, and the scientific community is speculating on the possible effects (or not) of nano-technology and nano-pollution. Here, we propose a mechanistic, hypothesis-led approach to rationalising the possible effects on nano-materials on fish and other aquatic animals. This includes a brief review of the microenvironment and surface chemistry of epithelia to indicate how we expect nano-materials to interact with the gills of aquatic animals. A mechanistic approach as to how nano-
S124
Abstracts / Comparative Biochemistry and Physiology, Part A 143 (2006) S123 – S125
materials are absorbed includes aspects of diffusion, and the ability of nano-particles to use ion or water channels on biological membranes. Concerns at the cellular level include the accumulation of nanoparticles in cells, and subsequent inflammation reactions or responses of the immune system. The role of blood circulation in distributing nanoparticles to internal organs (target organ toxicity) is discussed, and mechanisms of clearance from the body. Finally, based on the above, the likely health effects on animals that may be diagnostic of nano pollutant exposure.
roughness or highly defined topography yield enriched osteoblast populations, the addition of nanodisorder (T 50 nm) give the optimal performance in regard to in vitro osteogenesis. Acknowledgements: MJD is a BBSRC David Phillips Fellow, NG is a Royal Society of Edinburgh Fellow, DS is supported by EC grant Nanocues. RT is supported by BBSRC and ROC is supported by BBSRC and EPSRC. We also thank Prof Adam Curtis.
C2.6 Nanoscale investigations of forces produced by cells C2.4 Artificial micro-and nanostructured surfaces in biomedicine
A. Curtis, M. Dalby, N. Gadegaard, M. Riehle, L. Csaderova, (University of Glasgow)
F. Walboomers, C. Bruckmann, W. Loesberg, K. Matsuzaka, J. Parker, J. Jansen, (Radboud University)
Abstract not submitted.
The materials used to produce medical devices are also referred to as Fbiomaterials_. To improve biomaterial performance, research is aiming to improve overall design, mechanical properties, and surface chemical/physicochemical properties. Especially the use of surface topography is a proven powerful tool to influence the behaviour of adherent cells. Already since the beginning of last century biologists study substrate surface topography (i.e. both nonspecific roughnesses as well as dedicated textures). It is well known that applying a micrometer pattern of grooves and ridges on a culturing substrate will induce adherent cells to spread out and migrate in accordance with the pattern. This phenomenon is also known as Fcontact guidance_. In this presentation, a number of current topics in the field of contact guidance will be discussed. First, the patterns used nowadays more and more are designed to mimic the micro and nano designs of nature itself. For instance, it has been shown that fibroblasts derived from the periodontal ligament produce more extracellular matrix, when cultured on substrate surfaces resembling the micrometer-sized pits as present on the dentin. Second, also culture conditions become more and more complex. Recent literature shows examples of researchers studying the interaction between surface topography-driven processes, and mechanical stimuli resembling the movements in a living organism. Third, with the advances in machining techniques, it is evident that the boundaries of contact guidance are challenged, gradually moving form micro- to nanometer-sized patters. Finally, the translation of all such cell culture models to application in medical devices will be discussed.
C2.7 Function of toe pad cell nano-structuring in adhesion in tree frogs
C2.5 Nanotopographical stimulation of mesenchymal stem cells
J. Smith, M. Riehle, J. Barnes, R. Downie, (University of Glasgow) Tree frogs are noted for their ability to climb on smooth vertical surfaces. Consensus is that they are able to do this via wet adhesion, facilitated by disc-like toe pads, which have mucosal glands opening to their ventral surface. Across many families in which arboreal frogs are found, toe pads differ to only a small degree in their gross morphology but the specialised epithelial cells that cover them are remarkably similar. Cells are columnar and divided at their apices, which are hexagonal. This apical surface is further divided into hexagonal sub-units by nano-structural Fpegs_. These are often considered to be residual artefacts. However, use of interference reflection microscopy (IRM), has allowed us to observe how toe pad cells contact the surface and has suggested that these features may be of key importance to adhesive ability. Across much of the toe pad, cells are in much closer contact than would be expected for a wet adhesive system. This is particularly the case for the nano-structural features, where typical contact distances are between 0 – 20 nm. This suggests direct contact between cell and surface at these junctions, which has significant implications for our understanding of the adhesive system. Furthermore, using IRM over a range of orientations shows that as frogs approach the vertical, the numbers of nano-structural pegs in close contact increase, thereby creating greater friction and adhesion. Sylgard models with nano-structured surfaces will be investigated in an attempt to recreate tree frogs’ sticking ability to assess the biomimetic potential of their adhesive system.
M. Dalby, N. Gadegaard, M. O. Riehle, C. Wilkinson, (University of Glasgow); D. Sutherland, (Chalmers University of Technology); S. Affrossman, (University of Strathclyde)
C2.8 How the hinge angle of the integrin headpiece is switched open: a hypothesis of integrin activation
Due to rapidly developing nanofabrication technology, biologists are being able to observe the scale of possible cellular reactions to their nanoenvironment. One area of particular interest is that of nanocues in stem cell differentiation. We have tested osteogenesis of primary human mesenchymal stem cells using microarray and histological analysis. Our results show that whilst traditional approaches of use of random
E. Faucher, V. Vogel, (Swiss Federal Institute of Technology); M. Gao, K. Schulten, (University of Illinois) Experiments have shown that ligand binding to integrins causes the hA/hybrid domain hinge angle in the integrin headpiece to switch from closed to open and this conformational change has been linked to
Abstracts / Comparative Biochemistry and Physiology, Part A 143 (2006) S123 – S125
the switch from low to high ligand binding affinity. However, the ligand-bound avh3 integrin crystal structure, which was formed by soaking RGD-ligand into preformed unliganded integrin crystals, displays the closed hinge angle conformation. Here we demonstrate through molecular dynamics (MD) simulations that when the legless headpiece of this structure is docked to fibronectin’s 10 th type III module (FnIII10) and allowed to equilibrate, the closed hinge angle spontaneously opens. These simulations reveal how a minor structural perturbation at the ligand binding site can be amplified by elastic
S125
distortions propagated across the hA domain to the hA/hybrid domain interface. Changes within the hA domain that are characteristic of the transition from the closed to the open hinge angle conformation are described. We present this dynamical picture, together with currently available experimental data, in the framework of a two-state hA domain model. Finally, we show how force can accelerate the opening of the closed hinge angle in steered molecular dynamics (SMD) simulations and so extend our model to include the potential role of mechanical force in integrin activation.