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Biomedical subjects

Adam S G Curtis

Publications and source records attributed to Adam S G Curtis.

At least 19 recordsLinked to original sources
PubMed2007

Nanotopographical stimulation of mechanotransduction and changes in interphase centromere positioning.

We apply a recently developed method for controlling the spreading of cultured cells using electron beam lithography (EBL) to create polymethylmethacrylate (PMMA) substrata with repeating nanostructures. There are indications that the reduced cell spreading on these substrata, compared with planar PMMA, results from a reduced adhesivity since there are fewer adhesive structures and fewer of their associated stress fibres. The reduced cell spreading also results in a reduced nuclear area and a closer spacing of centrosomes within the nucleus, suggesting that the tension applied to the nucleus is reduced as would be expected from the reduction in stress fibres. In order to obtain further evidence for this, we have used specific inhibitors of components of the cytoskeleton and have found effects comparable with those induced by the new substrata. We have also obtained evidence that these subtrata result in downregulation of gene expression which suggests that this may be due to the changed tension on the nucleus: an intriguing possibility that merits further investigation.

Cell Adhesion↗
PubMed2006

Nanoprinting onto cells.

Growing cells on surfaces bearing nanotopography signals makes many changes in cell gene expression and downstream changes in phenotype but the mechanisms for this have, so far, been obscure. We consider the question of whether the topography directly nanoimprints onto the cell as a component of the signal transduction system. Evidence we present from SEM, TEM and fluorescence detection of the arrangements of cytoskeletal components is consistent with the possibility that cells are nanoimprinted by the substrate. The nanoprinting does not interfere with integrin-mediated adhesion processes and may perhaps work through them. Time-lapse video studies of cells moving from areas bearing nanotopography to flat areas and vice versa suggests that the nanoimprinting takes 1-6h to appear on the cell and a similar time to disappear when the cell moves from a flat surface to a nanotopographic one and back. This nanoprinting of cells would appear to be a novel type of cell signalling.

Cell Movement↗
PubMed2006

Nanoparticle targeting at cells.

Gold nanoparticles have been used for analytical and biomedical purposes for many years. In fact, the labeling of targeting molecules with nanoparticles has revolutionized the visualization of cellular or tissue components by electron microscopy. We report in this study the derivatization of tiopronin-protected nanoparticles with ethylenediamine and poly(ethylene glycol) bis(3-aminopropyl) terminated and their functionalization with the GRGDSP peptide sequence by a straightforward and economical methodology. The particles were subsequently tested in vitro with a human fibroblast cell line to determine the biocompatibility, and the cell-particle interactions, using fluorescence and scanning electron microscopies. The results indicate that tiopronin gold nanoparticles aggregate due to culture medium proteins, whereas the tiopronin gold nanoparticles derivatized with ethylenediamine induce endocytosis, and the same nanoparticles derivatized with poly(ethylene glycol) derivative promote particle-cell adhesion.

Cell Adhesion↗
PubMed2006

The effects of colloidal nanotopography on initial fibroblast adhesion and morphology.

Colloidal lithography offers a simple, inexpensive method of producing irregular nanotopographies, a pattern not easily attainable utilizing conventional serial writing processes. Colloids with 20- or 50-nm diameter were utilized to produce such an irregular topography and were characterized by calculating the percentage area coverage of particles. Interparticle and nearest neighbor spacing were also assessed for the individual colloids in the pattern. Two-way analysis of variance (ANOVA) indicated significant differences between the number of fibroblasts adhering to planar, 20-, and 50-nm-diameter colloidal topographies, the number of fibroblasts adhering to the substrates at the time intervals studied, namely 20 min, 1 h, and 3 h and significant interaction between time and topography on fibroblast adhesion (P < 0.01). Tukey tests were utilized for sensitive identification of the differences between the sample means and compounded ANOVA results. Cytoskeletal and general cell morphology were investigated on planar and colloidal substrates, and indicated cells in contact with irregular nanotopographies exhibit many peripheral protrusions while such protrusions are absent in cells on planar control surfaces. These protrusions are rich in microtubules on 20-nm-diameter colloidal surfaces while microfilaments are prevalent on 50-nm-diameter surfaces. Moreover, by 3 h, cells on the colloidal substrates initiate cell-cell adhesions, also absent in controls.

Cell Adhesion↗
PubMed2005

Human fibroblast reactions to standard and electropolished titanium and Ti-6Al-7Nb, and electropolished stainless steel.

Stainless steel (SS), titanium (cpTi), and Ti-6Al-7Nb (TAN) are frequently used metals in orthopedic internal fracture fixation. Although reactivity to SS and cpTi are noted in reference, the soft tissue compatibility of TAN has not been comprehensively studied. This study focuses on the in vitro soft tissue compatibility of TAN in comparison to SS and cpTi using a human fibroblast model. The industrial standard surface finishes of these three materials vary considerably in view of their use in similar applications. To distinguish between material parameters of topography and chemistry, we have included electropolished (e.p) counterparts of the standard preparations of cpTi and TAN in the study (standard SS is e.p). All materials were characterized using atomic force microscopy, profilometry, and scanning electron microscopy. Our findings demonstrate that cell morphology and growth rate was similar for SS, and e.p. cpTi and TAN, with cells well spread and forming a confluent monolayer by 10 days. Cell growth on standard cpTi was similar to the electropolished samples; however, they showed a less spread morphology with more filopodia and surface ruffling present. Cell morphology on standard TAN was rounded or elongated and proliferation was inhibited at all time points, with possible cell necrosis by day 10. We found evidence of endocytosis of beta-phase particles originating from the standard TAN surface. We believe that the particle uptake coupled with the characteristic surface topography contribute to the noncytocompatibility of fibroblasts on standard TAN.

Biocompatible Materials↗
PubMed2005

Neutralisation of TGF beta or binding of VLA-4 to fibronectin prevents rat tendon adhesion following transection.

Following tendon injury, severe loss of function often occurs either as a result of obliteration of the synovial canal with fibrous scar tissue or from rupture of the repaired tendon. The role of cell engineering in tendon repair is to promote strong and rapid healing of tendon whilst at the same time facilitating rapid reconstitution of the synovial canal. Modification of the immediate inflammatory response around healing tendon has been found to be of value. Experimentally this has been achieved by neutralisation of transforming growth factor-beta over the first 3 days following injury, or by blockade of inflammatory cell binding to the CS-1 locus on fibronectin with an anti-VLA-4 antibody, or with the synthetic VLA-4 inhibitor, CS-1 peptide, in a rat model of tendon transection. It is concluded from this pilot study that the treatments described hold promise in improving outcomes of the common clinical problem of tendon injury in man.

Animals↗
PubMed2004

Fibroblast response to a controlled nanoenvironment produced by colloidal lithography.

It is thought that by understanding how cells respond to topography, that better tissue engineering may be achievable. An important consideration in the cellular environment is topography. The effects of microtopography have been well documented, but the effects of nanotopography are less well known. Previously, methods of nanofabrication have been costly and time-consuming, but research by engineers, physicists, and chemists is starting to allow the production of nanostructures using low-cost techniques. In this report, nanotopography is specifically considered. Controlled patterns of 160 nm high nanocolumns were produced for in vitro cell culture using colloidal lithography. By studying cell adhesion with time and cytoskeletal (actin, tubulin, and vimentin) maturity, insight has been gained as to how fibroblasts adhere to these nanofeatures.

Actins↗
PubMed2004

Attempted endocytosis of nano-environment produced by colloidal lithography by human fibroblasts.

Control of the cells' nanoenvironment is likely to be important in the future of cell and tissue engineering. Microtopography has been shown to provide cues to cells that elicit a large range of cell responses, including control of adhesion, morphology, apoptosis and gene regulations. Now, researchers are focusing on nanotopography as techniques such as colloidal and electron beam lithography and polymer demixing have become available. In this study, human fibroblast response to nanocolumns (160-nm high, 100-nm diameter, 230-nm centre-centre spacing) produced by colloidal lithography are considered. Using electron microscopy and immunofluorescence to image the cytoskeleton, clathrin and dynamin, it was observed that the cells try to endocytose the nanocolumns. It also appeared that a small population of the cells changed to unusual morphologies with macrophage-like processes and highly disrupted cytoskeleton. These observations could have implications for nanomaterials science in areas such as cell transfection and drug delivery.

Actins↗
PubMed2004

Effect of cellular uptake of gelatin nanoparticles on adhesion, morphology and cytoskeleton organisation of human fibroblasts.

The aim of present study was to prepare nanometer sized particles of gelatin via water-in-oil microemulsion system for drug and gene delivery applications. In this study, cross-linked gelatin nanoparticles encapsulating a fluorescent marker molecule fluorescein isothiocyanate-dextran (FITC-Dex, Mol. Wt. 19.3kDa) have been prepared, characterized and their influence on human fibroblasts has been assessed in terms of cell adhesion, cytotoxicity, light microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and observation of cytoskeleton organisation. Gelatin nanoparticles were prepared inside the aqueous cores of sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/n-hexane reverse micelles. Transmission electron microscopy image showed that the particles are spherical in shape with size of 37+/-0.84 nm diameter. The release of FITC-Dex from the nanoparticles in phosphate buffer saline (pH 7.4) is found to increase with time and about 80% of the encapsulated dye is released in 6 h. Cell adhesion studies with human fibroblasts have shown that gelatin nanoparticles do not affect the number of cells adhered to glass as compared to control cells with no particles. Standard cell viability assay demonstrated that cells incubated with gelatin nanoparticles remained more than 100% viable at concentration as high as 500 microg/ml. From SEM image, it was observed that the nanoparticles were internalised and the fibroblasts exhibited vacuoles in the cell body with cell membrane abnormalities. Endocytosis of nanoparticles was confirmed from TEM studies and it resulted in disruption of F-actin and beta-tubulin cytoskeleton. These studies show that the gelatin nanoparticles prepared by water-in-oil microemulsion systems are endocytosed by the fibroblasts without being toxic to cells even at high concentration of nanoparticles.

Actins↗
PubMed2004

The influence of transferrin stabilised magnetic nanoparticles on human dermal fibroblasts in culture.

Magnetic nanoparticles have been used for bio-medical purposes including drug delivery, cell destruction and as MRI contrast agents for several years. A more recent biological application has focused on targeted drug delivery. To this end, a wide variety of iron oxide nanoparticles have been synthesised. This study involves the use of magnetic nanoparticles synthesised and derivatised with human transferrin, compared to identical underivatised particles. Human fibroblasts were used, representative of a tissue cell-type. The influence in vitro was determined using light and fluorescence microscopy, scanning and transmission electron microscopy, and 1718 gene microarray. The results indicate that the transferrin derivatised particles appear to localise to the cell membrane without instigating receptor-mediated endocytosis, and also induce up-regulation in the cells for many genes, particularly in the area of cytoskeleton and cell signalling. The microscopy results further support these findings.

Antigens, CD↗
PubMed2004

Investigating filopodia sensing using arrays of defined nano-pits down to 35 nm diameter in size.

In order for cells to react to topography, they must be able to sense shape. When considering nano-topography, these shapes are much smaller than the cell, but still strong responses to nano-topography have been seen. Filopodia, or microspikes, presented by cells at their leading edges are thought to be involved in gathering of special information. In order to investigate this, and to develop an understanding of what size of feature can be sensed by cells, morphological observation (electron and fluorescent microscopy) of fibroblasts reacting to nano-pits with 35, 75 and 120 nm diameters has been used in this study. The nano-pits are especially interesting because unlike many of the nanofeatures cited in the literature, they have no height for the cells to react to. The results showed that cell filopodia, and retraction fibres, interacted with all pit sizes, although direct interaction was hard to image on the 35 nm pits. This suggests that cells are extremely sensitive to their nanoevironment and that should be taken in to consideration when designing next-generation tissue engineering materials. We suggest that this may occur through nanocontact guidance as filopodia are moved over the pits.

Cells, Cultured↗
PubMed2004

Lactoferrin and ceruloplasmin derivatized superparamagnetic iron oxide nanoparticles for targeting cell surface receptors.

Tissue and cell-specific drug targeting can be achieved by employing nanoparticle coatings or carrier-drug conjugates that contain a ligand recognized by a receptor on the target cell. Superparamagnetic iron oxide nanoparticles have been used for many years in various biomedical applications. In this study, superparamagnetic nanoparticles with specific shape and size have been prepared and coupled to various proteins. These particles are characterized in vitro and their influence on human dermal fibroblasts is assessed in terms of cell adhesion, viability, morphology and cytoskeleton organization using various techniques to observe cell-nanoparticle interaction, including light, fluorescence, scanning and transmission electron microscopy. The results showed that each nanoparticle type with different surface characteristics caused a distinctly different cell response. The underivatized magnetic particles were internalized by the fibroblasts probably due to endocytosis, which resulted in disruption of the cell membrane and disorganized cell cytoskeleton. In contradiction, lactoferrin or ceruloplasmin coated nanoparticles attached to the cell membrane, most likely to the cell expressed receptors and were not endocytosed. One major problem with uncoated magnetic nanoparticles has been the endocytosis of particles leading to irreversible entry. These experiments provide a route to prevent this problem, suggesting that cell response can be directed via specifically engineered particle surfaces.

Biocompatible Materials↗
PubMed2004

Cell response to dextran-derivatised iron oxide nanoparticles post internalisation.

Magnetic nanoparticles have been used for many years as magnetic resonance imaging contrast agents. Despite the fact that there are currently several dextran-coated iron oxide nanoparticles are in preclinical and clinical use, there is very little information available concerning the influence such particles have on cells in culture. The prerequisite for particles employed as contrast agents is capture and subsequent uptake by cells. This study involved the use of magnetic nanoparticles synthesised and derivatised with dextran, compared to similar underivatised plain particles. The influence in vitro was assessed using human dermal fibroblasts and various techniques to observe cell-particles interaction, including light and fluorescence microscopy, scanning and transmission electron microscopy. The results indicate that although both the uncoated and the dextran-derivatised particles are uptaken into the cell, the derivatised particles induce alterations in cell behaviour and morphology distinct from the plain particles, suggesting that cell response is dependent on the particles coating.

Adsorption↗
PubMed2004

Changes in fibroblast morphology in response to nano-columns produced by colloidal lithography.

In designing new biomaterials, specific chemical and topographical cues will be important in guiding cell response. Filopodia are actin-driven structures produced by cells and speculated to be involved in cell sensing of the three-dimensional environment. This report quantifies filopodia response to cylindrical nano-columns (100 nm diameter, 160 nm high) produced by colloidal lithography. Also observed were actin cytoskeleton morphology by fluorescence microscopy and filopodia morphology by electron microscopy (scanning and transmission). The results showed that the fibroblasts used produced more filopodia per microm of cell perimeter and that filopodia could often be seen to interact with the cells' nano-environment. By understanding as to which features evoke spatial reactions in cells, it may be possible to design better biomaterials.

Biocompatible Materials↗
PubMed2004

The influence of microscale topography on fibroblast attachment and motility.

The ability of a cell to attach and migrate on a substrate or scaffold is important in the field of tissue engineering and biomaterials, and is thus extensively studied. When considering tissue-engineering applications, a highly porous scaffold is required to guide cell growth and proliferation in three dimensions. However existing scaffolds are less than ideal for actual applications, not only as they lack mechanical strength due to pore size and have regular distribution, but also they do not ensure cell attachment, in-growth and organisation. In this study, microfabrication technology was used to create regular arrays of pits on a two-dimensional quartz surface (7, 15 and 25 microm diameter, 20 and 40 microm spacing). The patterned surface thus exhibited spatially separated mechanical edges akin to the basic structural element of a three-dimensional network, and was used as a model system for studying the effects of substrate microgeometry on fibroblast attachment and motility. Results clearly showed that fibroblast interaction with the pit edges depended on both diameter, and therefore angle of circumference, and inter pit spacing, with the largest diameter permitting cells to enter the pits. Interestingly, the highest cell proliferation rates were recorded on the smaller pits. Such information may provide details on possible pore sizes for use in synthetic tissue engineering scaffolds that aim to support fibroblast in-growth and subsequent proliferation.

Biocompatible Materials↗
PubMed2004

Surface modified superparamagnetic nanoparticles for drug delivery: interaction studies with human fibroblasts in culture.

The concept of drug delivery using magnetic nanoparticles greatly benefit from the fact that nanotechnology has developed to a stage that it makes possible not only to produce magnetic nanoparticles in a very narrow size distribution range with superparamagnetic properties but also to engineer particle surfaces to provide site specific delivery of drugs. The size and surface characteristics of the nanoparticles are crucial factors that determine the success of the particles when used in vivo. The aim of this study was to modify the surfaces of the magnetic nanoparticles with PEG to improve the biocompatibility of the nanoparticles by resisting protein adsorption and increasing their intracellular uptake. In this study, the poly(ethyleneglycol) (PEG) modified superparamagnetic iron oxide nanoparticles have been prepared and their influence on human dermal fibroblasts is assessed in terms of cell adhesion/viability, morphology, particle uptake and cytoskeletal organisation studies. Various techniques have been used to determine nanoparticle-cell interactions including light, fluorescence, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The modification of nanoparticle surface induced alterations in cell behaviour distinct from the unmodified particles, suggesting that cell response can be directed via specifically engineered particle surfaces.

Cell Adhesion↗
PubMed2004

Use of nanotopography to study mechanotransduction in fibroblasts--methods and perspectives.

The environment around a cell during in vitro culture is unlikely to mimic those in vivo. Preliminary experiments with nanotopography have shown that nanoscale features can strongly influence cell morphology, adhesion, proliferation and gene regulation, but the mechanisms mediating this cell response remain unclear. In this perspective article, we attempt to illustrate that a possible mechanism is direct transmittal of forces encountered by cells during spreading to the nucleus via the cytoskeleton. We further try to illustrate that this 'self-induced' mechanotransduction may alter gene expression by changing interphase chromosome positioning. Whilst the observations described here to show how we think nanotopography can be developed as a tool to look at mechanotransduction are preliminary, we feel they indicate that topography may give cell biologists a non-invasive tool with which to investigate in vitro cellular mechanisms.

Animals↗