Laser-guided direct writing for applications in biotechnology.

Laser-induced optical forces can be used to guide and deposit 100 nm - 10 microm-diameter particles onto solid surfaces in a process we call 'laser-guided direct writing'. Nearly any particulate material, including both biological and electronic materials, can be manipulated and deposited on surfaces with micrometer accuracy. Potential applications include three-dimensional cell patterning for tissue engineering, hybrid biological-electronic-device construction, and biochip-array fabrication.     

Mechanostasis in apoptosis and medicine

Mechanostasis describes a complex and dynamic process where cells maintain equilibrium in response to mechanical forces. Normal physiological loading modes and magnitudes contribute to cell proliferation, tissue growth, differentiation and development. However, cell responses to abnormal forces include compensatory apoptotic mechanisms that may contribute to the development of tissue disease and pathological conditions. Mechanotransduction mechanisms tightly regulate the cell response through discrete signaling pathways. Here, we provide an overview of links between pro- and anti-apoptotic signaling and mechanotransduction signaling pathways, and identify potential clinical applications for treatments of disease by exploiting mechanically-linked apoptotic pathways.     

Rational engineering and applications of functional bioadhesives in biomedical engineering

For the past decades, several bioadhesives have been developed to replace conventional wound closure medical tools such as sutures, staples, and clips. The bioadhesives are easy to use and can minimize tissue damage. They are designed to provide strong adhesion with stable mechanical support on tissue surfaces. However, this monofunctionality of the bioadhesives hinders their practical applications. In particular, a bioadhesive can lose its intended function under harsh tissue environments or delay tissue regeneration during wound healing. Based on several natural and synthetic biomaterials, functional bioadhesives have been developed to overcome the aforementioned limitations. The functional bioadhesives are designed to have specific characteristics such as antimicrobial, cell infiltrative, stimuli-responsive, electrically conductive, and self-healing to ensure stability under harsh tissue conditions, facilitate tissue regeneration, and effectively monitor biosignals. Herein, we thoroughly review the functional bioadhesives from their fundamental background to recent progress with their practical applications for the enhancement of tissue healing and effective biosignal sensing. Furthermore, the future perspectives on the applications of functional bioadhesives and current challenges in their commercialization are also discussed.     

Electrical, chemical, and topological addressing of mammalian cells with microfabricated systems

This communication describes our work in electrical, topological, and chemical micromodification of surfaces to modulate cellular form and function. We have addressed the surface physico-chemico-mechano properties of cell culture substrates that play a role in modulating cellular behavior. Single factorial model systems have been built using techniques adapted from microlithography. The tools and techniques of microfabrication, if harnessed and used correctly, can be enabling in elucidating the underlying principles and fundamental forces driving the cell-substrate interface. Additionally, the long-term practical applications of microfabrication in medicine and biomaterial/tissue engineering lie in enabling "communication" with living cells/tissues at the cellular and subcellular levels.     

Laser microfabricated model surfaces for controlled cell growth

The relatively recent applications of microelectronics technology into the biological sciences arena has drastically revolutionized the field. New foreseeable applications include miniaturized, multiparametric biosensors for high performance multianalyte assays or DNA sequencing, biocomputers, and substrates for controlled cell growth (i.e. tissue engineering). The objectives of this work were to investigate a new method combining microphotolithographical techniques with laser excimer beam technology to create surfaces with well defined 3-D microdomains in order to delineate critical microscopic surface features governing material-cell interaction. Another obvious application of this study pertains to the fabrication of cell-based biosensors. Microfabricated surfaces were obtained with micron resolution, by "microsculpturing" polymer model surfaces using a laser excimer KrF beam coupled with a microlithographic projection technique. The laser beam after exiting a mask was focused onto the polymer target surface via an optical setup allowing for a 10-fold reduction of the mask pattern. Various 3-D micropatterned features were obtained at the micron level. Reproducible submicron features could also be obtained using this method. Subsequently, model osteoblast-like cells were plated onto the laser microfabricated surfaces in order to study the effects of particular surface microtopography on preferential cell deposition and orientation. Preferential cell deposition was observed on surfaces presenting "smooth" microtopographical transitions. This system may provide an interesting model for further insights into correlations between 3-D surface microtopography and cell response with new applications in the field biosensor, biomaterial and pharmaceutical engineering sciences (e.g. new cell based biosensors, controlled synthesis of immobilized cell derived active ingredients).     

Inhibition of fibroblast proliferation in cardiac myocyte cultures by surface microtopography

Cardiac myocyte cultures usually require pharmacological intervention to prevent overproliferation of contaminating nonmyocytes. Our aim is to prevent excessive fibroblast cell proliferation without the use of cytostatins. We have produced a silicone surface with 10-µm vertical projections that we term "pegs," to which over 80% of rat neonatal cardiac fibroblasts attach within 48 h after plating. There was a 50% decrease in cell proliferation by 5 days of culture compared with flat membranes (P < 0.001) and a concomitant 60% decrease (P < 0.01) in cyclin D1 protein levels, suggesting a G₁/S₁ cell cycle arrest due to microtopography. Inhibition of Rho kinase with 5 or 20 µM Y-27632 reduced attachment of fibroblasts to the pegs by over 50% (P < 0.001), suggesting that this signaling pathway plays an important role in the process. Using mobile and immobile 10-µm polystyrene spheres, we show that reactive forces are important for inhibiting fibroblast cell proliferation, because mobile spheres failed to reduce cell proliferation. In primary myocyte cultures, pegs also inhibit fibroblast proliferation in the absence of cytostatins. The ratio of aminopropeptide of collagen protein from fibroblasts to myosin from myocytes was significantly reduced in cultures from pegged surfaces (P < 0.01), suggesting an increase in the proportion of myocytes on the pegged surfaces. Connexin43 protein expression was also increased, suggesting improved myocyte-myocyte interaction in the presence of pegs. We conclude that this microtextured culture system is useful for preventing proliferation of fibroblasts in myocyte cultures and may ultimately be useful for tissue engineering applications in vivo. tissue engineering; cell culture; cell cycle     

Synthesis, patterning and applications of star-shaped poly(ethylene glycol) biofunctionalized surfaces

Poly(ethylene) glycol (PEG) is an excellent material to modify surfaces to resist non-specific protein adsorption. Linear PEG has been extensively studied both theoretically and experimentally and it has been found that resistance of PEG-coated surfaces to protein adsorption depends mainly on the molecular weight of the polymer and the surface grafting density. End-functionalized star-shaped PEGs allow for interpolymer crosslinking to form a dense layer. An excellent example of such a system consists of a 6-arm PEG/PPG (4 : 1) star polymer functionalized with isocyanate using IPDI. The end functionalization may be further biofunctionalized to recognize specific biomolecules such as streptavidin, His-tagged proteins, amino-terminated oligonucleotides and cell receptors. This functionalization may be patterned into specific geometries using stamping techniques or randomly distributed by statistical reaction of the end group with the biofunctional molecule in solution. The surface preparation uses simple spin-, dip- or spray-coating and produces smooth layers with low background fluorescence. These properties, together with the advantageous chemical properties of PEG, render the surfaces ideal for immobilizing proteins on surfaces with detection limits down to the single molecule level. Proteins immobilized on such surfaces are able to maintain their folded, functional form and are able to completely refold if temporarily exposed to denaturing conditions. Immobilized enzyme molecules were able to perform their function with the same activity as the enzyme in solution. Future directions of using surfaces coated with such crosslinked star polymers in highly sensitive and robust biotechnology applications will be discussed.     

Ultrastructural localization of heparan sulfate and chondroitin sulfates associated with granulopoiesis in embryonic chick bone marrow

Sulfated glycoconjugates were ultrastructurally localized within embryonic chick marrow by using the high iron diamine-silver proteinate stain. Stain was concentrated in the extravascular, granulopoietic compartment, indicating that granulopoiesis, but not erythropoiesis, proceeded in a highly sulfated environment. It was likely that most of the stainable material represented sulfated proteoglycans since staining was abrogated by predigesting tissue with enzymes and other treatments known to degrade specific glycosaminoglycan chains. Chondroitinase/hyaluronidase digestion resulted in the removal of most of the stainable material associated with the extracellular matrix and a portion of the stainable material associated with fibroblastic cell surfaces. Unaffected material lay in close proximity to fibroblastic cell membranes. Heparitinase/heparinase digestion had essentially the opposite effect. Sulfated material associated with matrix components was largely unaffected, but the fibroblastic plasmalemmal material was now absent. These results suggest that there are at least two categories of sulfated proteoglycans in the granulopoietic compartment, each differentially distributed. The plasmalemmal material likely represented heparan sulfate which in this tissue appeared to be associated in a uniform layer with fibroblastic stromal cell membranes and not with blood or endothelial cell membranes. Material identified as chondroitin sulfates was found within patches of amorphous matrix that was located on fibroblastic stromal cell surfaces and that was interspersed with fibrils in the extracellular matrix. Chondroitin sulfates were sparsely distributed on granulocytic cell surfaces.     

Guidance of Cell Migration by Substrate Dimension

There is increasing evidence to suggest that physical parameters, including substrate rigidity, topography, and cell geometry, play an important role in cell migration. As there are significant differences in cell behavior when cultured in 1D, 2D, or 3D environments, we hypothesize that migrating cells are also able to sense the dimension of the environment as a guidance cue. NIH 3T3 fibroblasts were cultured on micropatterned substrates where the path of migration alternates between 1D lines and 2D rectangles. We found that 3T3 cells had a clear preference to stay on 2D rather than 1D substrates. Cells on 2D surfaces generated stronger traction stress than did those on 1D surfaces, but inhibition of myosin II caused cells to lose their sensitivity to substrate dimension, suggesting that myosin-II-dependent traction forces are the determining factor for dimension sensing. Furthermore, oncogene-transformed fibroblasts are defective in mechanosensing while generating similar traction forces on 1D and 2D surfaces. Dimension sensing may be involved in guiding cell migration for both physiological functions and tissue engineering, and for maintaining normal cells in their home tissue.     

Application of a multiple surface tissue culture propagator for the production of cell monolayers,virus, and biochemicals

A new concept of tissue culture equipment and procedures was developed for the mass-scale growth of several types of animal tissue cells in monolayers on multiple glass surfaces. Continuous, cell lines, primary and diploid cell strains were grown in this equipment. Cells studied include primary bovine kidney, human diploid WI-38, human foreskin, and mouse CCL1 cells. Photomicrographic comparisons of cells grown by these techniques indicate they are morphologically identical to tissue culture cells grown in glass bottles or tubes. The growth of the tissue culture cells in the propagator was monitored by carbohydrate Utilization and acid production. Large-scale production of viruses and biochemicals on cells grown in the multiple-plate tissue culture propagator was accomplished. Virus titers were equal to those obtained from conventional bottle or tube cultures for several strains of influenza, parainfluenza, and respiratory syneytial viruses. High-titred mouse interferon was also produced in this system. In addition to tissue culture cell production, Eaton agent, Mycoplasma pneumoniae was grown on the multiple glass surfaces on a mass scale.     

Hematopoiesis on cellulose ester membranes (CEM). IV. Effect of variation in membrane composition and pore size

Artificial membranes composed of mixed cellulose nitrate and acetate esters (MCEM), polytetrafluoroethylene (Mitex), cellulose acetate (Celotate), polyvinylchloride (Polyvic), polycarbonate (Nuclepore), or linear polyethylene with pore sizes ranging from 0.45 mu to 70 mu were coated on their inner surfaces with a paste of bone marrow, diaphyseal bone, or cells proliferating in vitro on the inner surfaces of curetted femurs obtained from syngeneic mice. Squares of these membranes were folded into thirds, stapled to form an open-ended, tube-like structure, and implanted i.p. into mice. At intervals of 6-12 months, the membranes were removed and studied histologically. Control membranes not coated with any substance were also studied. Trilineal hematopoiesis, bone formation, sinusoidal structures, fat cells, and hemosiderin-laden macrophages were found with all three coatings in the tubes formed from the MCEM or Mitex membranes. The Celotate and Polyvic membranes coated with bone cells and the Polyvic membranes coated with marrow also developed trilineal hematopoiesis and new bone tissue. The remaining membranes essentially had only fibrous tissue, except for rare foci of hematopoiesis which developed on an occasional 30 mu (but not 70 mu) pore size linear polyethylene membrane. The results of these studies suggest that hematopoiesis and bone cell formation on these artificial membranes is closely linked, and that there may be some interaction between membrane constituents and the cells destined to form the hematopoietic support layer in the fostering of hematopoiesis. Whether this interaction is stimulatory or inhibitory was not defined by these studies.     

Mechanical force characterization in manipulating live cells with optical tweezers

Laser trapping with optical tweezers is a noninvasive manipulation technique and has received increasing attentions in biological applications. Understanding forces exerted on live cells is essential to cell biomechanical characterizations. Traditional numerical or experimental force measurement assumes live cells as ideal objects, ignoring their complicated inner structures and rough membranes. In this paper, we propose a new experimental method to calibrate the trapping and drag forces acted on live cells. Binding a micro polystyrene sphere to a live cell and moving the mixture with optical tweezers, we can obtain the drag force on the cell by subtracting the drag force on the sphere from the total drag force on the mixture, under the condition of extremely low Reynolds number. The trapping force on the cell is then obtained from the drag force when the cell is in force equilibrium state. Experiments on numerous live cells demonstrate the effectiveness of the proposed force calibration approach.     

Bacterial cellulose modified using recombinant proteins to improve neuronal and mesenchymal cell adhesion

A wide variety of biomaterials and bioactive molecules have been applied as scaffolds in neuronal tissue engineering. However, creating devices that enhance the regeneration of nervous system injuries is still a challenge, due the difficulty in providing an appropriate environment for cell growth and differentiation and active stimulation of nerve regeneration. In recent years, bacterial cellulose (BC) has emerged as a promising biomaterial for biomedical applications because of its properties such as high crystallinity, an ultrafine fiber network, high tensile strength, and biocompatibility. The small signaling peptides found in the proteins of extracellular matrix are described in the literature as promoters of adhesion and proliferation for several cell lineages on different surfaces. In this work, the peptide IKVAV was fused to a carbohydrate-binding module (CBM3) and used to modify BC surfaces, with the goal of promoting neuronal and mesenchymal stem cell (MSC) adhesion. The recombinant proteins IKVAV-CBM3 and (19)IKVAV-CBM3 were successfully expressed in E. coli, purified through affinity chromatography, and stably adsorbed to the BC membranes. The effect of these recombinant proteins, as well as RGD-CBM3, on cell adhesion was evaluated by MTS colorimetric assay. The results showed that the (19)IKVAV-CBM3 was able to significantly improve the adhesion of both neuronal and mesenchymal cells and had no effect on the other cell lineages tested. The MSC neurotrophin expression in cells grown on BC membranes modified with the recombinant proteins was also analyzed.     

Theoretical model for the formation of caveolae and similar membrane invaginations

We study a physical model for the formation of bud-like invaginations on fluid lipid membranes under tension, and apply this model to caveolae formation. We demonstrate that budding can be driven by membrane-bound proteins, provided that they exert asymmetric forces on the membrane that give rise to bending moments. In particular, caveolae formation does not necessarily require forces to be applied by the cytoskeleton. Our theoretical model is able to explain several features observed experimentally in caveolae, where proteins in the caveolin family are known to play a crucial role in the formation of caveolae buds. These include 1), the formation of caveolae buds with sizes in the 100-nm range and 2), that certain N- and C-termini deletion mutants result in vesicles that are an order-of-magnitude larger. Finally, we discuss the possible origin of the morphological striations that are observed on the surfaces of the caveolae.     

Modulation of cell adhesion, proliferation and differentiation on materials designed for body implants

The interaction of cells and tissues with artificial materials designed for applications in biotechnologies and in medicine is governed by the physical and chemical properties of the material surface. There is optimal cell adhesion to moderately hydrophilic and positively charged substrates, due to the adsorption of cell adhesion-mediating molecules (e.g. vitronectin, fibronectin) in an advantageous geometrical conformation, which makes specific sites on these molecules (e.g. specific amino acid sequences) accessible to cell adhesion receptors (e.g. integrins). Highly hydrophilic surfaces prevent the adsorption of proteins, or these molecules are bound very weakly. On highly hydrophobic materials, however, proteins are adsorbed in rigid and denatured forms, hampering cell adhesion. The wettability of the material surface, particularly in synthetic polymers, can be effectively regulated by physical treatments, e.g. by irradiation with ions, plasma or UV light. The irradiation-activated material surface can be functionalized by various biomolecules and nanoparticles, and this further enhances its attractiveness for cells and its effectiveness in regulating cell functions. Another important factor for cell-material interaction is surface roughness and surface topography. Nanostructured substrates (i.e. substrates with irregularities smaller than 100nm), are generally considered to be beneficial for cell adhesion and growth, while microstructured substrates behave more controversially (e.g. they can hamper cell spreading and proliferation but they enhance cell differentiation, particularly in osteogenic cells). A factor which has been relatively less investigated, but which is essential for cell-material interaction, is material deformability. Highly soft and deformable substrates cannot resist the tractional forces generated by cells during cell adhesion, and cells are not able to attach, spread and survive on such materials. Local variation in the physical and chemical properties of the material surface can be advantageously used for constructing patterned surfaces. Micropatterned surfaces enable regionally selective cell adhesion and directed growth, which can be utilized in tissue engineering, in constructing microarrays and in biosensorics. Nanopatterned surfaces are an effective tool for manipulating the type, number, spacing and distribution of ligands for cell adhesion receptors on the material surface. As a consequence, these surfaces are able to control the size, shape, distribution and maturity of focal adhesion plaques on cells, and thus cell adhesion, proliferation, differentiation and other cell functions.     

Substrate rigidity regulates the formation and maintenance of tissues

The ability of cells to form tissues represents one of the most fundamental issues in biology. However, it is unclear what triggers cells to adhere to one another in tissues and to migrate once a piece of tissue is planted on culture surfaces. Using substrates of identical chemical composition but different flexibility, we show that this process is controlled by substrate rigidity: on stiff substrates, cells migrate away from one another and spread on surfaces, whereas on soft substrates they merge to form tissue-like structures. Similar behavior was observed not only with fibroblastic and epithelial cell lines but also explants from neonatal rat hearts. Cell compaction on soft substrates involves a combination of weakened adhesions to the substrate and myosin II-dependent contractile forces that drive cells toward one another. Our results suggest that tissue formation and maintenance is regulated by differential mechanical signals between cell-cell and cell-substrate interactions, which in turn elicit differential contractile forces and adhesions to determine the preferred direction of cell migration and association.     

Painting and printing living bacteria: engineering nanoporous biocatalytic coatings to preserve microbial viability and intensify reactivity

Latex biocatalytic coatings containing approximately 50% by volume of microorganisms stabilize, concentrate and preserve cell viability on surfaces at ambient temperature. Coatings can be formed on a variety of surfaces, delaminated to generate stand-alone membranes or formulated as reactive inks for piezoelectric deposition of viable microbes. As the latex emulsion dries, cell preservation by partial desiccation occurs simultaneously with the formation of pores and adhesion to the substrate. The result is living cells permanently entrapped, surrounded by nanopores generated by partially coalesced polymer particles. Nanoporosity is essential for preserving microbial viability and coating reactivity. Cryo-SEM methods have been developed to visualize hydrated coating microstructure, confocal microscopy and dispersible coating methods have been developed to quantify the activity of the entrapped cells, and FTIR methods are being developed to determine the structure of vitrified biomolecules within and surrounding the cells in dry coatings. Coating microstructure, stability and reactivity are investigated using small patch or strip coatings where bacteria are concentrated 102- to 103-fold in 5-75 microm thick layers with pores formed by carbohydrate porogens. The carbohydrate porogens also function as osmoprotectants and are postulated to preserve microbial viability by formation of glasses inside the microbes during coat drying; however, the molecular mechanism of cell preservation by latex coatings is not known. Emerging applications include coatings for multistep oxidations, photoreactive coatings, stabilization of hyperthermophiles, environmental biosensors, microbial fuel cells, as reaction zones in microfluidic devices, or as very high intensity (>100 g.L-1 coating volume.h-1) industrial or environmental biocatalysts. We anticipate expanded use of nanoporous adhesive coatings for prokaryotic and eukaryotic cell preservation at ambient temperature and the design of highly reactive "living" paints and inks.     

Characterizing the effect of polymyxin B antibiotics to lipopolysaccharide on Escherichia coli surface using atomic force microscopy

Lipopolysaccharide (LPS) on gram‐negative bacterial outer membranes is the first target for antimicrobial agents, due to their spatial proximity to outer environments of microorganisms. To develop antibacterial compounds with high specificity for LPS binding, the understanding of the molecular nature and their mode of recognition is of key importance. In this study, atomic force microscopy (AFM) and single molecular force spectroscopy were used to characterize the effects of antibiotic polymyxin B (PMB) to the bacterial membrane at the nanoscale. Isolated LPS layer and the intact bacterial membrane were examined with respect to morphological changes at different concentrations of PMB. Our results revealed that 3 hours of 10 μg/mL of PMB exposure caused the highest roughness changes on intact bacterial surfaces, arising from the direct binding of PMB to LPS on the bacterial membrane. Single molecular force spectroscopy was used to probe specific interaction forces between the isolated LPS layer and PMB coupled to the AFM tip. A short range interaction regime mediated by electrostatic forces was visible. Unbinding forces between isolated LPS and PMB were about 30 pN at a retraction velocity of 500 nm/s. We further investigated the effects of the polycationic peptide PMB on bacterial outer membranes and monitored its influences on the deterioration of the bacterial membrane structure. Polymyxin B binding led to rougher appearances and wrinkles on the outer membranes surface, which may finally lead to lethal membrane damage of bacteria. Our studies indicate the potential of AFM for applications in pathogen recognition and nano‐resolution approaches in microbiology.     

Isolation of separate apical,lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

The tissue used in this study was the midgut of the tobacco hornworm larva, Manduca sexta. The midgut epithelium is a single layer of cells resting on a thin basal lamina and underlying discontinuous muscle layer. The epithelial cells are of two main types, goblet and columnar cells, joined together by the septate junctions characteristic of insect epithelia. From this tissue we were able to isolate four distinct plasma membrane fractions; the lateral membranes, the columnar cell apical membrane, the goblet cell apical membrane and a preparation of basal membranes from both cell types. The lateral membranes were isolated by density gradient centrifugation following gentle homogenization of the midgut hypotonic medium, which caused the cells to rupture at their apical and basal surfaces, releasing long segments of lateral membranes still joined by their septate junctions. For isolation of apical and basal membranes the tissue was disrupted by ultrasound, based on the light microscopic observation that carefully controlled ultrasound can be used to disrupt each cell in layers starting at the apical surface. The top layer contained the columnar cell apical membrane, which consists of microvilli forming a brush border covering the lumenal surface of the epithelium. The second layer contained the goblet cell apical membrane, which is invaginated to form a cavity occupying the apical half of the cell, and the third layer contained the basal membranes. As each layer was stripped off the epithelium it was collected and the plasma membrane purified by differential or density gradient centrifugation. For all four membrane fractions, the isolation procedure was designed to preserve the original structure of the membrane as far as possible. This allowed electron microscopy to be used to follow each step in the isolation procedure, and to identify the constituents of each subcellular preparation. Although developed specifically for M. sexta midgut, these techniques could readily be modified for use on other epithelia.     

Preparation and characterization of novel beta-chitin-hydroxyapatite composite membranes for tissue engineering applications

Beta-chitin is a biopolymer principally found in shells of squid pen. It has the properties of biodegradability, biocompatibility, chemical inertness, wound healing, antibacterial and anti-inflammatory activities. Hydroxyapatite (HAp) is a natural inorganic component of bone and teeth and has osteoconductive property. In this work, beta-chitin-HAp composite membranes were prepared by alternate soaking of beta-chitin membranes in CaCl2 (pH 7.4) and Na2HPO4 solutions for 2 h in each solution. After 1, 3 and 5 cycles of immersion, beta-chitin membranes were characterized using the SEM, FT-IR, EDS and XRD analyses. The results showed the presence of apatite layer on surface of beta-chitin membranes, and the amounts of size and deposition of apatite layers were increased with increasing number of immersion cycles. Human mesenchymal stem cells (hMSCs) were used for evaluation of the biocompatibility of pristine as well as composite membranes for tissue engineering applications. The presence of apatite layers on the surface of beta-chitin membranes increased the cell attachment and spreading suggesting that beta-chitin-HAp composite membranes can be used for tissue engineering applications.