Phenolics appear to be everywhere: they dominate the organic content of our landscape (humics and lignins); they impart color to plants and animals (flavonoids and melanins); they enable biology to harvest energy (quinone redox couples in respiration and photosynthesis); and they may enhance our health (dietary antioxidants). Biology enlists the diverse physicochemical properties of phenolics to perform a variety of functions that are not yet fully understood or appreciated. In this review, the synthesis, structures and functions of biological phenols is briefly sketched while an array of examples is provided to illustrate their diverse functions. The focus of this review however is recent studies which show that the biomimetic incorporation of phenolics into thin chitosan films can controllably impart mechanical, optical and redox properties. These studies demonstrate the potential for accessing nature's rich diversity of phenolics to impart functionality to soft matter.

Phenolics appear to be everywhere: they dominate the organic content of our landscape (humics and lignins); they impart color to plants and animals (flavonoids and melanins); they enable biology to harvest energy (quinone redox couples in respiration and photosynthesis); and they may enhance our health (dietary antioxidants). Biology enlists the diverse physicochemical properties of phenolics to perform a variety of functions that are not yet fully understood or appreciated. In this review, the synthesis, structures and functions of biological phenols is briefly sketched while an array of examples is provided to illustrate their diverse functions. The focus of this review however is recent studies which show that the biomimetic incorporation of phenolics into thin chitosan films can controllably impart mechanical, optical and redox properties. These studies demonstrate the potential for accessing nature's rich diversity of phenolics to impart functionality to soft matter.

PURPOSE:Regenerative medicine will benefit from technologies capable of fabricating soft matter to have appropriate architectures and that provide the necessary physical, chemical and biological cues to recruit cells and guide their development. The goal of this report is to review an emerging set of biofabrication techniques and suggest how these techniques could be applied for the fabrication of scaffolds for tissue engineering.METHODS:Electrical potentials are applied to submerged electrodes to perform cathodic and anodic reactions that direct stimuli-responsive film-forming polysaccharides to assemble into hydrogel films. Standard methods are used to microfabricate electrode surfaces to allow the electrical signals to be applied with spatial and temporal control. The enzymes mushroom tyrosinase and microbial transglutaminase are used to catalyze macromolecular grafting and crosslinking of proteins.RESULTS:Electrodeposition of the polysaccharides chitosan and alginate allow hydrogel films to be formed in response to localized electrical signals. Co-deposition of various components (e.g., proteins, vesicles and cells), and subsequent electrochemical processing allow the physical, chemical and biological activities of these films to be tailored. Enzymatic processing allows for the generation of stimuli-responsive protein conjugates that can also be directed to assemble in response to imposed electrical signals. Further, enzyme-catalyzed crosslinking of gelatin allows replica molding of soft matter to create hydrogel films with topological structure.CONCLUSIONS:Biofabrication with biological materials and mechanisms provides new approaches for soft matter construction. These methods may enable the formation of tissue engineering scaffolds with appropriate architectures, assembled cells, and spatially organized physical, chemical and biological cues.

The potential of an in situ gel-forming adhesive was examined as a hemostatic surgical sealant. The gel-forming mechanism for this adhesive mimics the last stages of blood coagulation but uses nonblood proteins. Specifically, gelatin is used as the structural protein and a calcium-independent microbial transglutaminase (mTG) is used as the crosslinking enzyme. lit vitro burst pressure tests with porcine skin demonstrate that the gelatin-mTG adhesive forms a gel within 30 min under moist conditions and this gel can restrain pressures of 200 mmHg. In vivo tests with a rat liver wound model showed that the gelatin-mTG adhesive achieves complete hemostasis in 2.5 min and the gel (i.e., the biomimetic clot) offers substantial adhesive and cohesive strength. Complete hemostasis was also observed in 2.5 min after the gelatin-mTG adhesive was applied to a briskly bleeding rat femoral artery wound. In a large animal porcine model, a femoral artery wound that resulted in extensive bleeding was sealed in 4 min by (i) clamping the artery for temporary hemostasis, (ii) removing excess blood, and (iii) applying the gelatin-mTG adhesive. Thus, the biomimetic gelatin-mTG adhesive may provide a simple, safe, and cost-effective surgical sealant. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 91B: 5-16, 2009

An alternative route to protein assembly at surfaces based on using the unique capabilities of biological materials for the spatially selective assembly of proteins is described. Specifically, the stimuli-responsive properties of aminopolysaccharide chitosan are combined with the molecular-recognition capabilities of biotin-streptavidin binding. Biotinylated chitosan retains its stimuli-responsive properties and is capable of electrodepositing at specific electrode addresses. Once deposited, it is capable of binding streptavidin, which can mediate the subsequent assembly of biotinylated proteins. Spatially selective protein assembly using biotinylated Protein A and fluorescently-labeled antibodies is demonstrated.

Gelatin is one of the most commonly used biomaterials for creating cellular scaffolds due to its innocuous nature. In order to create stable gelatin hydrogels at physiological temperatures (37 degrees C), chemical crosslinking agents such as glutaraldehyde are typically used. To circumvent potential problems with residual amounts of these crosslinkers in vivo and create scaffolds that are both physiologically robust and biocompatible, a microbial transglutaminase (mTG) was used in this study to enzymatically crosslink gelatin solutions. HEK293 cells encapsulated in mTG-crosslinked gelatin proliferated at a rate of 0.03 day(-1). When released via proteolytic degradation with trypsin, the cells were able to recolonize tissue culture flasks, suggesting that cells for therapeutic purposes could be delivered in vivo using an mTG-crosslinked gelatin construct. Upon submersion in a saline solution at 37 degrees C, the mTG-crosslinked gelatin exhibited no mass loss, within experimental error, indicating that the material is thermally stable. The proteolytic degradation rate of mTG-crosslinked gelatin at RT was slightly faster than that of thermally-cooled (physically-crosslinked) gelatin. Thermally-cooled gelatin that was subsequently crosslinked with mTG resulted in hydrogels that were more resistant to proteolysis. Degradation rates were found to be tunable with gelatin content, an attribute that may be useful for either long-time cell encapsulation or time-released regenerative cell delivery. Further investigation showed that proteolytic degradation was controlled by surface erosion.

Microelectronic devices employ electrons for signaling whereas the nervous system signals using ions and chemicals. Bridging these signaling differences would benefit applications that range from biosensing to neuroprosthetics. Here, we report the use of localized electrical signals to perform an operation common to chemical signaling in the nervous system. Specifically, we employ electrical signals to restrain vesicles reversibly. We perform this operation using the stimuli-responsive aminopolysaccharide chitosan that is able to electrodeposit onto cathode surfaces in response to localized electrical stimuli. We show that surfactant-vesicles and liposomes can be co-deposited with chitosan and are entrapped (i.e., restrained) within the deposited film's matrix. Vesicle co-deposition could be controlled spatially and temporally using microfabricated wafers with independent electrode addresses. Finally, we show that vesicles restrained within the deposited chitosan matrix can be mobilized under mildly acidic conditions (pH < 6.5) that resolubilize chitosan. Potentially, the ability to restrain and mobilize chemical signals that are segregated within vesicles may allow microfluidic systems to access the rich diversity offered by chemical signaling.

While microcantilevers offer exciting opportunities for mechano-detection, they often suffer from limitations in either sensitivity or selectivity. To address these limitations, we electrodeposited a chitosan film onto a cantilever surface and mechano-transduced detection events through the chitosan network. Our first demonstration was the detection of nucleic acid hybridization. In this instance, we electrodeposited the chitosan film onto the cantilever, biofunctionalized the film with oligonucleotide probe, and detected target DNA hybridization by cantilever bending in solution (static mode) or resonant frequency shifts in air (dynamic mode). In both detection modes, we observed a two-order of magnitude increase in sensitivity compared to values reported in literature for DNA immobilized on self-assembled monolayers. In our second demonstration, we coupled electrochemical and mechanical modes to selectively detect the neurotransmitter dopamine. A chitosan-coated cantilever was biased to electrochemically oxidize dopamine solution. Dopamine's oxidation products react with the chitosan film and create a tensile stress of approximately 1.7 MPa, causing substantial cantilever bending. A control experiment was performed with ascorbic acid solution. It was shown that the electrochemical oxidation of ascorbic acid does not lead to reactions with chitosan and does not change cantilever bending. These results suggest that chitosan can confer increased sensitivity and selectivity to microcantilever sensors.

Phenol reaction cascades are commonly used in nature to create crosslinked materials that perform mechanical functions. These processes are mimicked by electrochemically initiating a reaction cascade to examine if the mechanical properties of a biopolymer film can be predictably altered. Specifically, thin films (approximate to 30-45 mu m) of the polysaccharide chitosan are cast onto gold-coated silicon wafers, the chitosan-coated wafers are immersed in catechol-containing solutions, and the phenol is anodically oxidized. The product of this oxidation is highly reactive and undergoes reaction with chitosan chains adjacent to the anode. After reaction, the flexible chitosan film can be peeled from the wafer. Chemical and physical evidence support the conclusion that electrochemically initiated reactions crosslink chitosan. When gold is patterned onto the wafer, the electrochemical crosslinking reactions are spatially localized and impart anisotropic mechanical properties to the chitosan film. Further, deswelling of chitosan films can reversibly transduce environmental stimuli into contractile forces. Films patterned to have spatial variations in crosslinking respond to such environmental stimuli by undergoing reversible changes in shape. These results suggest the potential to enlist electrochemically initiated reaction cascades to engineer chitosan films for actuator functions.

Biological systems routinely use phenols to construct complex materials with diverse functions. Typically, these phenolic materials are generated using oxidative enzymes to initiate a cascade of uncatalyzed reactions. We mimic these processes to micro-pattern films of aminopolysaccharide chitosan. Specifically, we microfabric silicon wafers to have gold patterns, cast a chitosan film onto the patterned wafers, and commence pattern transfer by polarizing the underlying gold surfaces to electro-chemically initiate the phenol reaction cascade. The electrochemically initiated reactions lead to modification of the chitosan film's chemistry, structure, and fluorescence. Further, electrochemically initiated modification of the chitosan film is localized to the interfacial region between the film and the anode, with resolution in the lateral direction of at least 20 mum. These results demonstrate that electrochemical pattern transfer provides a promising new method for micropatterning flexible films.

The signal-guided and sequential assembly of biomolecules onto patterned surfaces is demonstrated. Readily transmittable electric signals are used to guide spatially selective deposition of the pH-responsive polysaccharide, chitosan, and functionalized chitosan conjugates, by generating localized pH gradients. The nucleophilic primary amine groups of chitosan enable facile conjugation of proteins and nucleic acids by two approaches, one an enzymatic approach and the other a standard chemical modification, thus providing flexibility when sequentially assembling biomolecules in a spatially selective manner. Moreover, we developed an agarose gel "biomask" for the sequential assembly of single-stranded DNA and confirmed its functionality through nucleic acid hybridization assays.

The traditional motivation for integrating biological components into microfabricated devices has been to create biosensors that meld the molecular recognition capabilities of biology with the signal processing capabilities of electronic devices. However, a different motivation is emerging; biological components are being explored to radically change how fabrication is achieved at the micro- and nanoscales. Here we review biofabrication, the use of biological materials for fabrication, and focus on three specific biofabrication approaches: directed assembly, where localized external stimuli are employed to guide assembly; enzymatic assembly, where selective biocatalysts are enlisted to build macromolecular structure; and self-assembly, where information internal to the biological material guides its own assembly. Also reviewed are recent results with the aminopolysaccharide chitosan, a material that offers a combination of properties uniquely suited for biofabrication. In particular, chitosan can be directed to assemble in response to locally applied electrical signals, and the chitosan backbone provides sites that can be employed for the assembly of proteins, nucleic acids, and virus particles.

A nucleic acid hybridization assay was assembled onto a robust and readily addressable silicon-based chip using polysaccharide chitosan as a scaffold for the covalent coupling of probe DNA to the chip's surface. Chitosan is a unique polymer, ideally suited for this application because its net charge and solubility are pH dependent. Specifically in this work, gold-patterned electrodes were created using standard photolithographic techniques, chitosan was electrodeposited in a spatially resolved manner onto the polarized electrodes, probe DNA was covalently assembled onto the chitosan, and both DNA: DNA and DNA:mRNA hybridization detection schemes were evaluated. Hybridization of target nucleic acid was quantifiable, reproducible, and robust; the surface was regenerated and rehybridized up to eight times without loss of signal. Finally, transcriptional upregulation of the Escherichia coli chaperone, DnaK, which is an indicator of cellular stress, was observed using the hybridization chip sandwich assay. Thus, this method enables rapid and facile monitoring of gene expression in a format that is reusable and requires minimal reagent quantities.