Quantitative Real-Time Analysis of Living Materials by Stimulated Raman Scattering Microscopy.

Composite materials built in part from living organisms have the potential to exhibit useful autonomous, adaptive, and self-healing behavior. The physicochemical, biological, and mechanical properties of such materials can be engineered through the genetic manipulation of their living components. Successful development of living materials will require not only new methods for design and preparation but also new analytical tools that are capable of real-time noninvasive mapping of chemical compositions. Here, we establish a strategy based on stimulated Raman scattering microscopy to monitor phosphatase-catalyzed mineralization of engineered bacterial films in situ. Real-time label-free imaging elucidates the mineralization process, quantifies both the organic and inorganic components of the material as functions of time, and reveals spatial heterogeneity at multiple scales. In addition, we correlate the mechanical performance of films with the extent of mineralization. This work introduces a promising strategy for quantitatively analyzing living materials, which should contribute to the accelerated development of such materials in the future.

Crimping-induced structural gradients explain the lasting strength of poly l-lactide bioresorbable vascular scaffolds during hydrolysis.

Biodegradable polymers open the way to treatment of heart disease using transient implants (bioresorbable vascular scaffolds, BVSs) that overcome the most serious complication associated with permanent metal stents-late stent thrombosis. Here, we address the long-standing paradox that the clinically approved BVS maintains its radial strength even after 9 mo of hydrolysis, which induces a ∼40% decrease in the poly l-lactide molecular weight (Mn). X-ray microdiffraction evidence of nonuniform hydrolysis in the scaffold reveals that regions subjected to tensile stress during crimping develop a microstructure that provides strength and resists hydrolysis. These beneficial morphological changes occur where they are needed most-where stress is localized when a radial load is placed on the scaffold. We hypothesize that the observed decrease in Mn reflects the majority of the material, which is undeformed during crimping. Thus, the global measures of degradation may be decoupled from the localized, degradation-resistant regions that confer the ability to support the artery for the first several months after implantation.

Multiplicity of morphologies in poly (l-lactide) bioresorbable vascular scaffolds.

Poly(l-lactide) (PLLA) is the structural material of the first clinically approved bioresorbable vascular scaffold (BVS), a promising alternative to permanent metal stents for treatment of coronary heart disease. BVSs are transient implants that support the occluded artery for 6 mo and are completely resorbed in 2 y. Clinical trials of BVSs report restoration of arterial vasomotion and elimination of serious complications such as late stent thrombosis. It is remarkable that a scaffold made from PLLA, known as a brittle polymer, does not fracture when crimped onto a balloon catheter or during deployment in the artery. We used X-ray microdiffraction to discover how PLLA acquired ductile character and found that the crimping process creates localized regions of extreme anisotropy; PLLA chains in the scaffold change orientation from the hoop direction to the radial direction on micrometer-scale distances. This multiplicity of morphologies in the crimped scaffold works in tandem to enable a low-stress response during deployment, which avoids fracture of the PLLA hoops and leaves them with the strength needed to support the artery. Thus, the transformations of the semicrystalline PLLA microstructure during crimping explain the unexpected strength and ductility of the current BVS and point the way to thinner resorbable scaffolds in the future.

Visible-light-initiated thiol-acrylate photopolymerization of heparin-based hydrogels.

An in situ heparin-based forming hydrogel that cures under visible-light is formulated using eosin Y as a photoinitiator with triethanolamine as an electron donor to initiate reaction of thiolated-heparin with acrylate-ended poly(ethylene glycol). Formulations and irradiation conditions are presented for control of heparin content (1.6 to 3.3% w/v), modulus (100-10,000 Pa), and gelation time (30-600 s). Encapsulation of 3T3 fibroblasts in the hydrogel gave over 96% viability for all conditions examined. In vitro characterization of epidermal growth factor released from the hydrogel confirmed that the growth factor remains bioactive. The ability to deliver growth factors, fast gelation kinetics under visible light, and independent control of physical and biochemical properties makes this system a promising candidate for use in regenerative medicine. In particular, irradiation conditions that achieve gelation in 150s are compatible with the stringent light exposure limits of the retina, which affords a wide safety margin for use with other tissues.

Living Material with Temperature-Dependent Light Absorption.

Engineered living materials (ELMs) exhibit desirable characteristics of the living component, including growth and repair, and responsiveness to external stimuli. Escherichia coli (E. coli) are a promising constituent of ELMs because they are very tractable to genetic engineering, produce heterologous proteins readily, and grow exponentially. However, seasonal variation in ambient temperature presents a challenge in deploying ELMs outside of a laboratory environment because E. coli growth rate is impaired both below and above 37 °C. Here, a genetic circuit is developed that controls the expression of a light-absorptive chromophore in response to changes in temperature. It is demonstrated that at temperatures below 36 °C, the engineered E. coli increase in pigmentation, causing an increase in sample temperature and growth rate above non-pigmented counterparts in a model planar ELM. On the other hand, at above 36 °C, they decrease in pigmentation, protecting the growth compared to bacteria with temperature-independent high pigmentation. Integrating the temperature-responsive circuit into an ELM has the potential to improve living material performance by optimizing growth and protein production in the face of seasonal temperature changes.

Tunable Temperature-Sensitive Transcriptional Activation Based on Lambda Repressor.

Temperature is a versatile input signal for the control of engineered cellular functions. Sharp induction of gene expression with heat has been established using bacteria- and phage-derived temperature-sensitive transcriptional repressors with tunable switching temperatures. However, few temperature-sensitive transcriptional activators have been reported that enable direct gene induction with cooling. Such activators would expand the application space for temperature control. In this technical note, we show that temperature-dependent versions of the Lambda phage repressor CI can serve as tunable cold-actuated transactivators. Natively, CI serves as both a repressor and activator of transcription. Previously, thermolabile mutants of CI, known as the TcI family, were used to repress the cognate promoters PR and PL. We hypothesized that TcI mutants can also serve as temperature-sensitive activators of transcription at CI's natural PRM promoter, creating cold-inducible operons with a tunable response to temperature. Indeed, we demonstrate temperature-responsive activation by two variants of TcI with set points at 35.5 and 38.5 °C in E. coli. In addition, we show that TcI can serve as both an activator and a repressor of different genes in the same genetic circuit, leading to opposite thermal responses. Transcriptional activation by TcI expands the toolbox for control of cellular function using globally or locally applied thermal inputs.

Tungsten disulfide nanotubes enhance flow-induced crystallization and radio-opacity of polylactide without adversely affecting in vitro toxicity.

Treatment of vascular disease, from peripheral ischemia to coronary heart disease (CHD), is poised for transformation with the introduction of transient implants designed to "scaffold" regeneration of blood vessels and ultimately leave nothing behind. Improved materials could expand the use of these devices. Here, we examine one of the leading polymers for bioresorbable scaffolds (BRS), polylactide (PLA), as the matrix of nanocomposites with tungsten disulfide (WS2) nanotubes (WSNT), which may provide mechanical reinforcement and enhance radio-opacity. We evaluate in vitro cytotoxicity using vascular cells, flow-induced crystallization and radio-opacity of PLA-WSNT nanocomposites at low WSNT concentration. A small amount of WSNT (0.1 wt%) can effectively promote oriented crystallization of PLA without compromising molecular weight. And radio-opacity improves significantly: as little as 0.5 to 1 wt% WSNT doubles the radio-opacity of PLA-WSNT relative to PLA at 17 keV. The results suggest that a single component, WSNT, has the potential to increase the strength of BRS to enable thinner devices and increase radio-opacity to improve intraoperative visualization. The in vitro toxicity results indicate that PLA-WSNT nanocomposites are worthy of investigation in vivo. Although substantial further preclinical studies are needed, PLA-WSNT nanocomposites may provide a complement of material properties that may improve BRS and expand the range of lesions that can be treated using transient implants. STATEMENT OF SIGNIFICANCE: Bioresorbable Scaffolds (BRSs) support regeneration of arteries without permanent mechanical constraint. Poly-L-lactide (PLLA) is the structural material of the first approved BRS for coronary heart disease (ABSORB BVS), withdrawn due to adverse events in years 1-3. Here, we examine tungsten disulfide (WS2) nanotubes (WSNT) in PLA to address two contributors to early complications: (1) reinforce PLLA (enable thinner BRS), and (2) increase radiopacity (provide intraoperative visibility). For BRS, it is significant that WSNT disperse, remain dispersed, reduce friction and improve mechanical properties without additional chemicals or surface modifications. Like WS2 nanospheres, bare WSNT and PLA-WSNT nanocomposites show low cytotoxicity in vitro. PLA-WSNT show enhanced flow-induced crystallization relative to PLA, motivating future study of the processing behavior and strength of these materials.

Competition between CO2-philicity and Mixing Entropy Leads to CO2 Solubility Maximum in Polyether Polyols.

In carbon dioxide-blown polymer foams, the solubility of carbon dioxide (CO2) in the polymer profoundly shapes the structure and, consequently, the physical properties of the foam. One such foam is polyurethane-commonly used for thermal insulation, acoustic insulation, and cushioning-which increasingly relies on CO2 to replace environmentally harmful blowing agents. Polyurethane is produced through the reaction of isocyanate and polyol, of which the polyol has the higher capacity for dissolving CO2. While previous studies have suggested the importance of the effect of hydroxyl end groups on CO2 solubility in short polyols (<1000 g/mol), their effect in polyols with higher molecular weight (≥1000 g/mol) and higher functionality (>2 hydroxyls per chain)-as are commonly used in polyurethane foams-has not been reported. Here, we show that the solubility of CO2 in polyether polyols decreases with molecular weight above 1000 g/mol and decreases with functionality using measurements performed by gravimetry-axisymmetric drop-shape analysis. The nonmonotonic effect of molecular weight on CO2 solubility results from the competition between effects that reduce CO2 solubility (lower mixing entropy) and effects that increase CO2 solubility (lower ratio of hydroxyl end groups to ether backbone groups). To generalize our measurements, we modeled the CO2 solubility using a perturbed chain-statistical associating fluid theory (PC-SAFT) model, which we validated by showing that a density functional theory model based on the PC-SAFT free energy accurately predicted the interfacial tension.

Interaction of Poly L-Lactide and Tungsten Disulfide Nanotubes Studied by in Situ X-ray Scattering during Expansion of PLLA/WS2NT Nanocomposite Tubes.

In situ synchrotron X-ray scattering was used to reveal the transient microstructure of poly(L-lactide) (PLLA)/tungsten disulfide inorganic nanotubes (WS2NTs) nanocomposites. This microstructure is formed during the blow molding process ("tube expansion") of an extruded polymer tube, an important step in the manufacturing of PLLA-based bioresorbable vascular scaffolds (BVS). A fundamental understanding of how such a microstructure develops during processing is relevant to two unmet needs in PLLA-based BVS: increasing strength to enable thinner devices and improving radiopacity to enable imaging during implantation. Here, we focus on how the flow generated during tube expansion affects the orientation of the WS2NTs and the formation of polymer crystals by comparing neat PLLA and nanocomposite tubes under different expansion conditions. Surprisingly, the WS2NTs remain oriented along the extrusion direction despite significant strain in the transverse direction while the PLLA crystals (c-axis) form along the circumferential direction of the tube. Although WS2NTs promote the nucleation of PLLA crystals in nanocomposite tubes, crystallization proceeds with largely the same orientation as in neat PLLA tubes. We suggest that the reason for the unusual independence of the orientations of the nanotubes and polymer crystals stems from the favorable interaction between PLLA and WS2NTs. This favorable interaction leads WS2NTs to disperse well in PLLA and strongly orient along the axis of the PLLA tube during extrusion. As a consequence, the nanotubes are aligned orthogonally to the circumferential stretching direction, which appears to decouple the orientations of PLLA crystals and WS2NTs.

Efficient synthesis of narrowly dispersed brush copolymers and study of their assemblies: the importance of side chain arrangement.

Efficient, one-pot preparation of synthetically challenging, high molecular weight (MW), narrowly dispersed brush block copolymers and random copolymers in high conversions was achieved by ring-opening metathesis (co)polymerization (ROMP) of various macromonomers (MMs) using the highly active, fast-initiating ruthenium olefin metathesis catalyst (H(2)IMes)(pyr)(2)(Cl)(2)RuCHPh. A series of random and block copolymers were prepared from a pair of MMs containing polylactide (PLA) and poly(n-butyl acrylate) (PnBA) side chains at similar MWs. Their self-assembly in the melt state was studied by small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM). In brush random copolymers containing approximately equal volume fractions of PLA and PnBA, the side chains segregate into lamellae with domain spacing of 14 nm as measured by SAXS, which was in good agreement with the lamellar thickness measured by AFM. The domain spacings and order-disorder transition temperatures of brush random copolymers were insensitive to the backbone length. In contrast, brush block copolymers containing approximately equal volume fractions of these MMs self-assembled into highly ordered lamellae with domain spacing over 100 nm. Their assemblies suggested that the brush block copolymer backbone adopted an extended conformation in the ordered state.

Ring-expansion metathesis polymerization: catalyst-dependent polymerization profiles.

Ring-expansion metathesis polymerization (REMP) mediated by recently developed cyclic Ru catalysts has been studied in detail with a focus on the polymer products obtained under varied reaction conditions and catalyst architectures. Depending upon the nature of the catalyst structure, two distinct molecular weight evolutions were observed. Polymerization conducted with catalysts bearing six-carbon tethers displayed rapid polymer molecular weight growth which reached a maximum value at ca. 70% monomer conversion, resembling a chain-growth polymerization mechanism. In contrast, five-carbon-tethered catalysts led to molecular weight growth that resembled a step-growth mechanism with a steep increase occurring only after 95% monomer conversion. The underlying reason for these mechanistic differences appeared to be ready release of five-carbon-tethered catalysts from growing polymer rings, which competed significantly with propagation. Owing to reversible chain transfer and the lack of end groups in REMP, the final molecular weights of cyclic polymers was controlled by thermodynamic equilibria. Large ring sizes in the range of 60-120 kDa were observed at equilibrium for polycyclooctene and polycyclododecatriene, which were found to be independent of catalyst structure and initial monomer/catalyst ratio. While six-carbon-tethered catalysts were slowly incorporated into the formed cyclic polymer, the incorporation of five-carbon-tethered catalysts was minimal, as revealed by ICP-MS. Further polymer analysis was conducted using melt-state magic-angle spinning (13)C NMR spectroscopy of both linear and cyclic polymers, which revealed little or no chain ends for the latter topology.

Well-defined liquid crystal gels from telechelic polymers.

Well-defined liquid crystal networks with controlled molecular weight between cross-links and cross-link functionality were prepared by "click" cross-linking of telechelic polymers produced by ring-opening metathesis polymerization (ROMP). The networks readily swell in a small molecule liquid crystal, 5CB, to form LC gels with high swelling ratios. These gels exhibit fast, reversible, and low-threshold optic switching under applied electric fields when they are unconstrained between electrodes. For a given electric field, the LC gels prepared from shorter telechelic polymers showed a reduced degree of switching than their counterparts made from longer polymer strands. The reported approach provides control over important parameters for LC networks, such as the length of the network strands between cross-links, cross-linker functionality, and mesogen density. Therefore, it allows a detailed study of relationships between molecular structure and macroscopic properties of these scientifically and technologically interesting networks.

Cyclic ruthenium-alkylidene catalysts for ring-expansion metathesis polymerization.

A series of cyclic Ru-alkylidene catalysts have been prepared and evaluated for their efficiency in ring-expansion metathesis polymerization (REMP). The catalyst structures feature chelating tethers extending from one N-atom of an N-heterocyclic carbene (NHC) ligand to the Ru metal center. The catalyst design is modular in nature, which provided access to Ru complexes having varying tether lengths, as well as electronically different NHC ligands. Structural impacts of the tether length were unveiled through (1)H NMR spectroscopy as well as single-crystal X-ray analyses. Catalyst activities were evaluated via polymerization of cyclooctene, and key data are provided regarding propagation rates, intramolecular chain transfer, and catalyst stabilities, three areas necessary for the efficient synthesis of cyclic poly(olefin)s via REMP. From these studies, it was determined that while increasing the tether length of the catalyst leads to enhanced rates of polymerization, shorter tethers were found to facilitate intramolecular chain transfer and release of catalyst from the polymer. Electronic modification of the NHC via backbone saturation was found to enhance polymerization rates to a greater extent than did homologation of the tether. Overall, cyclic Ru complexes bearing 5- or 6-carbon tethers and saturated NHC ligands were found to be readily synthesized, bench-stable, and highly active catalysts for REMP.

Rheological properties of the vitreous and the role of hyaluronic acid.

Coordinated rheological and biochemical measurements provide the linear and nonlinear mechanical properties of the vitreous and demonstrate the structural role of hyaluronic acid. "Cleated" tools are used to overcome wall slip and avoid tissue compression during measurements of the dynamic moduli of fresh porcine and bovine vitreous. Shear moduli decreased five-fold from initial to steady-state values in the first hour after dissection. Steady-state values (porcine: G'=2.8+/-0.9Pa, n=9; bovine: G'=7.0+/-2.0Pa, n=17) are significantly greater than previously reported. The decrease in modulus after removal from the eye correlates with a decrease in mass: even in the absence of external driving forces, porcine vitreous expels approximately 5% of its mass within 5min and continues to decay to a steady-state mass approximately 10% lower than its initial mass. The expelled fluid has a substantial hyaluronan concentration, but very low protein content. These results indicate that the vitreous network is under tension at its native volume and its high initial modulus results from this state of tension. We hypothesize that hyaluronan plays a role in sustaining the "internal tension" by Donnan swelling.

Tube Expansion Deformation Enables In Situ Synchrotron X-ray Scattering Measurements during Extensional Flow-Induced Crystallization of Poly l-Lactide Near the Glass Transition.

Coronary Heart Disease (CHD) is one of the leading causes of death worldwide, claiming over seven million lives each year. Permanent metal stents, the current standard of care for CHD, inhibit arterial vasomotion and induce serious complications such as late stent thrombosis. Bioresorbable vascular scaffolds (BVSs) made from poly l-lactide (PLLA) overcome these complications by supporting the occluded artery for 3⁻6 months and then being completely resorbed in 2⁻3 years, leaving behind a healthy artery. The BVS that recently received clinical approval is, however, relatively thick (~150 µm, approximately twice as thick as metal stents ~80 µm). Thinner scaffolds would facilitate implantation and enable treatment of smaller arteries. The key to a thinner scaffold is careful control of the PLLA microstructure during processing to confer greater strength in a thinner profile. However, the rapid time scales of processing (~1 s) defy prediction due to a lack of structural information. Here, we present a custom-designed instrument that connects the strain-field imposed on PLLA during processing to in situ development of microstructure observed using synchrotron X-ray scattering. The connection between deformation, structure and strength enables processing⁻structure⁻property relationships to guide the design of thinner yet stronger BVSs.

Director dynamics in liquid-crystal physical gels.

Nematic liquid-crystal (LC) elastomers and gels have a rubbery polymer network coupled to the nematic director. While LC elastomers show a single, non-hydrodynamic relaxation mode, dynamic light-scattering studies of self-assembled liquid-crystal gels reveal orientational fluctuations that relax over a broad time scale. At short times, the relaxation dynamics exhibit hydrodynamic behavior. In contrast, the relaxation dynamics at long times are non-hydrodynamic, highly anisotropic, and increase in amplitude at small scattering angles. We argue that the slower dynamics arise from coupling between the director and the physically associated network, which prevents director orientational fluctuations from decaying completely at short times. At long enough times the network restructures, allowing the orientational fluctuations to fully decay. Director dynamics in the self-assembled gels are thus quite distinct from those observed in LC elastomers in two respects: they display soft orientational fluctuations at short times, and they exhibit at least two qualitatively distinct relaxation processes.

Fabrication of Active Surfaces with Metastable Microgel Layers Formed during Breath Figure Templating.

Patterned porous surfaces with responsive functionalities are fabricated by a thermoresponsive microgel-assisted breath figure (BF) process. When water droplets submerge into a polystyrene (PS) solution during formation of a porous surface by the bottom-up BF process, poly(N-isopropylacrylamide)-co-acrylic acid (PNIPAm-co-AA) microgels dispersed in the solution spontaneously assemble at the water-organic interfaces like "Pickering emulsions", reinforced by capillary flow. The conformal layer of PNIPAm-co-AA microgels lining the pores appears in images from a scanning electron microscope (SEM) either as a smooth surface layer (L) or as an array of domelike protrusions (D), depending on the conditions at which the sample was dried for SEM. The change between L and D morphology correlates with the volume phase transition behavior of the microgels freely suspended: drying at a temperature below the volume phase transition temperature (VPTT) gives L, and the D morphology is formed by drying at a temperature greater than the VPTT of PNIPAm-co-AA microgels. The morphological transition is shown to accompany a significant change in surface contact angle (CA) relative to a corresponding pore layer made of PS, with L having a CA that is reduced by 85° relative to PS, while the decrease is only 22° for D. Porous structures with morphologically responsive surfaces could find application in biocatalysis or tissue engineering, for example, with functional enzymes sequestered when microgels are collaped and accessible when the microgels are swollen.

Structure and mechanical properties of artificial protein hydrogels assembled through aggregation of leucine zipper peptide domains.

Artificial protein hydrogels made from a triblock protein (designated AC10A, where A is an acidic zipper domain and C10 comprises 10 repeats of the nonapeptide sequence exhibit normalized plateau storage moduli (G'∞/nkT) less than 0.13 at all concentrations, pH values, and ionic strengths examined. These gels are surprisingly soft due to loop formation at the expense of bridges between physical junctions. Molecular-level evidence of loop formation is provided by strong fluorescence energy transfer (FRET) between distinct chromophores placed at the C- and N-termini of labelled chains diluted in an excess of unlabelled chains. The tendency to form loops originates from the compact size of the random coil midblock (mean RH(C10)≈ 20 Å, determined from quasi-elastic light scattering of C10), and is facilitated by the ability of the leucine zipper domains to form antiparallel aggregates. Although the aggregation number of the leucine zipper domains is small (tetrameric, determined from multi-angle static light scattering of AC10 diblock), the average center-to-center distance between aggregates is roughly 1.5 times the average end-to-end distance of the C10 domain in a 7% w/v network. To avoid stretching the C10 domain, the chains tend to form loops. Changes in pH or ionic strength that expand the polyelectrolyte midblock favor bridging, leading to greater G'∞ as long as leucine zipper endblocks do not dissociate. Understanding of the network structure provided successful design strategies to increase the rigidity of these hydrogels. In contrast to intuitive design concepts for rubber and gel materials, it was shown that increasing either the length or the charge density of the midblock increases rigidity, because fewer chains are wasted in loop formation.

Rheological study of structural transitions in triblock copolymers in a liquid crystal solvent.

Rheological properties of triblock copolymers dissolved in a nematic liquid crystal (LC) solvent demonstrate that their microphase separated structure is heavily influenced by changes in LC order. Nematic gels were created by swelling a well-defined, high molecular weight ABA block copolymer with the small-molecule nematic LC solvent 4-pentyl-4'-cyanobiphenyl (5CB). The "B" midblock is a side-group liquid crystal polymer (SGLCP) designed to be soluble in 5CB and the "A" endblocks are polystyrene, which is LC-phobic and microphase separates to produce a physically cross-linked, thermoreversible, macroscopic polymer network. At sufficiently low polymer concentration a plateau modulus in the nematic phase, characteristic of a gel, abruptly transitions to terminal behavior when the gel is heated into its isotropic phase. In more concentrated gels, endblock aggregates persist into the isotopic phase. Dramatic changes in network structure are observed over small temperature windows (as little as 1 °C) due to tccche rapidly changing LC order near the isotropization point. The discontinuous change in solvent quality produces an abrupt change in viscoelastic properties for three polymers having different pendant mesogenic groups and matched block lengths.

Sustained release of human growth hormone from in situ forming hydrogels using self-assembly of fluoroalkyl-ended poly(ethylene glycol).

Poly(ethylene glycol)s modified with fluorocarbon end groups are capable of in situ transition from an injectable liquid to a viscoelastic hydrogel by hydrophobic interaction of the end groups; this class of materials is useful for a variety of biomedical applications, including sustained protein release. The hydrogel state can be transformed into an injectable state by the addition of a toxicologically acceptable organic solvent, such as N-methyl pyrrolidone; after injection, this solution quickly returns to a gel state by diffusion of the water-miscible organic solvent into the surrounding environment. In vitro characterization of sustained release of human growth hormone (hGH) using this injectable depot shows that hGH remains stable inside the hydrogel formed, and demonstrates more than 2 weeks of prolonged release of hGH complexed with Zn(2+) ions without protein aggregation or initial burst.