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Engineered living materials (ELMs) constitute a novel class of functional materials that contain living organisms. The mechanical properties of many such systems are dominated by the polymeric matrices used to encapsulate the cellular components of the material, making it hard to tune the mechanical behavior through genetic manipulation. To address this issue, we have developed living materials in which mechanical properties are controlled by the cell-surface display of engineered proteins. Here, we show that engineered Esherichia coli cells outfitted with surface-displayed elastin-like proteins (ELPs, designated E6) grow into soft, cohesive bacterial films with biaxial moduli around 14 kPa. When subjected to bulge-testing, such films yielded at strains of approximately 10%. Introduction of a single cysteine residue near the exposed N-terminus of the ELP (to afford a protein designated CE6) increases the film modulus 3-fold to 44 kPa and eliminates the yielding behavior. When subjected to oscillatory stress, films prepared from E. coli strains bearing CE6 exhibit modest hysteresis and full strain recovery; in E6 films much more significant hysteresis and substantial plastic deformation are observed. CE6 films heal autonomously after damage, with the biaxial modulus fully restored after a few hours. This work establishes an approach to living materials with genetically programmable mechanical properties and a capacity for self-healing. Such materials may find application in biomanufacturing, biosensing, and bioremediation.

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.

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., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

In carbon dioxide-blown polymer foams, the solubility of carbon dioxide (CO 2 ) 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 CO 2 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 CO 2 . While previous studies have suggested the importance of the effect of hydroxyl end groups on CO 2 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 CO 2 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 CO 2 solubility results from the competition between effects that reduce CO 2 solubility (lower mixing entropy) and effects that increase CO 2 solubility (lower ratio of hydroxyl end groups to ether backbone groups). To generalize our measurements, we modeled the CO 2 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., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Background and Objective: To explore the association between best-corrected visual acuity (BCVA) improvement and changes in microperimetry (MP) and color vision in patients with nonexudative age-related macular degeneration following administration of two 1.0-mg intravitreal doses of risuteganib., Patients and Methods: In a phase 2a, prospective, double-masked, sham-controlled study, eyes with nonexudative age-related macular degeneration and Early Treatment Diabetic Retinopathy Study BCVA between 20/40 and 20/200 were randomized to intravitreal risuteganib (1.0 mg) or sham injection. The risuteganib group received a second 1.0-mg dose, and patients in the sham group crossed over to receive 1.0 mg of risuteganib at week 16. Exploratory endpoints included changes in color vision and mesopic MP., Results: Thirty-nine patients (risuteganib, n = 25; sham, n = 14) completed the study. There was a significant ( P < .05) correlation between BCVA and the total error score (TES) for both Lanthony and Hue Style. Confusion index was close to the criterion for significance ( P = .056) in the risuteganib group. All color vision metrics demonstrated a trend toward improvement in risuteganib responders (BCVA letter gain ≥8 letters) and no change in the nonresponders, with significant differences seen in confusion index between the risuteganib and control group ( P = .0493) and between responders and nonresponders ( P = .0478). MP showed that risuteganib responders improved in mean sensitivity and change in number of loci ≤11 dB and ≤0 dB, whereas nonresponders worsened., Conclusion: All color vision and MP parameters tested trended toward improvement in risuteganib-treated patients and risuteganib responders. Statistically significant improvement was evident in two metrics: confusion index (in risuteganib-treated patients and responders) and number of loci with decreased sensitivity (in responders). A significant correlation between BCVA and both TES Lanthony and TES Hue Style in risuteganib patients provides concurrent evidence of objective and subjective improvement of retinal function. [ Ophthalmic Surg Lasers Imaging Retina 2022;53:430-438.] .

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.

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 (WS 2 ) 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 (WS 2 ) 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 WS 2 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background and Objective: To evaluate the safety and efficacy of 1.0 mg risuteganib in subjects with nonexudative age-related macular degeneration (AMD)., Patients and Methods: This was a phase 2a, prospective, double-masked, sham-controlled study. Eyes with nonexudative (dry) AMD and Early Treatment Diabetic Retinopathy Study (ETDRS) best-corrected visual acuity (BCVA) between 20/40 and 20/200 were included. Subjects were randomized to intravitreal 1.0 mg risuteganib or sham injection. At Week 16, subjects in the risuteganib group received a second 1.0-mg dose and the sham group crossed over to receive a dose of 1.0 mg risuteganib and were evaluated at Week 28. The primary endpoint was proportion of subjects with 8 letters ETDRS or more BCVA gain from baseline to Week 28 in the risuteganib group versus baseline to Week 12 for the sham group. BCVA was tested and subjects were observed for adverse events (AEs) every 4 weeks until completion of the study at 32 weeks., Results: Forty-five subjects (risuteganib, n = 29; sham, n = 16) were enrolled in the study, of whom 39 (risuteganib, n = 25; sham, n = 14) completed the study and were included in the per protocol efficacy analysis. At baseline, mean age was 78.8 and 75.9 years and mean BCVA was 67.1 and 64.4 letters in the sham and risuteganib groups, respectively. The primary endpoint was met by 48% of the risuteganib group at Week 28 and 7% of the sham group at Week 12 ( P = .013). Of the risuteganib subjects, 20% gained 15 letters or more at Week 28, whereas no patients in the sham group at Week 12 achieved this visual acuity gain. The only ocular treatment-related treatment-emergent AE was vitreous floaters, which spontaneously recovered without sequelae. No drug-related serious AE was reported., Conclusions: Risuteganib demonstrated significant BCVA improvement in patients with non-exudative AMD. No drug-related AEs were seen during a 32-week observation period. [ Ophthalmic Surg Lasers Imaging Retina . 2021;52:327-335.] .

In situ synchrotron X-ray scattering was used to reveal the transient microstructure of poly(L-lactide) (PLLA)/tungsten disulfide inorganic nanotubes (WS 2 NTs) 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 WS 2 NTs and the formation of polymer crystals by comparing neat PLLA and nanocomposite tubes under different expansion conditions. Surprisingly, the WS 2 NTs 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 WS 2 NTs 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 WS 2 NTs. This favorable interaction leads WS 2 NTs 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 WS 2 NTs.

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., Competing Interests: Conflict of interest statement: M.B.K. and J.P.O. are employees of Abbott Vascular. Funding for this research was provided by Abbott Vascular.

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., Competing Interests: The authors declare no conflict of interest.

Presence of an ultra high molecular weight (UHMw) fraction in flowing polymer melts is known to facilitate formation of oriented crystalline structures significantly. The UHMw fraction manifests itself as a minor tail in the molar mass distribution and is hardly detectable in the canonical characterization methods. In this study, alternatively, we demonstrate how the nonlinear extensional rheology reveals to be a very sensitive characterization tool for investigating the effect of the UHMw-tail on the structural ordering mechanism. Samples containing a UHMw-tail relative to samples without, exhibit a clear increase in extensional stress that is directly correlated with the crystalline orientation of the quenched samples. Extensional rheology, particularly, in combination with linear creep measurements, thus, enables the conformational evolution of the UHMw-tail to be studied and linked to the enhanced formation of oriented structures.

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.

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., Competing Interests: M.B.K. and J.P.O. are employees of Abbott Vascular. Funding for this research was provided by Abbott Vascular.

We used statistical mechanics to design polymers that defy conventional wisdom by self-assembling into "megasupramolecules" (≥5000 kg/mol) at low concentration (≤0.3 weight percent). Theoretical treatment of the distribution of individual subunits—end-functional polymers—among cyclic and linear supramolecules (ring-chain equilibrium) predicts that megasupramolecules can form at low total polymer concentration if, and only if, the backbones are long (>400 kg/mol) and end-association strength is optimal. Viscometry and scattering measurements of long telechelic polymers having polycyclooctadiene backbones and acid or amine end groups verify the formation of megasupramolecules. They control misting and reduce drag in the same manner as ultralong covalent polymers. With individual building blocks short enough to avoid hydrodynamic chain scission (weight-average molecular weights of 400 to 1000 kg/mol) and reversible linkages that protect covalent bonds, these megasupramolecules overcome the obstacles of shear degradation and engine incompatibility., (Copyright © 2015, American Association for the Advancement of Science.)

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.

Ligno(hemi)cellulosics (L(h)Cs) as sugarcane bagasse and loblolly pine sawdust are currently being used to produce biofuels such as bioethanol and biobutanol through fermentation of free sugars that are often obtained enzymatically. However, this bioconversion requires a pretreatment to solubilize the hemicellulose fractions, thus facilitating the action of the cellulolytic enzymes. Instead of the main free monosaccharides used in these current models, the modulation of thermopressurized orthophosphoric acid as a pretreatment, in the ranges of 3-12 atm and pH 1.5-2.5, can produce nondigestible oligosaccharides (NDOS) such as xylo-oligosaccharides (XOS) because heteroxylan is present in both types of hardwood and softwood hemicelluloses. A comparative thin-layer chromatographic analysis of the hydrolytic products showed the best conditions for NDOS production to be 7 atm/water, pH 2.25 and 2.50, and 8.5 atm/water for both sources. Particular hydrolysates from 7 atm (171 °C) at pHs 2.25 and 2.50 both for cane bagasse and pine sawdust, with respective oligosaccharide contents of 57 and 59 %, once mixed in a proportion of 1:1 for each plant source, were used in vitro as carbon sources for Bifidobacterium or Lactobacillus. Once both bacteria attained the stationary phase of growth, an unforeseen feature emerged: the preference of B. animalis for bagasse hydrolysates and, conversely, the preference of L. casei for pine hydrolysates. Considering the fact that nutraceutical oligosaccharides from both hemicelluloses correspond to higher value-added byproducts, the technology using a much diluted thermopressurized orthophosphoric acid pretreatment becomes an attractive choice for L(h)Cs.

The usual aggregation and precipitation driven by crystallization of nascent PE during homogeneous polymerization of ultra-high molecular weight polyethylene (UHMWPE) is inhibited by including linear low-density polyethylene (LLDPE) in the catalyst solution prior to addition of ethylene monomer. Co-crystallization of newly formed PE and dissolved LLDPE creates a polymer brush on the fold surfaces of the nascent crystallites. Consequently, aggregation is inhibited by steric stabilization. Scanning electron microscopy (SEM) images show that individual lamellae (approximately 10-20 nm thick) typically have lateral dimensions of 0.5 μm × 3.5 μm and form "bowtie" shaped stacks that are approximately 200-500 nm thick. This simple method for stabilizing nascent crystals against precipitation is enabling fundamental studies of their metastable "disentangled" state and may open scalable routes to compounding UHMWPE.

A method for evaluating strength of adhesives for hydrogels and soft tissues is presented. Quantitative measurements of shear strength for applications in tissue engineering and biomedicine are performed in torsion using a rheometer. Small, disk shaped specimens of soft biological tissues and/or hydrogels (8 mm diameter, 1-2 mm thick) are mounted onto rheometer tools and then bonded together using the adhesive to be tested. The torsional loading geometry imposes simple shear without deforming the planar adhesive bond, in contrast to the lap-shear test. It retains the advantages of the napkin ring test while reducing artifacts due to cutting and handling soft specimens. The method is demonstrated by measuring the shear strength of two types of biomedical adhesives (cyanoacrylate and polyethylene glycol-based) between model hydrogels (gelatin) and tissues (corneal stroma and skin)., (Copyright © 2012 Wiley Periodicals, Inc.)

R(f)-PEG (fluoroalkyl double-ended poly(ethylene glycol)) hydrogel is potentially useful as a drug delivery depot due to its advanced properties of sol-gel two-phase coexistence and low surface erosion. In this study, (1)H molecular diffusion nuclear magnetic resonance (NMR) and (19)F spin diffusion NMR were used to probe the drug loading and diffusion properties of the R(f)-PEG hydrogel for small anticancer drugs, 5-fluorouracil (FU) and its hydrophobic analog, 1,3-dimethyl-5-fluorouracil (DMFU). It was found that FU has a larger apparent diffusion coefficient than that of DMFU, and the diffusion of the latter was more hindered. The result of (19)F spin diffusion NMR for the corresponding freeze-dried samples indicates that a larger portion of DMFU resided in the R(f) core/IPDU intermediate-layer region (where IPDU refers to isophorone diurethane, as a linker to interconnect the R(f) group and the PEG chain) than that of FU while the opposite is true in the PEG-water phase. To understand the experimental data, a diffusion model was proposed to include: (1) hindered diffusion of the drug molecules in the R(f) core/IPDU-intermediate-layer region; (2) relatively free diffusion of the drug molecules in the PEG-water phase (or region); and (3) diffusive exchange of the probe molecules between the above two regions. This study also shows that molecular diffusion NMR combined with spin diffusion NMR is useful in studying the drug loading and diffusion properties in hydrogels for the purpose of drug delivery applications.

Injectable hydrogels show substantial promise for use in minimally invasive tissue engineering and drug delivery procedures.1,2 A new injectable hydrogel material, developed from recombinant telechelic proteins expressed in E. coli, demonstrates shear thinning by three orders of magnitude at large strains. Large amplitude oscillatory shear illustrates that shear thinning is due to yielding within the bulk of the gel, and the rheological response and flow profiles are consistent with a shear-banding mechanism for yielding. The sharp yielding transition and large magnitude of the apparent shear thinning allow gels to be injected through narrow gauge needles with only gentle hand pressure. After injection the gels reset to full elastic strength in seconds due to rapid reformation of the physical network junctions, allowing self-supporting structures to be formed. The shear thinning and recovery behavior is largely independent of the midblock length, enabling genetic engineering to be used to control the equilibrium modulus of the gel without loss of the characteristic yielding behavior. The shear-banding mechanism localizes deformation during flow into narrow regions of the gels, allowing more than 95% of seeded cells to survive the injection process.

Purpose: To measure the tissue mechanical response to elevated intraocular pressure (IOP) using intact globe expansion of rabbit eyes. This method examined rabbit kit (2-3 weeks old) eyes as a model for weakened tissue and evaluated riboflavin/UVA and glyceraldehyde cross-linking treatments., Methods: The ocular shape of enucleated eyes was photographed during a 24-hour period while a controlled IOP was imposed (either low IOP = 22 mm Hg or high IOP = 85 mm Hg). Untreated controls consisted of kit eyes tested at both low- and high IOP and adult eyes tested at high IOP. Treated kit eyes (dextran controls, riboflavin/UVA treatment of the cornea, and glyceraldehyde treatment of the entire globe) were tested at high IOP., Results: Low IOP elicited negligible creep of the sclera and very gradual creep of the cornea. In contrast, high IOP induced up to an 8% strain in the sclera and a 15% strain in the cornea of rabbit kit eyes. The expansion of adult eyes was less than one third that of kit eyes at the same, high IOP. Riboflavin/UVA treatment of corneas reduced expansion compared with that in both dextran-treated and untreated control corneas. Glyceraldehyde treatment prevented expansion of the cornea and sclera., Conclusions: The intact globe expansion method (GEM) imposes a loading geometry comparable to in vivo conditions and can quantify changes in mechanical stability as a function of testing conditions (e.g., IOP, tissue maturation, and therapeutic cross-linking) with small sample sizes and small variability. Rabbit kit eyes provide a model of weak tissue suitable for screening treatments that strengthen the cornea and sclera.

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 (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.

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.

An experimental configuration that combines the powerful capabilities of a short-term shearing apparatus with simultaneous optical and X-ray scattering techniques is demonstrated, connecting the earliest events that occur during shear-induced crystallization of a polymer melt with the subsequent kinetics and morphology development. Oriented precursors are at the heart of the great effects that flow can produce on polymer crystallization (strongly enhanced kinetics and formation of highly oriented crystallites), and their creation is highly dependent on material properties and the level of stress applied. The sensitivity of rheo-optics enables the detection of these dilute shear-induced precursors as they form during flow, before X-ray techniques are able to reveal them. Then, as crystallization occurs from these precursors, X-ray scattering allows detailed quantification of the characteristics and kinetics of growth of the crystallites nucleated by the flow-induced precursors. This simultaneous combination of techniques allows unambiguous correlation between the early events that occur during shear and the evolution of crystallization after flow has stopped, eliminating uncertainties that result from the extreme sensitivity of flow-induced crystallization to small changes in the imposed stress and the material. Experimental data on a bimodal blend of isotactic polypropylenes are presented.

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.

Rf-IPDU-PEGs belong to a class of fluoroalkyl-ended poly(ethylene glycol) polymers (Rf-PEGs), where the IPDU (isophorone diurethane) functions as a linker to connect each end of the PEG chain to a fluoroalkyl group. The Rf-IPDU-PEGs form hydrogels in water with favorable sol-gel coexistence properties. Thus, they are promising for use as drug delivery agents. In this study, we introduce an electron-spin induced 19F relaxation NMR technique to probe the location and drug-loading capacity for an electron-spin labeled hydrophobic drug, CT (chlorambucil-tempol adduct), enclosed in the Rf-IPDU-PEG micelle. With the assistance of molecular dynamics simulations, a clear idea regarding the structures of the Rf-IPDU-PEG micelle and its CT-loaded micelle was revealed. The significance of this research lies in the finding that the hydrophobic drug molecules were loaded within the intermediate IPDU shells of the Rf-IPDU-PEG micelles. The molecular structures of IPDU and that of CT are favorably comparable. Consequently, it appears that this study opens a window to modify the linker between the Rf group and the PEG chain for achieving customized structure-based drug-loading capabilities for these hydrogels, while the advantage of the strong affinity among the Rf groups to hold individual micelles together and to interconnect the micellar network is still retained in hopes of maintaining the sol-gel coexistence of the Rf-PEGs.

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.

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.

In the rich and long-standing literature on the flow-induced formation of oriented precursors to polymer crystallization, it is often asserted that the longest, most extended chains are the dominant molecular species in the "shish" of the "shish-kebab" formation. We performed a critical examination of this widely held view, using deuterium labeling to distinguish different chain lengths within an overall distribution. Small-angle neutron-scattering patterns of the differently labeled materials showed that long chains are not overrepresented in the shish relative to their concentration in the material as a whole. We observed that the longest chains play a catalytic role, recruiting other chains adjacent to them into formation of the shish.

The advancing contact angle (thetaadv) of water on thin films ( approximately 1 microm) of poly(ethylene glycol) (PEG) with fluoroalkyl endgroups (6 kg/mol PEG with 10-carbon fluoroalkyl, denoted 6KC10) changes strongly with relative humidity (RH). Films of 6KC10 on silicon wafers pretreated with a fluorinated alkylsilane (TFOS) display thetaadv increasing from 75 degrees at 12% RH to 95 degrees at 94% RH. The surprising transition to nonwetting character at high humidity is attributed to fluoroalkyl groups ordering at the air-hydrogel interface when they are liberated by dissolution of PEG crystallites above 85% RH. When water is withdrawn from a drop on 6KC10, the contact line does not recede. This extreme hysteresis is attributed to restructuring of the gel to bury the fluoroalkyl groups when in contact with water.

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.

In our pursuit to find potent anticancer drugs, we have covalently bonded free radical tempol to chlorambucil giving a chlorambucil-tempol (CT) adduct in which both of the anticancer active sites in tempol and chlorambucil were left intact. Analysis using NMR, Maldi-TOF, and EPR verified the designed chemical structure. Because the CT adduct is more hydrophobic than chlorambucil, its delivery also was investigated using fluoroalkyl double-ended poly (ethylene glycol) (Rf-PEG) micelles. Results from EPR spectra and(19) F and(1) H NMR spin lattice relaxation times show that the Rf-PEG micelles are able to encapsulate CT into the Rf cores of the micelles.

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.

In a nematic gel we observe a low-energy buckling deformation arising from soft and semisoft elastic modes. We prepare the self-assembled gel by dissolving a coil-side-group liquid-crystalline polymer-coil copolymer in a nematic liquid crystal. The gel has long network strands and a precisely tailored structure, making it ideal for studying nematic rubber elasticity. Under polarized optical microscopy we observe a striped texture that forms when gels uniformly aligned at 35 degrees C are cooled to room temperature. We model the instability using the molecular theory of nematic rubber elasticity, and the theory correctly captures the change in pitch length with sample thickness and polymer concentration. This buckling instability is a clear example of a low-energy deformation that arises in materials where polymer network strains are coupled to the director orientation.

Erosion behaviour governs the use of physical hydrogels in biomedical applications ranging from controlled release to cell encapsulation. Genetically engineered protein hydrogels offer unique means of controlling the erosion rate by engineering their amino acid sequences and network topology. Here, we show that the erosion rate of such materials can be tuned by harnessing selective molecular recognition, discrete aggregation number and orientational discrimination of coiled-coil protein domains. Hydrogels formed from a triblock artificial protein bearing dissimilar helical coiled-coil end domains (P and A) erode more than one hundredfold slower than hydrogels formed from those bearing the same end domains (either P or A). The reduced erosion rate is a consequence of the fact that looped chains are suppressed because P and A tend not to associate with each other. Thus, the erosion rate can be tuned over several orders of magnitude in artificial protein hydrogels, opening the door to diverse biomedical applications.

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.

Liquid crystals are often combined with polymers to influence the liquid crystals' orientation and mechanical properties, but at the expense of reorientation speed or uniformity of alignment. We introduce a new method to create self-assembled nematic liquid-crystal gels using an ABA triblock copolymer with a side-group liquid-crystalline midblock and liquid-crystal-phobic endblocks. In contrast to in situ polymerized networks, these physical gels are homogeneous systems with a solubilized polymer network giving them exceptional optical uniformity and well-defined crosslink density. Furthermore, the unusually high-molecular-weight polymers used allow gels to form at lower concentrations than previously accessible. This enables these gels to be aligned by surface anchoring, shear, or magnetic fields. The high content of small-molecule liquid crystal (>/=95%) allows access to a regime of fast reorientation dynamics.

Purpose: To determine whether digital spatial intensity patterns can be developed to effect precise in vitro correction of myopic, hyperopic, and astigmatic refractive errors in a silicone light-adjustable lens (LAL). Also, to determine whether a new spatial intensity pattern for "lock-in" is effective in vitro., Methods: A digital interferometer/irradiation system was developed to irradiate LALs and measure the power change following irradiation. Light-adjustable lenses were mounted into a wet cell maintained at 35.0 +/- 0.5 degrees C (simulated ocular temperature) and allowed to equilibrate for a minimum of 2 hours. Ultraviolet light was then applied with spatial light intensity patterns to correct hyperopia, myopia, and astigmatism. Light-adjustable lenses were also treated to effect lock-in with a separate spatial light intensity pattern. Treated lenses were characterized for power change and optical quality. In the case of lock-in, exhaustive chemical extraction was also performed to determine the percentage of remaining macromer., Results: Appropriate digital irradiation spatial intensity patterns were created to develop nomograms for in vitro correction of myopia, hyperopia, and astigmatism in approximate 0.25 D steps. Power changes were reproducible and did not alter optical quality of the LALs. Further, lock-in dosing of the LALs did not alter optical quality or significantly change LAL power., Conclusions: In vitro nomograms have been developed for a silicone LAL that permit precise correction of myopia, hyperopia, and astigmatism. Furthermore, a spatial light intensity pattern has been devised that effects lock-in without significantly altering LAL power or optical quality.

Addition of a low concentration of a very long (430 kg/mol) side group liquid crystal polymer is shown to produce dramatic changes in the flow characteristics of a calamitic nematic liquid crystal. This polymer causes a typical flow-aligning nematic liquid crystal to align near the velocity gradient direction rather than near the velocity direction, corresponding to having a tumbling parameter lambda<-1, for concentrations greater than 7.5% polymer. Such flow-aligning behavior has not been reported previously in a calamitic nematic. The large molecular weight of the present polymer relative to those examined in the prior literature is responsible for these new phenomena.

The role of the highly reactive triose sugars glyceraldehyde and glyceraldehyde-3-phosphate in protein cross-linking and other amino acid modifications during the Maillard reaction was investigated. From the incubation of glyceraldehyde with N (alpha)-acetyl-L-lysine and N (alpha)-acetyl-L-arginine, we isolated four new Maillard reaction pyridinium compounds named 'triosidines'. Two of them, 'lys-hydroxy-triosidine' [1-(5-amino-5-carboxypentyl)-3-[(5-amino-5-carboxypentylamino)methyl]-5-hydroxypyridinium] and 'arg-hydroxy-triosidine' [2-(4-amino-4-carboxybutylamino)-8-(5-amino-5-carboxypentyl)-6-hydroxy-3,4-dihydro-pyrido[2,3-d]pyrimidin-8-ium] are fluorescent, UV-active Lys-Lys and Lys-Arg cross-links respectively. Their structures were identified by NMR and MS. In addition, two UV-active lysine adducts, 'trihydroxy-triosidine' [1-(5-amino-5-carboxypentyl)-3,4-dihydroxy-5-(hydroxymethyl)pyridinium] and 'triosidine carbaldehyde' [1-(5-amino-5-carboxypentyl)-3-formylpyridinium] were tentatively identified by MS. All structures involve six sugar-derived carbons as part of the heterocyclic ring. Of the two novel cross-links, only arg-hydroxy-triosidine was formed by glyceraldehyde-3-phosphate, an intermediate metabolite of the glycolytic pathway. Lys-hydroxy-triosidine and arg-hydroxy-triosidine were detected in human and porcine corneas treated with glyceraldehyde. The HPLC-fluorescence identification was confirmed by MS. Triosidines were also formed from dihydroxyacetone, a widely used artificial sun-tanning agent. Triosidines are expected to be useful tools in tissue engineering, where the utilization of highly reactive sugars is needed to stabilize the loose matrix. In addition, they are expected to be present in selected biological conditions, such as on consumption of a high fructose diet, and syndromes associated with high glyceraldehyde excretion, such as Fanconi Syndrome, fructose-1,6-diphosphatase deficiency and tyrosinaemia.

To eliminate persistent refractive errors after cataract and phakic IOL surgery, photosensitive silicone IOLs have been developed. These IOL formulations enable precise laser adjustment of IOL power to correct spherical and toric errors post-operatively, after wound and IOL stabilization. Initial experience with these laser adjustable IOLs indicate excellent biocompatability and adjustability of more than five diopters.

Recent two-dimensional (2D) 2H-NMR studies on nearly ideal mixtures are reviewed. The use of selective deuterium labeling allows the unambiguous observation of the individual segmental dynamics of each component in the blend. 2D exchange spectra provide both the mean motional rates and the motional distributions, which was necessary to discriminate among the disparate explanations of blend behavior. The components exhibit very different mean mobilities and broad mobility distributions near the glass transition. The broad macroscopic glass transition in the blends is attributed to both these two kinds of dynamic heterogeneities. The individual mean motional rates are used to define distinct glass transition temperatures, T(g,i) for each species, i. The separation between the T(g,i),s of the two species increases with the content of the high-T(g) component. The widths of their individual motional distributions also increase with the content of the high-T(g) component. These two effects combine to produce the increase in both the anomalously broad glass transition and the thermorheological complexity of these blends with fraction of the high-T(g) component.

The interplay of molecular weight, layer thickness, and thermal annealing in controlling molecular order in ultrathin Langmuir-Blodgett films is characterized with the use of polarization-modulation laser-scanning microscopy. The degree and direction of molecular alignment can be imaged rapidly and sensitively through the magnitude and orientation of linear dichroism in Langmuir-Blodgett films of rodlike poly(phthalocyaninatosiloxane) (PcPS). Images are presented for films as thin as two molecular layers ( approximately 44 angstroms). Molecular alignment along the transfer direction is much stronger for films of PcPS with approximately 25 repeat units ( approximately 10 nanometers long) than for those with approximately 50 repeat units ( approximately 20 nanometers long). Enhancement of alignment by thermal annealing is also much greater for PcPS-25 than PcPS-50. Intimate interaction with the substrate suppresses improvement in alignment by annealing, evident by an anomalously small increase in anisotropic absorption of the first two layers.