Experimental observation of near-wall effects during the puncture of soft solids.

Performing conventional mechanical characterization techniques on soft materials can be challenging due to issues such as limited sample volumes and clamping difficulties. Deep indentation and puncture is a promising alternative as it is an information-rich measurement with the potential to be performed in a high-throughput manner. Despite its promise, the method lacks standardized protocols, and open questions remain about its possible limitations. Addressing these shortcomings is vital to ensure consistent methodology, measurements, and interpretation across samples and labs. To fill this gap, we examine the role of finite sample dimensions (and by extension, volume) on measured forces to determine the sample geometry needed to perform and unambiguously interpret puncture tests. Through measurements of puncture on a well-characterized elastomer using systematically varied sample dimensions, we show that the apparent mechanical response of a material is in fact sensitive to near-wall effects, and that additional properties, such as the sliding friction coefficient, can only be extracted in the larger dimension case where such effects are negligible.

Modular Synthesis and Patterning of High-Stiffness Networks by Postpolymerization Functionalization with Iron-Catechol Complexes.

Bioinspired iron-catechol cross-links have shown remarkable success in increasing the mechanical properties of polymer networks, in part due to clustering of Fe3+-catechol domains which act as secondary network reinforcing sites. We report a versatile synthetic procedure to prepare modular PEG-acrylate networks with independently tunable covalent bis(acrylate) and supramolecular Fe3+-catechol cross-linking. Initial control of network structure is achieved through radical polymerization and cross-linking, followed by postpolymerization incorporation of catechol units via quantitative active ester chemistry and subsequent complexation with iron salts. By tuning the ratio of each building block, dual cross-linked networks reinforced by clustered iron-catechol domains are prepared and exhibit a wide range of properties (Young's moduli up to ∼245 MPa), well beyond the values achieved through purely covalent cross-linking. This stepwise approach to mixed covalent and metal-ligand cross-linked networks also permits local patterning of PEG-based films through masking techniques forming distinct hard, soft, and gradient regions.

ACUVRA: Anion-Exchange Chromatography UV-Ratio Analysis-A QC-Friendly Method for Monitoring Adeno-Associated Virus Empty Capsid Content To Support Process Development and GMP Release Testing.

The genome content of adeno-associated virus (AAV) vectors is critical to the safety and potency of AAV-based gene therapy products. Empty capsids are considered a product-related impurity and a critical quality attribute (CQA) of the drug product, thus requiring characterization throughout the production process to demonstrate they are controlled to acceptable levels in the final drug product. Anion exchange chromatography has been used to achieve separation between empty and full capsids, but requires method development and gradient optimization for different serotypes and formulations. Here, we describe an alternative approach to quantitation that does not rely on achieving separation between empty and full capsids, but instead uses the well-established relationship between absorbance at UV A260/A280 and relation to DNA/protein content, in combination with anion-exchange chromatography to allow one to calculate the relative proportion of empty and full capsids in AAV samples from a single peak. We call this approach ACUVRA: Anion-exchange Chromatography UV-Ratio Analysis, and show the applicability of the method through a case study with recombinant AAV2 (rAAV2) process intermediates and drug substance. Method qualification and GMP validation in a quality control (QC) laboratory results show that ACUVRA is a fit-for-purpose method for process development support and characterization, while also being a QC-friendly option for GMP release testing at all stages of clinical development. Graphical abstract.

Strength of fluid-filled soft composites across the elastofracture length.

Materials that utilize heterogeneous microstructures to control macroscopic mechanical response are ubiquitous in nature. Yet, translating nature's lessons to create synthetic soft solids has remained challenging. This is largely due to the limited synthetic routes available for creating soft composites, particularly with submicron features, as well as uncertainty surrounding the role of such a microstructured secondary phase in determining material behavior. This work leverages recent advances in the development of photocrosslinkable thermogelling nanoemulsions to produce composite hydrogels with a secondary phase assembled at well controlled length scales ranging from tens of nm to tens of µm. Through analysis of the mechanical response of these fluid-filled composite hydrogels, it is found that the size scale of the secondary phase has a profound impact on the strength when at or above the elastofracture length. Moreover, this work shows that mechanical integrity of fluid-filled soft solids can be sensitive to the size scale of the secondary phase.

Bond strength regime dictates stress relaxation behavior.

Reconfigurable polymer networks are gaining interest for their potential applications as self-healing, recyclable, and stimuli-responsive smart materials. Relating the bond strength of dynamic interactions to material properties including stress relaxation time and modulus is crucial for smart material design. In this work, in situ crosslinked transition metal-terpyridine reconfigurable networks were utilized to modulate the characteristic network stress relaxation time, τR. The use of stress relaxation experiments rather than oscillatory frequency sweeps allowed for the measurement of network bond dynamics across a wider dynamic range than has been previously reported. The stress relaxation time was shown to be tunable by metal center, counterion, and crosslink density. Remarkably, the network crosslinked with covalent-like ruthenium chloride-terpyridine interaction, while having a longer τR, was qualitatively similar to the other metal-ligand networks. Furthermore, the relaxation time was independent of crosslink density in strongly bonded networks, allowing for independent tunability of modulus and τR. In contrast, increasing crosslink density reduced τR in networks crosslinked with weaker interactions.

Linking cavitation and fracture to molecular scale structural damage of model networks.

Rapid expansion of soft solids subjected to a negative hydrostatic stress can occur through cavitation or fracture. Understanding how these two mechanisms relate to a material's molecular structure is important to applications in materials characterization, adhesive design, and tissue damage. Here, a recently improved needle-induced cavitation (NIC) protocol is applied to a set of model end-linked PEG gels with quantitatively linked elastic and fracture properties. This quantitative link between molecular scale structure and macroscopic properties is exploited to experimentally probe the relationship between cavitation, fracture, and molecular scale damage. This work indicates that rational tuning of the elastofracture length relative to the crack geometry can be used to alter the expansion mechanism from cavitation to fracture during NIC.

Rational mechanochemical design of Diels-Alder crosslinked biocompatible hydrogels with enhanced properties.

An important but often overlooked feature of Diels-Alder (DA) cycloadditions is the ability for DA adducts to undergo mechanically induced cycloreversion when placed under force. Herein, we demonstrate that the commonly employed DA cycloaddition between furan and maleimide to crosslink hydrogels results in slow gelation kinetics and "mechanolabile" crosslinks that relate to reduced material strength. Through rational computational design, "mechanoresistant" DA adducts were identified by constrained geometries simulate external force models and employed to enhance failure strength of crosslinked hydrogels. Additionally, utilization of a cyclopentadiene derivative, spiro[2.4]hepta-4,6-diene, provided mechanoresistant DA adducts and rapid gelation in minutes at room temperature. This study illustrates that strategic molecular-level design of DA crosslinks can provide biocompatible materials with improved processing, mechanical durability, lifetime, and utility.

Fracture of model end-linked networks.

Advances in polymer chemistry over the last decade have enabled the synthesis of molecularly precise polymer networks that exhibit homogeneous structure. These precise polymer gels create the opportunity to establish true multiscale, molecular to macroscopic, relationships that define their elastic and failure properties. In this work, a theory of network fracture that accounts for loop defects is developed by drawing on recent advances in network elasticity. This loop-modified Lake-Thomas theory is tested against both molecular dynamics (MD) simulations and experimental fracture measurements on model gels, and good agreement between theory, which does not use an enhancement factor, and measurement is observed. Insight into the local and global contributions to energy dissipated during network failure and their relation to the bond dissociation energy is also provided. These findings enable a priori estimates of fracture energy in swollen gels where chain scission becomes an important failure mechanism.

Deep indentation and puncture of a rigid cylinder inserted into a soft solid.

Deep indentation and puncture can be used to characterize the large strain elastic and fracture properties of soft solids and biological tissues. While this characterization method is growing in application there are still open questions about deep indentation and puncture, including how the distribution of strains and stresses in the surrounding material relate to the resultant force exerted on the indenter. Direct quantification of the deformation field around a rigid indenter during penetration of a soft solid is necessary to substantiate the current qualitative understanding of these strains and increase the impact and usefulness of puncture tests. Here, the deformation field of a rigid cylinder inserted into a soft solid is quantified using digital image correlation (DIC). DIC measurements are validated by reconstituting the measured nominal force on the cylinder during deep indentation and puncture. The deformation field is used to map the strain field around the indenter during deep indentation and puncture. These measurements provide direct insight into the puncture process and show that while the resultant force mainly arises from the sheared region on the sides of the indenter, the compressed region below the tip is responsible for initiating failure.

Seeded laser-induced cavitation for studying high-strain-rate irreversible deformation of soft materials.

Characterizing the high-strain-rate and high-strain mechanics of soft materials is critical to understanding the complex behavior of polymers and various dynamic injury mechanisms, including traumatic brain injury. However, their dynamic mechanical deformation under extreme conditions is technically difficult to quantify and often includes irreversible damage. To address such challenges, we investigate an experimental method, which allows quantification of the extreme mechanical properties of soft materials using ultrafast stroboscopic imaging of highly reproducible laser-induced cavitation events. As a reference material, we characterize variably cross-linked polydimethylsiloxane specimens using this method. The consistency of the laser-induced cavitation is achieved through the introduction of laser absorbing seed microspheres. Based on a simplified viscoelastic model, representative high-strain-rate shear moduli and viscosities of the soft specimens are quantified across different degrees of crosslinking. The quantified rheological parameters align well with the time-temperature superposition prediction of dynamic mechanical analysis. The presented method offers significant advantages with regard to quantifying high-strain rate, irreversible mechanical properties of soft materials and tissues, compared to other methods that rely upon the cyclic dynamics of cavitation. These advances are anticipated to aid in the understanding of how damage and injury develop in soft materials and tissues.

Low-Voltage Reversible Electroadhesion of Ionoelastomer Junctions.

Electroadhesion provides a simple route to rapidly and reversibly control adhesion using applied electric potentials, offering promise for a variety of applications including haptics and robotics. Current electroadhesives, however, suffer from key limitations associated with the use of high operating voltages (>kV) and corresponding failure due to dielectric breakdown. Here, a new type of electroadhesion based on heterojunctions between iono-elastomer of opposite polarity is demonstrated, which can be operated at potentials as low as ≈1 V. The large electric field developed across the molecular-scale ionic double layer (IDL) when the junction is placed under reverse bias allows for strong adhesion at low voltages. In contrast, under forward bias, the electric field across the IDL is destroyed, substantially lowering the adhesion in a reversible fashion. These ionoelastomer electroadhesives are highly efficient with respect to the force capacity per electrostatic capacitive energy and are robust to defects or damage that typically lead to catastrophic failure of conventional dielectric electroadhesives. The findings provide new fundamental insight into low-voltage electroadhesion and broaden its possible applications.

Residual strain effects in needle-induced cavitation.

Needle-induced cavitation (NIC) locally probes the elastic and fracture properties of soft materials, such as gels and biological tissues. Current NIC protocols tend to overestimate properties when compared to traditional techniques. New NIC methods are needed in order to address this issue. NIC measurements consist of two distinct processes, namely (1) the needle insertion process and (2) the cavitation process. The cavitation process is hypothesized to be highly dependent on the initial needle insertion process due to the influence of residual strain below the needle. Retracting the needle before pressurization to a state in which a cylindrical, tube-like fracture is left below the needle tip is experimentally demonstrated to reduce the impact of residual strain on NIC. Verification of the critical cavitation pressure equation in this new geometry is necessary before implementing this retraction NIC protocol. Complementary modeling shows that the change in initial geometry has little effect on the critical cavitation pressure. Together, these measurements demonstrate that needle retraction is a viable experimental protocol for reducing the influence of residual strain, thus enabling the confident measurement of local elastic and fracture properties in soft gels and tissues.

Injections of Galanin-Like Peptide directly into the nucleus of the tractus solitarius (NTS) reduces food intake and body weight but increases metabolic rate and plasma leptin.

Galanin-Like Peptide (GALP) is a hypothalamic neuromediator of metabolism and reproduction. GALP is known to stimulate reproduction and alter food intake and body weight in multiple species. The regulation of body weight involves control of both energy intake and energy expenditure. Since GALP is known to alter food intake - possibly via the autonomic nervous system - we first hypothesized that GALP would increase metabolic rate. First, male Sprague-Dawley rats were implanted with intracerebroventricular (ICV) cannulae and abdominal radiotelemetry temperature transmitters. Following ICV injection with either 5nmol GALP or vehicle, the oxygen consumption of each rat was monitored for 8h. Food intake, core temperature, and general motor activity were monitored for 24h. GALP significantly increased oxygen consumption, an indirect estimator of metabolic rate, without having any significant effect on motor activity. Compared to controls, GALP increased core body temperature during the photophase and reduced food intake over the 24h period following injection. ICV GALP also increased plasma levels of luteinizing hormone (LH). A second group of male Sprague-Dawley rats were implanted with abdominal transmitters and given injections of GALP directly into the nucleus of the tractus solitarius (NTS). These injections resulted in a significant reduction in food intake, and a significant increase in both oxygen consumption and core body temperature compared to vehicle injections. Direct injections of GALP into the NTS compared to vehicle also resulted in a significant increase in plasma leptin levels, but not LH levels. GALP appears to increase energy expenditure in addition to decreasing energy input by actions within the NTS and thus may play an important role in the hypothalamic regulation of body weight.

Intermittent heat exposure and thirst in rats.

Adequate water intake, supporting both cardiovascular function and evaporative cooling, is a critical factor in mitigating the effects of heat waves, which are expected to increase with global warming. However, the regulation of water intake during periods of intermittent heat exposure is not well understood. In this study, the effects of access to water or no access during intermittent heat exposure were assessed using male Sprague-Dawley rats exposed to 37.5°C for 4 h/day. After 7 days of intermittent heat exposure, reductions in evaporative water loss were observed in all animals and reductions in water intake following heat exposure occurred as the days of heat exposure increased. Rats that were not allowed water during the 7 days of exposure had decreased rehydration levels, however, rats allowed access to water increased water intake during exposure and exhibited higher overall rehydration levels over the same time period. Peripheral administration of angiotensinII, mimicking activation of volemic thirst, or hypertonic saline solution, activating intracellular thirst, did not result in alteration of water intake in rats exposed to heat with access to water compared to control rats. In contrast, rats exposed to heat without access to water had reduced water intake after administration of hypertonic saline and increased water intake after administration of angiotensinIIcompared to control rats. These experiments demonstrate that thirst responses to intermittent heat exposure are altered by providing water during heat exposure and that intermittent heat exposure without access to water alters drinking responses to both intracellular and extracellular thirst challenges.

Heat acclimation and thirst in rats.

The effects of heat acclimation on water intake and urine output responses to thermal dehydration and other thirst stimuli were studied in male Sprague-Dawley rats. Rats were heat acclimated by continuous exposure to a 34°C environment for at least 6 weeks. Thermal dehydration-induced thirst was brought about by exposing the heat-acclimated rats and control rats housed at 24°C to a 37.5°C environment for 4 h without access to food or water. Heat acclimation reduced evaporative and urinary water losses and the increases in plasma sodium and osmolality during thermal dehydration, which led to a reduction in thermal dehydration-induced thirst. Heat acclimation reduced the rate of rehydration following thermal dehydration but did not alter the final rehydration level, indicating that heat acclimation does not alter the primary control of thermal dehydration-induced thirst. Heat acclimation did not alter water intake or urine output following administration of hypertonic saline, which selectively stimulates intracellular thirst, but led to greater water intake following administration of angiotensin II, which plays an important role in extracellular/volemic thirst, and following water deprivation, which activates both thirst pathways. Cardiovascular responses to angiotensin II were not altered by heat acclimation. Heat acclimation thus reduces water loss during heat exposure in rats, but does not have major effects on thermal dehydration-induced or extracellular thirst but does appear to alter volemic thirst.

Smooth Muscle Stiffness Sensitivity is Driven by Soluble and Insoluble ECM Chemistry.

Smooth muscle cell (SMC) invasion into plaques and subsequent proliferation is a major factor in the progression of atherosclerosis. During disease progression, SMCs experience major changes in their microenvironment, such as what integrin-binding sites are exposed, the portfolio of soluble factors available, and the elasticity and modulus of the surrounding vessel wall. We have developed a hydrogel biomaterial platform to examine the combined effect of these changes on SMC phenotype. We were particularly interested in how the chemical microenvironment affected the ability of SMCs to sense and respond to modulus. To our surprise, we observed that integrin binding and soluble factors are major drivers of several critical SMC behaviors, such as motility, proliferation, invasion, and differentiation marker expression, and these factors modulated the effect of stiffness on proliferation and migration. Overall, modulus only modestly affected behaviors other than proliferation, relative to integrin binding and soluble factors. Surprisingly, pathological behaviors (proliferation, motility) are not inversely related to SMC marker expression, in direct conflict with previous studies on substrates coupled with single extracellular matrix (ECM) proteins. A high-throughput bead-based ELISA approach and inhibitor studies revealed that differentiation marker expression is mediated chiefly via focal adhesion kinase (FAK) signaling, and we propose that integrin binding and FAK drive the transition from a migratory to a proliferative phenotype. We emphasize the importance of increasing the complexity of in vitro testing platforms to capture these subtleties in cell phenotypes and signaling, in order to better recapitulate important features of in vivo disease and elucidate potential context-dependent therapeutic targets.

New insights into the mechanism for VACM-1/cul5 expression in vascular tissue in vivo.

Vasopressin-activated calcium-mobilizing (VACM-1)/cul5 is the least conserved member of a cullin protein family involved in the formation of E3-specific ligase complexes that are responsible for delivering the ubiquitin protein to their target substrate proteins selected for ubiquitin-dependent degradation. This chapter summarizes work to date that has focused on VACM-1/cul5's tissue-specific expression in vivo and on its potential role in the control of specific cellular signaling pathways in those structures. As mammalian cells may contain hundreds of E3 ligases, identification VACM-1/cul5 as a specific subunit of the system that is expressed in the endothelium and in collecting tubules, structures known for their control of cellular permeability, may have significant implications when designing studies to elucidate the mechanism of water conservation. For example, VACM-1/cul5 expression is affected by water deprivation in some tissues and there is a potential relationship between neddylated VACM-1/cul5 and aquaporins.

Aquaporin-2 levels in vitro and in vivo are regulated by VACM-1, a cul 5 gene.

BACKGROUND: In the renal collecting duct, vasopressin regulates water permeability by a process that involves stimulation of adenylyl cyclase activity, cAMP production and subsequent translocation of water channel aquaporin-2 (AQP2) into the apical plasma membrane. We have previously shown that in cos 1 cells in vitro, both adenylyl cyclase activity and cAMP production can be regulated by VACM-1, a cul 5 gene that forms complexes involved in protein ubiquitination and subsequent degradation. METHODS: To extend these observations further, the effects of changes in hydration state on the expression of VACM-1 at the mRNA and the protein level were examined in rats deprived of water (WD) for 24 hrs. RESULTS: In the kidney of WD rats Western blot analyses of kidney tissue showed that the decrease in VACM-1 protein concentration was correlated with the increase in the AQP2 protein level. The immunostaining data suggested that VACM-1/cul5 may be decreased in renal collecting duct but increases in the vasculature of the inner medullary region in response to WD. To determine the possible consequences of the WD dependent decrease in VACM-1/cul5, we next examined the effects of VACM-1 expression on AQP2 protein in vitro. Immunocytochemistry and Western blot analyses data indicate that VACM-1/cul5 expression in MDCK line stably expressing AQP2 gene and in cos 1 cells co-transfected with the AQP2 and VACM-1/cul5 cDNAs decreased AQP2 protein concentration when compared to the vector transfected control groups. CONCLUSION: In summary, our data demonstrate that VACM-1 is involved in the regulation of AQP2 protein concentration and may play a role in regulating water balance.

VACM-1/cul5 expression in vascular tissue in vivo is induced by water deprivation and its expression in vitro regulates aquaporin-1 concentrations.

VACM-1, a cul5 gene product, when overexpressed in vitro, has an antiproliferative effect. In vivo, VACM-1/cul5 is present in tissues involved in the regulation of water balance. Neither proteins targeted for VACM-1/cul5-specific degradation nor factors that may regulate its expression in those tissues have been studied. To identify genes that may be misregulated by VACM-1 cDNA, we performed microarray analysis. Our results indicate that in cos-1 cells transfected with VACM-1 cDNA, mRNA levels for several genes, including AQP1, were decreased when compared to the control group. Our results also indicate that in cos-1 cells transfected with VACM-1 cDNA, endogenous AQP1 protein was decreased about 6-fold when compared to the controls. To test the hypothesis that VACM-1/cul5 may be regulated by conditions that compromise water homeostasis in vivo, we determined if 24 h of water deprivation affects VACM-1/cul5 levels or the effect of VACM-1/cul5 on AQP1. VACM-1 mRNA and protein levels were significantly higher in rat mesenteric arteries, skeletal muscle and the heart ventricle but not in the heart atrium from 24-h water-deprived rats when compared to the controls. Interestingly, 24 h of water deprivation increased modification of VACM-1 by an ubiquitin-like protein, Nedd8, essential for cullin-dependent E3 ligase activity. Although water deprivation did not significantly change AQP1 levels in the mesenteric arteries, AQP1 protein concentrations were inversely correlated with the ratio of the VACM-1 to Nedd8-modified VACM-1. These results suggest that VACM-1/cul5 may regulate endothelial AQP1 concentration both in vivo and in vitro.