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Monitoring of extracellular matrix (ECM) microstructure is essential in studying structure-associated cellular processes, improving cellular function, and for ensuring sufficient mechanical integrity in engineered tissues. This paper describes a novel method to study the microscale alignment of the matrix in engineered tissue scaffolds (ETS) that are usually composed of a variety of biomacromolecules derived by cells. First, a trained loading function was derived from Raman spectra of highly aligned native tissue via principal component analysis (PCA), where prominent changes associated with specific Raman bands (e.g., 1444, 1465, 1605, 1627-1660, and 1665-1689 cm-1) were detected with respect to the polarization angle. These changes were mainly caused by the aligned matrix of many compounds within the tissue relative to the laser polarization, including proteins, lipids, and carbohydrates. Hence this trained function was applied to quantify the alignment within ETS of various matrix components derived by cells. Furthermore, a simple metric called Amplitude Alignment Metric (AAM) was derived to correlate the orientation dependence of polarized Raman spectra of ETS to the degree of matrix alignment. It was found that the AAM was significantly higher in anisotropic ETS than isotropic ones. The PRS method revealed a lower p-value for distinguishing the alignment between these two types of ETS as compared to the microscopic method for detecting fluorescent-labeled protein matrices at a similar microscopic scale. These results indicate that the anisotropy of a complex matrix in engineered tissue can be assessed at the microscopic scale using a PRS-based simple metric, which is superior to the traditional microscopic method. This PRS-based method can serve as a complementary tool for the design and assessment of engineered tissues that mimic the native matrix organizational microstructures.
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Ingeniería de Tejidos , Andamios del Tejido , Espectrometría Raman/instrumentación , Espectrometría Raman/métodos , Ingeniería de Tejidos/métodos , MicroscopíaRESUMEN
Vibrational spectroscopy is a nondestructive technique commonly used in chemical and physical analyses to determine atomic structures and associated properties. However, the evaluation and interpretation of spectroscopic profiles based on human-identifiable peaks can be difficult and convoluted. To address this challenge, we present a reliable protocol based on supervised manifold learning techniques meant to connect vibrational spectra to a variety of complex and diverse atomic structure configurations. As an illustration, we examined a large database of virtual vibrational spectroscopy profiles generated from atomistic simulations for silicon structures subjected to different stress, amorphization, and disordering states. We evaluated representative features in those spectra via various linear and nonlinear dimensionality reduction techniques and used the reduced representation of those features with decision trees to correlate them with structural information unavailable through classical human-identifiable peak analysis. We show that our trained model accurately (over 97% accuracy) and robustly (insensitive to noise) disentangles the contribution from the different material states, hence demonstrating a comprehensive decoding of spectroscopic profiles beyond classical (human-identifiable) peak analysis.
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Near-ground growth offers low-statured plants many benefits but also exposes them to the risk of being overtopped and losing access to sunlight. Plant community development is often portrayed as a process of serial dominance by successively taller species, but here we describe a mechanism by which a low-growing rosette species alters community spatial structure. Elephantopus elatus (Asteraceae), an herbaceous savanna plant with low-growing leaves that emerge radially from a central bud, pushes neighboring plants away and thereby avoids being overtopped. Active pushing is possible because the leaves have stout petioles that are basally anchored rather than attached to flexible twigs or stems. This growth-mediated leaf pushing introduces a novel example of active plant interactions that is likely important for other rosette plants.
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Asteraceae , Hojas de la Planta , PlantasRESUMEN
The organization of proteins is an important determinant of functionality in soft tissues. However, such organization is difficult to monitor over time in soft tissue with complex compositions. Here, we establish a method to determine the alignment of proteins in soft tissues of varying composition by polarized Raman spectroscopy (PRS). Unlike most conventional microscopy methods, PRS leverages non-destructive, label-free sample preparation. PRS data from highly aligned muscle layers were utilized to derive a weighting function for aligned proteins via principal component analysis (PCA). This trained weighting function was used as a master loading function to calculate a principal component score (PC1 Score) as a function of polarized angle for tendon, dermis, hypodermis, and fabricated collagen gels. Since the PC1 Score calculated at arbitrary angles was insufficient to determine level of alignment, we developed an Amplitude Alignment Metric by fitting a sine function to PC1 Score with respect to polarized angle. We found that our PRS-based Amplitude Alignment Metric can be used as an indicator of level of protein alignment in soft tissues in a non-destructive manner with label-free preparation and has similar discriminatory capacity among isotropic and anisotropic samples compared to microscopy-based image processing method. This PRS method does not require a priori knowledge of sample orientation nor composition and appears insensitive to changes in protein composition among different tissues. The Amplitude Alignment Metric introduced here could enable convenient and adaptable evaluation of protein alignment in soft tissues of varying protein and cell composition. STATEMENT OF SIGNIFICANCE: Polarized Raman spectroscopy (PRS) has been used to characterize the of organization of soft tissues. However, most of the reported applications of PRS have been on collagen-rich tissues and reliant on intensities of collagen-related vibrations. This work describes a PRS method via a multivariate analysis to characterize alignment in soft tissues composed of varying proteins. Of note, the highly aligned muscle layer of mouse skin was used to train a master function then applied to other soft tissue samples, and the degree of anisotropy in the PRS response was evaluated to obtain the level of alignment in tissues. We have demonstrated that this method supports convenient and adaptable evaluation of protein alignment in soft tissues of varying protein and cell composition.
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Colágeno , Espectrometría Raman , Animales , Anisotropía , Geles , Ratones , TendonesRESUMEN
An analytical model is developed to predict shockwave propagation and attenuation in hydrogels by combining the classical method of shock characteristics and a solution for the shock front structure. To guide the development of the model, molecular dynamics (MD) simulations are performed. Specifically, a one-dimensional shock pulse in poly(ethylene glycol) diacrylate (PEGDA) hydrogels is simulated with the nonequilibrium MD method. The role of polymer concentration on the shock response is evaluated by constructing hydrogels with 20, 35, and 50 wt% PEGDA concentrations in an idealized crosslinked network. Steady-state pressure-density and shock-particle velocity relationships are established using the Murnaghan equation of state. Shock front structure is characterized by a power-law equation that relates the shock front thickness with shock pressure. These results are used as critical input for the shock propagation and attenuation model. The model is then evaluated via comparison with the classical method of characteristics. It shows significant improvement in accuracy and successfully captures salient features of shockwave attenuation, including the shock pressure amplitude, the velocities of the shock and release waves, and the attenuation timeline. Hydrogels with higher polymer concentrations exhibit a shorter attenuation time at all particle velocities studied. This behavior is attributed to differences in bulk properties and shock front structure in hydrogels with different polymer/water concentrations.
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Materiales Biocompatibles , Hidrogeles , Simulación de Dinámica Molecular , Polietilenglicoles , AguaRESUMEN
Raman spectroscopy has been used extensively to characterize the influence of mechanical deformation on microstructure changes in biomaterials. While traditional piezo-spectroscopy has been successful in assessing internal stresses of hard biomaterials by tracking prominent peak shifts, peak shifts due to applied loads are near or below the resolution limit of the spectrometer for soft biomaterials with moduli in the kilo- to mega-Pascal range. In this Review, in addition to peak shifts, other spectral features (e.g., polarized intensity and intensity ratio) that provide quantitative assessments of microstructural orientation and secondary structure in soft biomaterials and their strain dependence are discussed. We provide specific examples for each method and classify sensitive Raman characteristic bands common across natural (e.g., soft tissue) and synthetic (e.g., polymeric scaffolds) soft biomaterials upon mechanical deformation. This Review can provide guidance for researchers aiming to analyze micromechanics of soft tissues and engineered tissue constructs by Raman spectroscopy.
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Materiales Biocompatibles , Espectrometría Raman , Ingeniería de TejidosRESUMEN
The high strain rate behavior of nonideal poly(ethylene glycol) diacrylate hydrogels under uniaxial tension and transient-state shear deformations is investigated using molecular dynamics (MD) simulations. This work specifically focuses on the influence of first-order loop defects, including their effect on topological evolutions. Two approaches are proposed to systematically introduce first-order loops, allowing separate and controllable investigations of effective cross-link functionality and cross-link density. MD simulations confirm that first-order loop defects are elastically inactive, but the topological disruptions caused by the presence of loop defects influence mechanical behavior. For decreasing effective cross-link functionality but constant cross-link density, a weaker tensile stress-strain response and decreasing shear-thickening behavior are observed. This is due to nonaffine translation of cross-link junction positions during deformation. Hydrogels with lower cross-link density but constant effective functionality show a stronger stress-strain response and an earlier transition between entropic and enthalpic deformation regimes. This behavior is correlated to changes in mesh size caused by the introduction of loops within an elastically active network. However, the resulting range of cross-link densities is not sufficient to cause measurable changes in shear-thickening behavior. To conclude, reductions in effective cross-link functionality are more important to high strain rate behavior than reductions in cross-link density.
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Shockwave propagation in polyethylene glycol diacrylate (PEGDA) hydrogels is simulated for the first time using nonequilibrium molecular dynamics simulations. PEGDA hydrogel models are built using the "perfect network" approach such that each crosslink junction is comprised of six chain connections. The influence of PEGDA concentration (20-70â¯wt%) on shock behavior is investigated for a range of particle velocities (200-1000â¯m/s). In agreement with reported experimental results in the literature on gels with similar densities, shock velocity and pressure in PEGDA hydrogels are found to increase with polymer concentration, within a range bounded by pure water and pure polymer behaviors. Nonlinear relationships are observed for shock pressure and shock front thickness as a function of concentration, and a logarithmic equation is proposed to describe this behavior. In addition, the relationship between pressure and shock front thickness is compared with hydrodynamic theory. Deviation from hydrodynamic predictions is observed at high particle velocities and this deviation is found to be related to viscosity changes. A power-law relationship between strain rate and pressure in PEGDA hydrogels is identified, similar to that of metals. However, a power-law exponent of 1.4 is computed for all gel concentrations, whereas an exponent of 4 is typically reported for metals.
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Hidrogeles/química , Fenómenos Mecánicos , Simulación de Dinámica Molecular , Polietilenglicoles/química , Agua/química , Hidrodinámica , Conformación Molecular , Presión , ViscosidadRESUMEN
Multiscale damage due to cavitation is considered as a potential mechanism of traumatic brain injury (TBI) associated with explosion. In this study, we employed a TBI relevant hippocampal ex vivo slice model to induce bubble cavitation. Placement of single reproducible seed bubbles allowed control of size, number, and tissue location to visualize and measure deformation parameters. Maximum strain value was measured at 45 µs after bubble collapse, presented with a distinct contour and coincided temporally and spatially with the liquid jet. Composite injury maps combined this maximum strain value with maximum measured bubble size and location along with histological injury patterns. This facilitated the correlation of bubble location and subsequent jet direction to the corresponding regions of high strain which overlapped with regions of observed injury. A dynamic threshold strain range for tearing of cerebral cortex was estimated to be between 0.5 and 0.6. For a seed bubble placed underneath the hippocampus, cavitation induced damage was observed in hippocampus (local), proximal cerebral cortex (marginal) and the midbrain/forebrain (remote) upon histological evaluation. Within this test model, zone of cavitation injury was greater than the maximum radius of the bubble. Separation of apposed structures, tissue tearing, and disruption of cellular layers defined early injury patterns that were not detected in the blast-exposed half of the brain slice. Ultrastructural pathology of the neurons exposed to cavitation was characterized by disintegration of plasma membrane along with loss of cellular content. The developed test system provided a controlled experimental platform to study cavitation induced high strain deformations on brain tissue slice. The goal of the future studies will be to lower underpressure magnitude and cavitation bubble size for more sensitive evaluation of injury.
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Traumatismos por Explosión/patología , Lesiones Encefálicas/patología , Animales , Encéfalo/patología , Membrana Celular/patología , Masculino , Ratas Sprague-Dawley , SonicaciónRESUMEN
Exposure to explosive blasts can produce functional debilitation in the absence of brain pathology detectable at the scale of current diagnostic imaging. Transient (ms) overpressure components of the primary blast wave are considered to be potentially damaging to the brain. Astrocytes participate in neuronal metabolic maintenance, blood-brain barrier, regulation of homeostatic environment, and tissue remodeling. Damage to astrocytes via direct physical forces has the potential to disrupt local and global functioning of neuronal tissue. Using an ex vivo brain slice model, we tested the hypothesis that viable astrocytes within the slice could be injured simply by transit of a single blast wave consisting of overpressure alone. A polymer split Hopkinson pressure bar (PSHPB) system was adapted to impart a single positive pressure transient with a comparable magnitude to those that might be present inside the head. A custom built test chamber housing the brain tissue slice incorporated revised design elements to reduce fluid space and promote transit of a uniform planar waveform. Confocal microscopy, stereology, and morphometry of glial fibrillary acidic protein (GFAP) immunoreactivity revealed that two distinct astrocyte injury profiles were identified across a 4 hr post-test survival interval: (a) presumed conventional astrogliosis characterized by enhanced GFAP immunofluorescence intensity without significant change in tissue area fraction and (b) a process comparable to clasmatodendrosis, an autophagic degradation of distal processes that has not been previously associated with blast induced neurotrauma. Analysis of astrocyte branching revealed early, sustained, and progressive differences distinct from the effects of slice incubation absent overpressure testing. Astrocyte vulnerability to overpressure transients indicates a potential for significant involvement in brain blast pathology and emergent dysfunction. The testing platform can isolate overpressure injury phenomena to provide novel insight on physical and biological mechanisms.
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Astrocitos/patología , Traumatismos por Explosión/patología , Lesiones Encefálicas/patología , Encéfalo/patología , Animales , Modelos Animales de Enfermedad , Explosiones , Proteína Ácida Fibrilar de la Glía/análisis , Masculino , Presión/efectos adversos , Ratas Sprague-DawleyRESUMEN
Wave propagation characteristics in long cylindrical specimens of ballistic gelatine have been investigated using a high speed digital camera and hyper elastic constitutive models. The induced transient deformation is modelled with strain rate dependent Mooney-Rivlin parameters which are determined by modelling the stress-strain response of gelatine at a range of strain rates. The varying velocity of wave propagation through the gelatine cylinder is derived as a function of prestress or stretch in the gelatine specimen. A finite element analysis is conducted using the above constitutive model by suitably defining the impulse imparted by the polymer bar into the gelatine specimen. The model results are found to capture the experimentally observed wave propagation characteristics in gelatine effectively.
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Gelatina/química , Ensayo de Materiales , Elasticidad , Análisis de Elementos FinitosRESUMEN
Blast-induced traumatic brain injury (bTBI) has received increasing attention in recent years due to ongoing military operations in Iraq and Afghanistan. Sudden impacts or explosive blasts generate stress and pressure waves that propagate at high velocities and affect sensitive neurological tissues. The immediate soft tissue response to these stress waves is difficult to assess using current in vivo imaging technologies. However, these stress waves and resultant stretching and shearing of tissue within the nano- to microsecond time scale of blast and impact are likely to cause initial injury. To visualize the effects of stress wave loading, we have developed a new ex vivo model in which living tissue slices from rat brain, attached to a ballistic gelatin substrate, were subjected to high-strain-rate loads using a polymer split Hopkinson pressure bar (PSHPB) with real-time high-speed imaging. In this study, average peak fluid pressure within the test chamber reached a value of 1584±63.3 psi. Cavitation due to a trailing underpressure wave was also observed. Time-resolved images of tissue deformation were collected and large maximum eigenstrains (0.03-0.42), minimum eigenstrains (-0.33 to -0.03), maximum shear strains (0.09-0.45), and strain rates (8.4×10³/sec) were estimated using digital image correlation (DIC). Injury at 4 and 6 h was quantified using Fluoro-Jade C. Neuronal injury due to PSHPB testing was found to be significantly greater than injury associated with the tissue slice paradigm alone. While large pressures and strains were encountered for these tests, this system provides a controllable test environment to study injury to submerged brain slices over a range of strain rate, pressure, and strain loads.
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Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Modelos Animales de Enfermedad , Hipocampo/patología , Hipocampo/fisiopatología , Enfermedad Aguda , Animales , Lesiones Encefálicas/etiología , Degeneración Nerviosa/etiología , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Técnicas de Cultivo de Órganos , Presión/efectos adversos , Ratas , Ratas Sprague-Dawley , Estrés MecánicoRESUMEN
A new approach for characterization of agarose gel permeability under compression at different loading velocities is proposed. Uniaxial compression tests on thin agarose gel specimens in a rigid porous confinement cell immersed in a water bath are undertaken. The equilibrium response of the gel, which is assumed to be achieved under extremely low-loading velocity (of the order of tens nanometers per second) is considered to be the response of the hydrated gel scaffold. The water exudation behavior from the agarose gel was extracted from the load-displacement response under various loading velocities by subtracting the equilibrium response. It was found that the pressure on water in the gel is not a linear function of loading velocity or volume flow rate and therefore, the permeability of agarose gel was observed to vary with deformation and water flow velocity. In addition, it was inferred from the analysis that at low velocities and large strain levels the gel permeability dominates the compression behavior, and at higher velocities and small strain levels the viscosity of the hydrated matrix may contribute to the load. Finally, permeability variation in agarose gel at different loading velocities is attributed to the two states (free water and bound water) of water molecules in the gel.
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Fuerza Compresiva , Ensayo de Materiales/métodos , Sefarosa/química , Geles , Hidrodinámica , Permeabilidad , Porosidad , Estrés Mecánico , Agua/químicaRESUMEN
Gelatin is a popular tissue simulant used in biomedical applications. The uniaxial compressive stress-strain response of gelatin was determined at a range of strain rates. In the quasistatic regime, gelatin strength remained relatively constant. With increase in loading rate, the compressive strength increased from 3kPa at a strain rate of around 0.0013/s to 6MPa at a strain rate of around 3200/s. This dramatic increase in strength of gelatin at high rates is attributed to its shear-thickening behavior and is argued on the basis of hydrocluster formation mechanism and differences in internal energy dissipation mechanism under static and dynamic loading.