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Microscale deformation processes, such as reorientation, buckling, and sliding of collagen fibrils, determine the mechanical behavior and function of collagenous tissue. While changes in the structure and composition of tendon have been extensively studied, the deformation mechanisms that modulate the interaction of extracellular matrix (ECM) constituents are not well understood, partly due to the lack of appropriate techniques to probe the behavior. In particular, the role of glycosaminoglycans (GAGs) in modulating collagen fibril interactions has remained controversial. Some studies suggest that GAGs act as crosslinkers between the collagen fibrils, while others have not found such evidence and postulate that GAGs have other functions. Here, we introduce a new framework, relying on orientation-dependent indentation behavior of tissue and computational modeling, to evaluate the shear-mediated function of GAGs in modulating the collagen fibril interactions at a length scale more relevant to fibrils compared to bulk tests. Specifically, we use chondroitinase ABC to enzymatically deplete the GAGs in tendon; measure the orientation-dependent indentation response in transverse and longitudinal orientations; and infer the microscale deformation mechanisms and function of GAGs from a microstructural computational model and a modified shear-lag model. We validate the modeling approach experimentally and show that GAGs facilitate collagen fibril sliding with minimal crosslinking function. We suggest that the molecular reconfiguration of GAGs is a potential mechanism for their microscale, strain-dependent viscoelastic behavior. This study reveals the mechanisms that control the orientation-dependent indentation response by affecting the shear deformation and provides new insights into the mechanical function of GAGs and collagen crosslinkers in collagenous tissue.

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The spatial arrangement and interactions of the extracellular matrix (ECM) components control the mechanical behavior of tissue at multiple length scales. Changes in microscale deformation mechanisms affect tissue function and are often hallmarks of remodeling and disease. Despite their importance, the deformation mechanisms that modulate the mechanical behavior of collagenous tissue, particularly in indentation and compression modes of deformation, remain poorly understood. Here, we develop an integrated computational and experimental approach to investigate the deformation mechanisms of collagenous tissue at the microscale. While the complex deformation arising from indentation with a spherical probe is often considered a pitfall rather than an opportunity, we leverage this orientation-dependent deformation to examine the shear-regulated interactions of collagen fibrils and the role of crosslinks in modulating these interactions. We specifically examine tendon and cervix, two tissues rich in collagen with quite different microstructures and mechanical functions. We find that interacting, crosslinked collagen fibrils resist microscale longitudinal compressive forces, while widely used constitutive models fail to capture this behavior. The reorientation of collagen fibrils tunes the compressive stiffness of complex tissues like cervix. This study offers new insights into the mechanical behavior of collagen fibrils during indentation, and more generally, under longitudinal compressive forces, and illustrates the mechanisms that contribute to the experimentally observed orientation-dependent mechanical behavior. STATEMENT OF SIGNIFICANCE: Remodeling and disease can affect the deformation and interaction of tissue constituents, and thus mechanical function of tissue. Yet, the microscale deformation mechanisms are not well characterized in many tissues. Here, we develop a combined experimental-computational approach to infer the microscale deformation mechanisms of collagenous tissues with very different functions: tendon and cervix. Results show that collagen fibrils resist microscale forces along their length, though widely-used constitutive models do not account for this mechanism. This deformation process partially modulates the compressive stiffness of complex tissues such as cervix. Computational modeling shows that crosslink-mediated shear deformations are central to this unexpected behavior. This study offers new insights into the deformation mechanisms of collagenous tissue and the function of collagen crosslinkers.

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The cervix acts as a dynamic barrier between the uterus and vagina, retaining the fetus during pregnancy and allowing birth at term. Critical to this function, the physical properties of the cervix change, or remodel, but abnormal remodeling can lead to preterm birth (PTB). Although cervical remodeling has been studied, the complex 3D cervical microstructure has not been well-characterized. In this complex, dynamic, and heterogeneous tissue microenvironment, the microstructural changes are likely also heterogeneous. Using quantitative, 3D, second-harmonic generation microscopy, we demonstrate that rat cervical remodeling during pregnancy is not uniform across the cervix; the collagen fibers orient progressively more perpendicular to the cervical canals in the inner cervical zone, but do not reorient in other regions. Furthermore, regions that are microstructurally distinct early in pregnancy become more similar as pregnancy progresses. We use a finite element simulation to show that heterogeneous regional changes influence cervical funneling, an important marker of increased risk for PTB; the internal cervical os shows ∼6.5x larger radial displacement when fibers in the inner cervical zone are parallel to the cervical canals compared to when fibers are perpendicular to the canals. Our results provide new insights into the microstructural and tissue-level cervical changes that have been correlated with PTB and motivate further clinical studies exploring the origins of cervical funneling. STATEMENT OF SIGNIFICANCE: Cervical funneling, or dilation of the internal cervical os, is highly associated with increased risk of preterm birth. This study explores the 3D microstructural changes of the rat cervix during pregnancy and illustrates how these changes influence cervical funneling, assuming similar evolution in rats and humans. Quantitative imaging showed that microstructural remodeling during pregnancy is nonuniform across cervical regions and that initially distinct regions become more similar. We report, for the first time, that remodeling of the inner cervical zone can influence the dilation of the internal cervical os and allow the cervix to stay closed despite increased intrauterine pressure. Our results suggest a possible relationship between the microstructural changes of this zone and cervical funneling, motivating further clinical investigations.

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The structural and mechanical properties of tissue and the interplay between them play a critical role in tissue function. We introduce the optomechanogram, a combined quantitative and qualitative visualization of spatially co-registered measurements of the microstructural and micromechanical properties of any tissue. Our approach relies on the co-registration of two independent platforms, second-harmonic generation (SHG) microscopy for quantitative assessment of 3D collagen-fiber microstructural organization, and nanoindentation (NI) for local micromechanical properties. We experimentally validate our method by applying to uterine cervix tissue, which exhibits structural and mechanical complexity. We find statistically significant agreement between the micromechanical and microstructural data, and confirm that the distinct tissue regions are distinguishable using either the SHG or NI measurements. Our method could potentially be used for research in pregnancy maintenance, mechanobiological studies of tissues and their constitutive modeling and more generally for the optomechanical metrology of materials.

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The 2D optical trapping ability of larger-than average-particles is compared for three different beam types: a flat-top, a Gaussian beam, and a donut shaped beam. Optical force-displacement curves are calculated in four different size regimes of particle diameters (1.5-20 µm). We find that the trapping efficiency increases linearly with ratio of particle diameter to wavelength for all three beams. As the ratio reaches a specific threshold value, the flat-top focus exhibits the largest trapping efficiency compared to the other two beam types. We experimentally demonstrate that flat-top focusing provides the largest transverse trapping efficiency for particles as large as 20 µm in diameter for our given experimental conditions. The overall trend in our experimental results follows that observed in our simulation model. The results from this study could facilitate light manipulation of large particles.

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We present the application of Fourier transform-second-harmonic generation (FT-SHG) imaging to evaluate the arrangement of collagen fibers in five nonpregnant rat cervices. Tissue slices from the mid-cervix and near the external orifice of the cervix were analyzed in both two-dimensions (2D) and three-dimensions (3D). We validate that the cervical microstructure can be quantitatively assessed in three dimensions using FT-SHG imaging and observe collagen fibers oriented both in and out-of-plane in the outermost and the innermost layers, which cannot be observed using 2D FT-SHG analysis alone. This approach has the potential to be a clinically applicable method for measuring progressive changes in collagen organization during cervical remodeling in humans.

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The plasmon resonance-based optical trapping (PREBOT) method is used to achieve stable trapping of metallic nanoparticles of different shapes and composition, including Au bipyramids and Au/Ag core/shell nanorods. In all cases the longitudinal plasmon mode of these anisotropic particles is used to enhance the gradient force of an optical trap, thereby increasing the strength of the trap potential. Specifically, the trapping laser is slightly detuned to the long-wavelength side of the longitudinal plasmon resonance where the sign of the real component of the polarizability leads to an attractive gradient force. A second (femtosecond pulsed) laser is used to excite two-photon fluorescence for detection of the trapped nanoparticles. Two-photon fluorescence time trajectories are recorded for up to 20 minutes for single and multiple particles in the trap. In the latter case, a stepwise increase reflects sequential loading of single Au bipyramids. The nonlinearity of the amplitude and noise with step number are interpreted as arising from interactions or enhanced local fields amongst the trapped particles and fluctuations in the arrangements thereof.

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We present a novel interferometric technique for performing ellipsometric measurements. This technique relies on the use of a nonclassical optical source, namely, polarization-entangled twin photons generated by spontaneous parametric downconversion from a nonlinear crystal, in conjunction with a coincidence-detection scheme. Ellipsometric measurements acquired with this scheme are absolute; i.e., they do not require source and detector calibration.