Beyond 2D: Novel biomaterial approaches for modeling the placenta.

This review considers fully three-dimensional biomaterial environments of varying complexity as these pertain to research on the placenta. The developments in placental cell sources are first considered, along with the corresponding maternal cells with which the trophoblast interact. We consider biomaterial sources, including hybrid and composite biomaterials. Properties and characterization of biomaterials are discussed in the context of material design for specific placental applications. The development of increasingly complicated three-dimensional structures includes examples of advanced fabrication methods such as microfluidic device fabrication and 3D bioprinting, as utilized in a placenta context. The review finishes with a discussion of the potential for in vitro, three-dimensional placenta research to address health disparities and sexual dimorphism, especially in light of the exciting recent changes in the regulatory environment for in vitro devices.

Epigenetic Priming Enhances Chondrogenic Potential of Expanded Chondrocytes.

Expansion of chondrocytes presents a major obstacle in the cartilage regeneration procedure, such as matrix-induced autologous chondrocyte implantation. Dedifferentiation of chondrocytes during the expansion process leads to the emergence of a fibrotic (chondrofibrotic) phenotype that decreases the chondrogenic potential of the implanted cells. We aim to (1) determine the extent that chromatin architecture of H3K27me3 and H3K9me3 remodels during dedifferentiation and persists after the transfer to a three-dimensional (3D) culture; and (2) to prevent this persistent remodeling to enhance the chondrogenic potential of expanded bovine chondrocytes, used as a model system. Chromatin architecture remodeling of H3K27me3 and H3K9me3 was observed at 0 population doublings, 8 population doublings, and 16 population doublings (PD16) in a two-dimensional (2D) culture and after encapsulation of the expanded chondrocytes in a 3D hydrogel culture. Chondrocytes were treated with inhibitors of epigenetic modifiers (epigenetic priming) for PD16 and then encapsulated in 3D hydrogels. Chromatin architecture of chondrocytes and gene expression were evaluated before and after encapsulation. We observed a change in chromatin architecture of epigenetic modifications H3K27me3 and H3K9me3 during chondrocyte dedifferentiation. Although inhibiting enzymes that modify H3K27me3 and H3K9me3 did not alter the dedifferentiation process in 2D culture, applying these treatments during the 2D expansion did increase the expression of select chondrogenic genes and protein deposition of type II collagen when transferred to a 3D environment. Overall, we found that epigenetic priming of expanded bovine chondrocytes alters the cell fate when chondrocytes are later encapsulated into a 3D environment, providing a potential method to enhance the success of cartilage regeneration procedures.

Biomechanical Modeling of Cesarean Section Scars and Scar Defects.

Uterine rupture is an intrinsically biomechanical process associated with high maternal and fetal mortality. A previous Cesarean section (C-section) is the main risk factor for uterine rupture in a subsequent pregnancy due to tissue failure at the scar region. Finite element modeling of the uterus and scar tissue presents a promising method to further understand and predict uterine ruptures. Using patient dimensions of an at-term uterus, a C-section scar was modeled with an applied intrauterine pressure to study how scars affect uterine stress. The scar positioning and uterine thickness were varied, and a defect was incorporated into the scar region. The modeled stress distributions confirmed clinical observations as the increased regions of stress due to scar positioning, thinning of the uterine walls, and the presence of a defect are consistent with clinical observations of features that increase the risk of uterine rupture.

Mechanically induced alterations in chromatin architecture guide the balance between cell plasticity and mechanical memory.

Phenotypic plasticity, or adaptability, of a cell determines its ability to survive and function within changing cellular environments. Changes in the mechanical environment, ranging from stiffness of the extracellular matrix (ECM) to physical stress such as tension, compression, and shear, are critical environmental cues that influence phenotypic plasticity and stability. Furthermore, an exposure to a prior mechanical signal has been demonstrated to play a fundamental role in modulating phenotypic changes that persist even after the mechanical stimulus is removed, creating stable mechanical memories. In this mini review, our objective is to highlight how the mechanical environment alters both phenotypic plasticity and stable memories through changes in chromatin architecture, mainly focusing on examples in cardiac tissue. We first explore how cell phenotypic plasticity is modulated in response to changes in the mechanical environment, and then connect the changes in phenotypic plasticity to changes in chromatin architecture that reflect short-term and long-term memories. Finally, we discuss how elucidating the mechanisms behind mechanically induced chromatin architecture that lead to cell adaptations and retention of stable mechanical memories could uncover treatment methods to prevent mal-adaptive permanent disease states.

Mechanical memory stored through epigenetic remodeling reduces cell therapeutic potential.

Understanding how cells remember previous mechanical environments to influence their fate, or mechanical memory, informs the design of biomaterials and therapies in medicine. Current regeneration therapies, such as cartilage regeneration procedures, require 2D cell expansion processes to achieve large cell populations critical for the repair of damaged tissues. However, the limit of mechanical priming for cartilage regeneration procedures before inducing long-term mechanical memory following expansion processes is unknown, and mechanisms defining how physical environments influence the therapeutic potential of cells remain poorly understood. Here, we identify a threshold to mechanical priming separating reversible and irreversible effects of mechanical memory. After 16 population doublings in 2D culture, expression levels of tissue-identifying genes in primary cartilage cells (chondrocytes) are not recovered when transferred to 3D hydrogels, while expression levels of these genes were recovered for cells only expanded for eight population doublings. Additionally, we show that the loss and recovery of the chondrocyte phenotype correlates with a change in chromatin architecture, as shown by structural remodeling of the trimethylation of H3K9. Efforts to disrupt the chromatin architecture by suppressing or increasing levels of H3K9me3 reveal that only with increased levels of H3K9me3 did the chromatin architecture of the native chondrocyte phenotype partially return, along with increased levels of chondrogenic gene expression. These results further support the connection between the chondrocyte phenotype and chromatin architecture, and also reveal the therapeutic potential of inhibitors of epigenetic modifiers as disruptors of mechanical memory when large numbers of phenotypically suitable cells are required for regeneration procedures.

Dynamic biophysical responses of neuronal cell nuclei and cytoskeletal structure following high impulse loading.

Cells are continuously exposed to dynamic environmental cues that influence their behavior. Mechanical cues can influence cellular and genomic architecture, gene expression, and intranuclear mechanics, providing evidence of mechanosensing by the nucleus, and a mechanoreciprocity between the nucleus and environment. Force disruption at the tissue level through aging, disease, or trauma, propagates to the nucleus and can have lasting consequences on proper functioning of the cell and nucleus. While the influence of mechanical cues leading to axonal damage has been well studied in neuronal cells, the mechanics of the nucleus following high impulse loading is still largely unexplored. Using an in vitro model of traumatic neural injury, we show a dynamic nuclear behavioral response to impulse stretch (up to 170% strain per second) through quantitative measures of nuclear movement, including tracking of rotation and internal motion. Differences in nuclear movement were observed between low and high strain magnitudes. Increased exposure to impulse stretch exaggerated the decrease in internal motion, assessed by particle tracking microrheology, and intranuclear displacements, assessed through high-resolution deformable image registration. An increase in F-actin puncta surrounding nuclei exposed to impulse stretch additionally demonstrated a corresponding disruption of the cytoskeletal network. Our results show direct biophysical nuclear responsiveness in neuronal cells through force propagation from the substrate to the nucleus. Understanding how mechanical forces perturb the morphological and behavioral response can lead to a greater understanding of how mechanical strain drives changes within the cell and nucleus, and may inform fundamental nuclear behavior after traumatic axonal injury. STATEMENT OF SIGNIFICANCE: The nucleus of the cell has been implicated as a mechano-sensitive organelle, courting molecular sensors and transmitting physical cues in order to maintain cellular and tissue homeostasis. Disruption of this network due to disease or high velocity forces (e.g., trauma) can not only result in orchestrated biochemical cascades, but also biophysical perturbations. Using an in vitro model of traumatic neural injury, we aimed to provide insight into the neuronal nuclear mechanics and biophysical responses at a continuum of strain magnitudes and after repetitive loads. Our image-based methods demonstrate mechanically-induced changes in cellular and nuclear behavior after high intensity loading and have the potential to further define mechanical thresholds of neuronal cell injury.

The heterogeneous mechanical properties of adolescent growth plate cartilage: A study in rabbit.

The growth plate is a cartilaginous tissue that functions to lengthen bones in children. When fractured, however, the growth plate can lose this critical function. Our understanding of growth plate fracture and mechanobiology is currently hindered by sparse information on the growth plate's microscale spatial gradients in mechanical properties. In this study, we performed microindentation across the proximal tibia growth plate of 9-week-old New Zealand White rabbits (n = 15) to characterize spatial variations in mechanical properties using linear elastic and nonlinear poroelastic material models. Mean indentation results for Hertz reduced modulus ranged from 380 to 690 kPa, with a peak in the upper hypertrophic zone and significant differences (p < 0.05) between neighboring zones. Using a subset of these animals (n = 7), we characterized zonal structure and extracellular matrix content of the growth plate through confocal fluorescent microscopy and Raman spectroscopy mapping. Comparison between mechanical properties and matrix content across the growth plate showed that proteoglycan content correlated with compressive modulus. This study is the first to measure poroelastic mechanical properties from microindentation across growth plate cartilage and to discern differing mechanical properties between the upper and lower hypertrophic zones. This latter finding may explain the location of typical growth plate fractures. The spatial variation in our reported mechanical properties emphasize the heterogeneous structure of the growth plate which is important to inform future regenerative implant design and mechanobiological models.

Dedifferentiation alters chondrocyte nuclear mechanics during in vitro culture and expansion.

The biophysical features of a cell can provide global insights into diverse molecular changes, especially in processes like the dedifferentiation of chondrocytes. Key biophysical markers of chondrocyte dedifferentiation include flattened cellular morphology and increased stress-fiber formation. During cartilage regeneration procedures, dedifferentiation of chondrocytes during in vitro expansion presents a critical limitation to the successful repair of cartilage tissue. Our study investigates how biophysical changes of chondrocytes during dedifferentiation influence the nuclear mechanics and gene expression of structural proteins located at the nuclear envelope. Through an experimental model of cell stretching and a detailed spatial intranuclear strain quantification, we identified that strain is amplified and the distribution of strain within the chromatin is altered under tensile loading in the dedifferentiated state. Further, using a confocal microscopy image-based finite element model and simulation of cell stretching, we found that the cell shape is the primary determinant of the strain amplification inside the chondrocyte nucleus in the dedifferentiated state. Additionally, we found that nuclear envelope proteins have lower gene expression in the dedifferentiated state. This study highlights the role of cell shape in nuclear mechanics and lays the groundwork to design biophysical strategies for the maintenance and enhancement of the chondrocyte phenotype during cell expansion with a goal of successful cartilage tissue engineering.

Nuclear deformation guides chromatin reorganization in cardiac development and disease.

In cardiovascular tissues, changes in the mechanical properties of the extracellular matrix are associated with cellular de-differentiation and with subsequent functional declines. However, the underlying mechanoreceptive mechanisms are largely unclear. Here, by generating high-resolution, full-field strain maps of cardiomyocyte nuclei during contraction in vitro, complemented with evidence from tissues from patients with cardiomyopathy and from mice with reduced cardiac performance, we show that cardiomyocytes establish a distinct nuclear organization during maturation, characterized by the reorganization of H3K9me3-marked chromatin towards the nuclear border. Specifically, we show that intranuclear tension is spatially correlated with H3K9me3-marked chromatin, that reductions in nuclear deformation (through environmental stiffening or through the disruption of complexes of the linker of nucleoskeleton and cytoskeleton) abrogate chromatin reorganization and lead to the dissociation of H3K9me3-marked chromatin from the nuclear periphery, and that the suppression of H3K9 methylation induces chromatin reorganization and reduces the expression of cardiac developmental genes. Overall, our findings indicate that, by integrating environmental mechanical cues, the nuclei of cardiomyocytes guide and stabilize the fate of cells through the reorganization of epigenetically marked chromatin.

Nuclear Stiffness Decreases with Disruption of the Extracellular Matrix in Living Tissues.

Reciprocal interactions between the cell nucleus and the extracellular matrix lead to macroscale tissue phenotype changes. However, little is known about how the extracellular matrix environment affects gene expression and cellular phenotype in the native tissue environment. Here, it is hypothesized that enzymatic disruption of the tissue matrix results in a softer tissue, affecting the stiffness of embedded cell and nuclear structures. The aim is to directly measure nuclear mechanics without perturbing the native tissue structure to better understand nuclear interplay with the cell and tissue microenvironments. To accomplish this, an atomic force microscopy needle-tip probe technique that probes nuclear stiffness in cultured cells to measure the nuclear envelope and cell membrane stiffness within native tissue is expanded. This technique is validated by imaging needle penetration and subsequent repair of the plasma and nuclear membranes of HeLa cells stably expressing the membrane repair protein CHMP4B-GFP. In the native tissue environment ex vivo, it is found that while enzymatic degradation of viable cartilage tissues with collagenase 3 (MMP-13) and aggrecanase-1 (ADAMTS-4) decreased tissue matrix stiffness, cell and nuclear membrane stiffness is also decreased. Finally, the capability for cell and nucleus elastography using the AFM needle-tip technique is demonstrated. These results demonstrate disruption of the native tissue environment that propagates to the plasma membrane and interior nuclear envelope structures of viable cells.

TENSCell: Imaging of Stretch-Activated Cells Reveals Divergent Nuclear Behavior and Tension.

Mechanisms of cellular and nuclear mechanosensation are unclear, partially because of a lack of methods that can reveal dynamic processes. Here, we present a new concept for a low-cost, three-dimensionally printed device that enables high-magnification imaging of cells during stretch. We observed that nuclei of mouse embryonic skin fibroblasts underwent rapid (within minutes) and divergent responses, characterized by nuclear area expansion during 5% strain but nuclear area shrinkage during 20% strain. Only responses to low strain were dependent on calcium signaling, whereas actin inhibition abrogated all nuclear responses and increased nuclear strain transfer and DNA damage. Imaging of actin dynamics during stretch revealed similar divergent trends, with F-actin shifting away from (5% strain) or toward (20% strain) the nuclear periphery. Our findings emphasize the importance of simultaneous stimulation and data acquisition to capture mechanosensitive responses and suggest that mechanical confinement of nuclei through actin may be a protective mechanism during high mechanical stretch or loading.

Deformation Microscopy for Dynamic Intracellular and Intranuclear Mapping of Mechanics with High Spatiotemporal Resolution.

Structural heterogeneity is a hallmark of living cells that drives local mechanical properties and dynamic cellular responses. However, the robust quantification of intracellular mechanics is lacking from conventional methods. Here, we describe the development of deformation microscopy, which leverages conventional imaging and an automated hyperelastic warping algorithm to investigate strain history, deformation dynamics, and changes in structural heterogeneity within the interior of cells and cell nuclei. Using deformation microscopy, we found that partial or complete disruption of LINC complexes in cardiomyocytes in vitro and lamin A/C deficiency in myocytes in vivo abrogate dominant tensile loading in the nuclear interior. We also found that cells cultured on stiff substrates or in hyperosmotic conditions displayed abnormal strain burden and asymmetries at interchromatin regions, which are associated with active transcription. Deformation microscopy represents a foundational approach toward intracellular elastography, with the potential utility to provide mechanistic and quantitative insights in diverse mechanobiological applications.

In Vivo Multiscale and Spatially-Dependent Biomechanics Reveals Differential Strain Transfer Hierarchy in Skeletal Muscle.

Biological tissues have a complex hierarchical architecture that spans organ to subcellular scales and comprises interconnected biophysical and biochemical machinery. Mechanotransduction, gene regulation, gene protection, and structure-function relationships in tissues depend on how force and strain are modulated from macro to micro scales, and vice versa. Traditionally, computational and experimental techniques have been used in common model systems (e.g., embryos) and simple strain measures were applied. But the hierarchical transfer of mechanical parameters like strain in mammalian systems is largely unexplored in vivo. Here, we experimentally probed complex strain transfer processes in mammalian skeletal muscle tissue over multiple biological scales using complementary in vivo ultrasound and optical imaging approaches. An iterative hyperelastic warping technique quantified the spatially-dependent strain distributions in tissue, matrix, and subcellular (nuclear) structures, and revealed a surprising increase in strain magnitude and heterogeneity in active muscle as the spatial scale also increased. The multiscale strain heterogeneity indicates tight regulation of mechanical signals to the nuclei of individual cells in active muscle, and an emergent behavior appearing at larger (e.g. tissue) scales characterized by dramatically increased strain complexity.