Automatic segmentation, feature extraction and comparison of healthy and stroke cerebral vasculature.

Accurate segmentation of cerebral vasculature and a quantitative assessment of its morphology is critical to various diagnostic and therapeutic purposes and is pertinent to studying brain health and disease. However, this is still a challenging task due to the complexity of the vascular imaging data. We propose an automated method for cerebral vascular segmentation without the need of any manual intervention as well as a method to skeletonize the binary segmented map to extract vascular geometric features and characterize vessel structure. We combine a Hessian-based probabilistic vessel-enhancing filtering with an active-contour-based technique to segment magnetic resonance and computed tomography angiograms (MRA and CTA) and subsequently extract the vessel centerlines and diameters to calculate the geometrical properties of the vasculature. Our method was validated using a 3D phantom of the Circle-of-Willis region, demonstrating 84% mean Dice similarity coefficient (DSC) and 85% mean Pearson's correlation coefficient (PCC) with minimal modified Hausdorff distance (MHD) error (3 surface pixels at most), and showed superior performance compared to existing segmentation algorithms upon quantitative comparison using DSC, PCC and MHD. We subsequently applied our algorithm to a dataset of 40 subjects, including 1) MRA scans of healthy subjects (n = 10, age = 30 ± 9), 2) MRA scans of stroke patients (n = 10, age = 51 ± 15), 3) CTA scans of healthy subjects (n = 10, age = 62 ± 12), and 4) CTA scans of stroke patients (n = 10, age = 68 ± 11), and obtained a quantitative comparison between the stroke and normal vasculature for both imaging modalities. The vascular network in stroke patients compared to age-adjusted healthy subjects was found to have a significantly (p < 0.05) higher tortuosity (3.24 ± 0.88 rad/cm vs. 7.17 ± 1.61 rad/cm for MRA, and 4.36 ± 1.32 rad/cm vs. 7.80 ± 0.92 rad/cm for CTA), higher fractal dimension (1.36 ± 0.28 vs. 1.71 ± 0.14 for MRA, and 1.56 ± 0.05 vs. 1.69 ± 0.20 for CTA), lower total length (3.46 ± 0.99 m vs. 2.20 ± 0.67 m for CTA), lower total volume (61.80 ± 18.79 ml vs. 34.43 ± 22.9 ml for CTA), lower average diameter (2.4 ± 0.21 mm vs. 2.18 ± 0.07 mm for CTA), and lower average branch length (4.81 ± 1.97 mm vs. 8.68 ± 2.03 mm for MRA), respectively. We additionally studied the change in vascular features with respect to aging and imaging modality. While we observed differences between features as a result of aging, statistical analysis did not show any significant differences, whereas we found that the number of branches were significantly different (p < 0.05) between the two imaging modalities (201 ± 73 for MRA vs. 189 ± 69 for CTA). Our segmentation and feature extraction algorithm can be applied on any imaging modality and can be used in the future to automatically obtain the 3D segmented vasculature for diagnosis and treatment planning as well as to study morphological changes due to stroke and other cerebrovascular diseases (CVD) in the clinic.

Probing Collective Mechanoadaptation in Cardiomyocyte Development by Plasma Lithography Patterned Elastomeric Substrates.

Understanding how the mechanical microenvironment affects cardiomyocyte development is crucial to the creation of in vitro models for studying heart physiology and pathophysiology. This knowledge will also facilitate the design of biomaterials and tissue scaffolds utilized in the generation of functional tissue constructs for regenerative medicine and drug screening applications. Here, plasma lithography patterning of elastomeric substrates is exploited for creating microtissues composed of neonatal cardiomyocytes and investigating their attributes in different mechanical microenvironments. Restriction of the cellular outgrowth in line patterns results in cardiomyocytes developing into multicellular clusters and collectively adapting to geometric confinement and substrate stiffness. Immunofluorescence microscopy, video microscopy, and force spectroscopy show that the size and shape of the cardiomyocyte clusters, as well as sarcomere length, fiber alignment, beating amplitude, and beating frequency of the cardiomyocytes, are regulated by the microenvironmental cues. Computational analysis reveals that the mechanical stress at the cluster-substrate interface strongly correlates with the characteristics of the cardiomyocytes. Taken together, our results underscore a collective mechanoadaptation scheme in cardiac development.

Decreasing Wound Edge Stress Enhances Leader Cell Formation during Collective Smooth Muscle Cell Migration.

Collective cell migration is vital to tissue remodeling in wound repair, development, and cancer invasion. Nevertheless, studies on collective cell migration have largely focused on epithelial growth and repair mechanisms and have only recently expanded to explore coordinated metastatic cancer and smooth muscle cell behaviors. The regulatory mechanisms of smooth muscle cell collective migration, such as leader-follower organization and mechanosensitivity, remain poorly understood. In this study, we demonstrate the involvement of leader cells during collective smooth muscle cell migration using dynamic cell tracking and single cell gene expression analysis. Engineered wound models, including ingrowth, outgrowth, and straight edge geometries, along with traction force microscopy and finite element stress mapping reveal that smooth muscle leader cells are enhanced at the wound edge when the intercellular tension near the cell wound boundary is reduced. Pharmacological perturbation further supports the notion that mechanical force negatively regulates the formation of leader cells. The mechanical regulation of collective smooth muscle cell migration via the formation of leader cells may lead to novel treatment strategies for pathogenic smooth muscle cell conditions in the future.

Probing 3D Collective Cancer Invasion Using Double-Stranded Locked Nucleic Acid Biosensors.

Cancer is a leading cause of death worldwide and metastases are responsible for over 90% of human cancer deaths. There is an urgent need to develop novel therapeutics for suppressing cancer invasion, the initial step of metastasis. Nevertheless, the regulation of cancer invasion is poorly understood due to a paucity of tools for monitoring the invasion process in 3D microenvironments. Here, we report a double-stranded locked nucleic acid (dsLNA) biosensor for investigating 3D collective cancer invasion. By incorporating multiphoton microscopy and the dsLNA biosensor, we perform dynamic single cell gene expression analysis while simultaneously characterizing the biomechanical interaction between the invading sprouts and the extracellular matrix. Gene profiling of invasive leader cells and detached cells suggest distinctive signaling mechanisms involved in collective and individual invasion in the 3D microenvironment. Our results underscore the involvement of Notch signaling in 3D collective cancer invasion, which warrants further investigation toward antimetastasis therapy in the future.

Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement.

When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters.

Knockout of Lmod2 results in shorter thin filaments followed by dilated cardiomyopathy and juvenile lethality.

Leiomodin 2 (Lmod2) is an actin-binding protein that has been implicated in the regulation of striated muscle thin filament assembly; its physiological function has yet to be studied. We found that knockout of Lmod2 in mice results in abnormally short thin filaments in the heart. We also discovered that Lmod2 functions to elongate thin filaments by promoting actin assembly and dynamics at thin filament pointed ends. Lmod2-KO mice die as juveniles with hearts displaying contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy. Lmod2-null cardiomyocytes produce less contractile force than wild type when plated on micropillar arrays. Introduction of GFP-Lmod2 via adeno-associated viral transduction elongates thin filaments and rescues structural and functional defects observed in Lmod2-KO mice, extending their lifespan to adulthood. Thus, to our knowledge, Lmod2 is the first identified mammalian protein that functions to elongate actin filaments in the heart; it is essential for cardiac thin filaments to reach a mature length and is required for efficient contractile force and proper heart function during development.

Probing mechanoregulation of neuronal differentiation by plasma lithography patterned elastomeric substrates.

Cells sense and interpret mechanical cues, including cell-cell and cell-substrate interactions, in the microenvironment to collectively regulate various physiological functions. Understanding the influences of these mechanical factors on cell behavior is critical for fundamental cell biology and for the development of novel strategies in regenerative medicine. Here, we demonstrate plasma lithography patterning on elastomeric substrates for elucidating the influences of mechanical cues on neuronal differentiation and neuritogenesis. The neuroblastoma cells form neuronal spheres on plasma-treated regions, which geometrically confine the cells over two weeks. The elastic modulus of the elastomer is controlled simultaneously by the crosslinker concentration. The cell-substrate mechanical interactions are also investigated by controlling the size of neuronal spheres with different cell seeding densities. These physical cues are shown to modulate with the formation of focal adhesions, neurite outgrowth, and the morphology of neuroblastoma. By systematic adjustment of these cues, along with computational biomechanical analysis, we demonstrate the interrelated mechanoregulatory effects of substrate elasticity and cell size. Taken together, our results reveal that the neuronal differentiation and neuritogenesis of neuroblastoma cells are collectively regulated via the cell-substrate mechanical interactions.

Geometric control of capillary architecture via cell-matrix mechanical interactions.

Capillary morphogenesis is a multistage, multicellular activity that plays a pivotal role in various developmental and pathological situations. In-depth understanding of the regulatory mechanism along with the capability of controlling the morphogenic process will have direct implications on tissue engineering and therapeutic angiogenesis. Extensive research has been devoted to elucidate the biochemical factors that regulate capillary morphogenesis. The roles of geometric confinement and cell-matrix mechanical interactions on the capillary architecture, nevertheless, remain largely unknown. Here, we show geometric control of endothelial network topology by creating physical confinements with microfabricated fences and wells. Decreasing the thickness of the matrix also results in comparable modulation of the network architecture, supporting the boundary effect is mediated mechanically. The regulatory role of cell-matrix mechanical interaction on the network topology is further supported by alternating the matrix stiffness by a cell-inert PEG-dextran hydrogel. Furthermore, reducing the cell traction force with a Rho-associated protein kinase inhibitor diminishes the boundary effect. Computational biomechanical analysis delineates the relationship between geometric confinement and cell-matrix mechanical interaction. Collectively, these results reveal a mechanoregulation scheme of endothelial cells to regulate the capillary network architecture via cell-matrix mechanical interactions.

The effects of replacing collagen fibers with carbon nanotubes on the rate of bone remodeling process.

Application of carbon nanotubes (CNTs) instead of collagen fibers (CFs) in bone tissue is one of the proposed avenues for the enhancement of bone's mechanical properties. The mechanical behavior improvement caused by such a replacement is somehow guaranteed because of the superior mechanical properties of CNTs compared to those of CFs. But on the other side, bone is a very active and dynamic tissue, which is maintained through a lifelong coupled process of resorption and formation in order to reach an optimal configuration. Hence, the well accepted fact of the bone remodeling dependency on mechanical stimuli besides the differences in mechanical behavior of CNTs and CFs under loading can encourage one to hypothesize that such a replacement would cause an imbalance in the normal rate of bone remodeling process. Results of our finite element analysis indicate that the application of CNTs instead of CFs can cause a significant reduction in strain energy density, assumed here as the mechanical stimulus to initiate the bone remodeling process. Our results also show that this replacement may change the strain energy distribution within the bone. Based on a semi-mechanistic bone remodeling theory, it is speculated that this alteration in strain energy distribution in artificial bone can destabilize normal bone remodeling process, and therefore it is likely to cause some abnormalities in bone's mechanical and biological functions.

A cross-linking model for estimating Young's modulus of artificial bone tissue grown on carbon nanotube scaffold.

Carbon nanotubes (CNTs) provide a suitable environment for growth and proliferation of bone cells. The elastic properties exhibited by CNTs can enhance mechanical characteristics of bone mineral phase, hydroxyapatite (HAp), precipitated on such a scaffold. In this article, a simplified model for estimating the axial Young's modulus of a representative volume element (RVE) of CNT-HAp composite is presented. The model is based on the idea of HAp formation on functionalized sites on CNTs as cross-links between HAp matrix and CNT. Modeling results show that the reinforcement role contributed by CNT in the RVE causes a significant increase in the Young's modulus of the composite material which is a direct consequence of transferring stresses from the HAp matrix to the CNT through the cross-links. Similar conclusions may be suggested regarding the improvement of overall mechanical properties of the material. The prediction made by the model lies reasonably well within the limits proposed by conventional Rule-of-Mixtures, and sliding below Voigt's model. The Young's modulus predicted by the model lies adjacent to the Hashin-Shtrikman upper bound as a function of the RVE length (or equivalently CNT aspect ratio). The model simulation indicates that an increase in the CNT aspect ratio and/or number of cross-links in the RVE, results in the prediction to move closer to the estimation made by Voigt as the assumption of perfect bonding between composite phases is approached.