Quantifying Spatial Patterns of Tissue Stiffness Within the Embryonic Mouse Kidney.

Biophysical factors, including changes in mechanical stiffness, have been shown to influence the morphogenesis of developing organs. There is a lack of experimental techniques, however, that can probe the mechanical properties of embryonic tissues-especially those which are not mechanically or optically accessible, such as the visceral organs of the developing mouse embryo. Here, using the embryonic kidney as a model system, we describe a method to use microindentation to quantify tissue-level regional differences in the mechanical properties of an embryonic organ. This technique is generalizable and can be used to quantify patterns of tissue stiffness within other developing organ systems. Going forward, these data will enable new experimental studies of the role of biophysical cues during organogenesis.

Treatment with both TGF-ß1 and PDGF-BB disrupts the stiffness-dependent myofibroblast differentiation of corneal keratocytes.

During corneal wound healing, stromal keratocytes transform into a repair phenotype that is driven by the release of cytokines, like transforming growth factor-beta 1 (TGF-ß1) and platelet-derived growth factor-BB (PDGF-BB). Previous work has shown that TGF-ß1 promotes the myofibroblast differentiation of corneal keratocytes in a manner that depends on PDGF signaling. In addition, changes in mechanical properties are known to regulate the TGF-ß1-mediated differentiation of cultured keratocytes. While PDGF signaling acts synergistically with TGF-ß1 during myofibroblast differentiation, how treatment with multiple growth factors affects stiffness-dependent differences in keratocyte behavior is unknown. Here, we treated primary corneal keratocytes with PDGF-BB and TGF-ß1 and cultured them on polyacrylamide (PA) substrata of different stiffnesses. In the presence of TGF-ß1 alone, the cells underwent stiffness-dependent myofibroblast differentiation. On stiff substrata, the cells developed robust stress fibers, exhibited high levels of ⍺-SMA staining, formed large focal adhesions (FAs), and exerted elevated contractile forces, whereas cells in a compliant microenvironment showed low levels of ⍺-SMA immunofluorescence, formed smaller focal adhesions, and exerted decreased contractile forces. When the cultured keratocytes were treated simultaneously with PDGF-BB however, increased levels of ⍺-SMA staining and stress fiber formation were observed on compliant substrata, even though the cells did not exhibit elevated contractility or focal adhesion size. Pharmacological inhibition of PDGF signaling disrupted the myofibroblast differentiation of cells cultured on substrata of all stiffnesses. These results indicate that treatment with PDGF-BB can decouple molecular markers of myofibroblast differentiation from the elevated contractile phenotype otherwise associated with these cells, suggesting that crosstalk in the mechanotransductive signaling pathways downstream of TGF-ß1 and PDGF-BB can regulate the stiffness-dependent differentiation of cultured keratocytes. Statement of Significance: In vitro experiments have shown that changes in ECM stiffness can regulate the differentiation of myofibroblasts. Typically, these assays involve the use of individual growth factors, but it is unclear how stiffness-dependent differences in cell behavior are affected by multiple cytokines. Here, we used primary corneal keratocytes to show that treatment with both TGF-ß1 and PDGF-BB disrupts the dependency of myofibroblast differentiation on substratum stiffness. In the presence of both growth factors, keratocytes on soft substrates exhibited elevated ⍺-SMA immunofluorescence without a corresponding increase in contractility or focal adhesion formation. This result suggests that molecular markers of myofibroblast differentiation can be dissociated from the elevated contractile behavior associated with the myofibroblast phenotype, suggesting potential crosstalk in mechanotransductive signaling pathways downstream of TGF-ß1 and PDGF-BB.

Corneal keratocytes, fibroblasts, and myofibroblasts exhibit distinct transcriptional profiles in vitro.

Purpose: After stromal injury to the cornea, the release of growth factors and pro-inflammatory cytokines promotes the activation of quiescent keratocytes into a migratory fibroblast and/or fibrotic myofibroblast phenotype. Persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. This study aims to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which these phenotypic changes occur. Methods: Primary rabbit corneal keratocytes were cultured in either defined serum-free media (SF), fetal bovine serum (FBS) containing media, or in the presence of TGF-ß1 to induce keratocyte, fibroblast, or myofibroblast phenotypes, respectively. Bulk RNA sequencing followed by bioinformatic analyses was performed to identify significant differentially expressed genes (DEGs) and enriched biological pathways for each phenotype. Results: Genes commonly associated with keratocytes, fibroblasts, or myofibroblasts showed high relative expression in SF, FBS, or TGF-ß1 culture conditions, respectively. Differential expression and functional analyses revealed novel DEGs for each cell type, as well as enriched pathways indicative of differences in proliferation, apoptosis, extracellular matrix (ECM) synthesis, cell-ECM interactions, cytokine signaling, and cell mechanics. Conclusions: Overall, these data demonstrate distinct transcriptional differences among cultured corneal keratocytes, fibroblasts, and myofibroblasts. We have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to mechanotransduction and ECM biology. Our findings have revealed novel molecular markers for each cell type, as well as possible targets for modulating cell behavior and promoting physiological corneal wound healing.

Fabrication of Micropatterns of Aligned Collagen Fibrils.

Many tissues in vivo contain aligned structures such as filaments, fibrils, and fibers, which expose cells to anisotropic structural and topographical cues that range from the nanometer to micrometer scales. Understanding how cell behavior is regulated by these cues during physiological and pathological processes (e.g., wound healing, cancer invasion) requires substrates that can expose cells to anisotropic cues over several length scales. In this study, we developed a novel method of fabricating micropatterns of aligned collagen fibrils of different geometry onto PDMS-coated glass coverslips that allowed us to investigate the roles of topography and confinement on corneal cell behavior. When corneal cells were cultured on micropatterns of aligned collagen fibrils in the absence of confinement, the degree of cell alignment increased from 40 ± 14 to 82 ± 5% as the size of the micropattern width decreased from 750 to 50 µm. Although the cell area (∼2500 µm2), cell length (∼160 µm), and projected nuclear area (∼175 µm2) were relatively constant on the different micropattern widths, cells displayed an increased aspect ratio as the width of the aligned collagen fibril micropatterns decreased. We also observed that the morphology of cells adhering to the surrounding uncoated PDMS was dependent upon both the size of the aligned collagen fibril micropattern and the distance from the micropatterns. When corneal cells were confined to the micropatterns of aligned collagen fibrils by a Pluronic coating to passivate the surrounding area, a similar trend in increasing cell alignment was observed (35 ± 10 to 89 ± 2%). However, the projected nuclear area decreased significantly (∼210 to 130 µm2) as the micropattern width decreased from 750 to 50 µm. The development of this method allows for the deposition of aligned collagen fibril micropatterns of different geometries on a transparent and elastic substrate and provides an excellent model system to investigate the role of anisotropic cues in cell behavior.

Effects of Topography and PDGF on the Response of Corneal Keratocytes to Fibronectin-Coated Surfaces.

During corneal wound healing, corneal keratocytes are exposed to both biophysical and soluble cues that cause them to transform from a quiescent state to a repair phenotype. How keratocytes integrate these multiple cues simultaneously is not well understood. To investigate this process, primary rabbit corneal keratocytes were cultured on substrates patterned with aligned collagen fibrils and coated with adsorbed fibronectin. After 2 or 5 days of culture, keratocytes were fixed and stained to assess changes in cell morphology and markers of myofibroblastic activation by fluorescence microscopy. Initially, adsorbed fibronectin had an activating effect on the keratocytes as evidenced by changes in cell shape, stress fiber formation, and expression of alpha-smooth muscle actin (α-SMA). The magnitude of these effects depended upon substrate topography (i.e., flat substrate vs aligned collagen fibrils) and decreased with culture time. When keratocytes were simultaneously exposed to adsorbed fibronectin and soluble platelet-derived growth factor-BB (PDGF-BB), the cells elongated and had reduced expression of stress fibers and α-SMA. In the presence of PDGF-BB, keratocytes plated on the aligned collagen fibrils elongated in the direction of the fibrils. These results provide new information on how keratocytes respond to multiple simultaneous cues and how the anisotropic topography of aligned collagen fibrils influences keratocyte behavior.

Quantifying endodermal strains during heart tube formation in the developing chicken embryo.

In the early avian embryo, the developing heart forms when bilateral fields of cardiac progenitor cells, which reside in the lateral plate mesoderm, move toward the embryonic midline, and fuse above the anterior intestinal portal (AIP) to form a straight, muscle-wrapped tube. During this process, the precardiac mesoderm remains in close contact with the underlying endoderm. Previous work has shown that the endoderm around the AIP actively contracts to pull the cardiac progenitors toward the midline. The morphogenetic deformations associated with this endodermal convergence, however, remain unclear, as do the signaling pathways that might regulate this process. Here, we fluorescently labeled populations of endodermal cells in early chicken embryos and tracked their motion during heart tube formation to compute time-varying strains along the anterior endoderm. We then determined how the computed endodermal strain distributions are affected by the pharmacological inhibition of either myosin II or fibroblast growth factor (FGF) signaling. Our data indicate that a mediolateral gradient in endodermal shortening is present around the AIP, as well as substantial convergence and extension movements both anterior and lateral to the AIP. These active endodermal deformations are disrupted if either actomyosin contractility or FGF signaling are inhibited pharmacologically. Taken together, these results demonstrate how active deformations along the anterior endoderm contribute to heart tube formation within the developing embryo.

Focal sources of FGF-10 promote the buckling morphogenesis of the embryonic airway epithelium.

During airway branching morphogenesis, focal regions of FGF-10 expression in the pulmonary mesenchyme are thought to provide a local guidance cue, which promotes chemotactically the directional outgrowth of the airway epithelium. Here, however, we show that an ectopic source of FGF-10 induces epithelial buckling morphogenesis and the formation of multiple new supernumerary buds. FGF-10-induced budding can be modulated by altered epithelial tension and luminal fluid pressure. Increased tension suppresses the formation of ectopic branches, while a collapse of the embryonic airway promotes more expansive buckling and additional FGF-10-induced supernumerary buds. Our results indicate that a focal source of FGF-10 can promote epithelial buckling and suggest that the overall branching pattern cannot be explained entirely by the templated expression of FGF-10. Both FGF-10-mediated cell behaviors and exogenous mechanical forces must be integrated to properly shape the bronchial tree.

Signaling Downstream of Focal Adhesions Regulates Stiffness-Dependent Differences in the TGF-ß1-Mediated Myofibroblast Differentiation of Corneal Keratocytes.

Following injury and refractive surgery, corneal wound healing can initiate a protracted fibrotic response that interferes with ocular function. This fibrosis is related, in part, to the myofibroblast differentiation of corneal keratocytes in response to transforming growth factor beta 1 (TGF-ß1). Previous studies have shown that changes in the mechanical properties of the extracellular matrix (ECM) can regulate this process, but the mechanotransductive pathways that govern stiffness-dependent changes in keratocyte differentiation remain unclear. Here, we used a polyacrylamide (PA) gel system to investigate how mechanosensing via focal adhesions (FAs) regulates the stiffness-dependent myofibroblast differentiation of primary corneal keratocytes treated with TGF-ß1. Soft (1 kPa) and stiff (10 kPa) PA substrata were fabricated on glass coverslips, plated with corneal keratocytes, and cultured in defined serum free media with or without exogenous TGF-ß1. In some experiments, an inhibitor of focal adhesion kinase (FAK) activation was also added to the media. Cells were fixed and stained for F-actin, as well as markers for myofibroblast differentiation (α-SMA), actomyosin contractility phosphorylated myosin light chain (pMLC), focal adhesions (vinculin), or Smad activity (pSmad3). We also used traction force microscopy (TFM) to quantify cellular traction stresses. Treatment with TGF-ß1 elicited stiffness-dependent differences in the number, size, and subcellular distribution of FAs, but not in the nuclear localization of pSmad3. On stiff substrata, cells exhibited large FAs distributed throughout the entire cell body, while on soft gels, the FAs were smaller, fewer in number, and localized primarily to the distal tips of thin cellular extensions. Larger and increased numbers of FAs correlated with elevated traction stresses, increased levels of α-SMA immunofluorescence, and more prominent and broadly distributed pMLC staining. Inhibition of FAK disrupted stiffness-dependent differences in keratocyte contractility, FA patterning, and myofibroblast differentiation in the presence of TGF-ß1. Taken together, these data suggest that signaling downstream of FAs has important implications for the stiffness-dependent myofibroblast differentiation of corneal keratocytes.

Toward Measuring the Mechanical Stresses Exerted by Branching Embryonic Airway Epithelial Explants in 3D Matrices of Matrigel.

Numerous organs in the bodies of animals, including the lung, kidney, and mammary gland, contain ramified networks of epithelial tubes. These structures arise during development via a process known as branching morphogenesis. Previous studies have shown that mechanical forces directly impact this process, but the patterns of mechanical stress exerted by branching embryonic epithelia are not well understood. This is, in part, owing to a lack of experimental tools. Traditional traction force microscopy assays rely on the use of compliant hydrogels with well-defined mechanical properties. Isolated embryonic epithelial explants, however, have only been shown to branch in three-dimensional matrices of reconstituted basement membrane protein, or Matrigel, a biomaterial with poorly characterized mechanical behavior, especially in the regime of large deformations. Here, to compute the traction stresses generated by branching epithelial explants, we quantified the finite-deformation constitutive behavior of gels of reconstituted basement membrane protein subjected to multi-axial mechanical loads. We then modified the mesenchyme-free assay for the ex vivo culture of isolated embryonic airway epithelial explants by suspending fluorescent microspheres within the surrounding gel and tracking their motion during culture. Surprisingly, the tracked bead motion was non-zero in regions of the gel far away from the explants, suggestive of passive swelling deformations within the matrix. To compute accurate traction stresses, these swelling deformations must be decomposed from those generated by the branching explants. We thus tracked the motion of beads suspended within cell-free matrices and quantified spatiotemporal patterns of gel swelling. Taken together, these passive swelling data can be combined with the measured mechanical properties of the gel to compute the traction forces exerted by intact embryonic epithelial explants.

ECM stiffness modulates the proliferation but not the motility of primary corneal keratocytes in response to PDGF-BB.

During corneal wound healing, keratocytes present within the corneal stroma become activated into a repair phenotype upon the release of growth factors, such as transforming growth factor-beta 1 (TGF-ß1) and platelet-derived growth factor-BB (PDGF-BB). The process of injury and repair can lead to changes in the mechanical properties of the tissue, and previous work has shown that the TGF-ß1-mediated myofibroblast differentiation of corneal keratocytes depends on substratum stiffness. It is still unclear, however, if changes in stiffness can modulate keratocyte behavior in response to other growth factors, such as PDGF-BB. Here, we used a polyacrylamide (PA) gel system to determine whether changes in stiffness influence the proliferation and motility of primary corneal keratocytes treated with PDGF-BB. In the presence of PDGF-BB, cells on stiffer substrata exhibited a more elongated morphology and had higher rates of proliferation than cells in a more compliant microenvironment. Using a freeze-injury to assay cell motility, however, we did not observe any stiffness-dependent differences in the migration of keratocytes treated with PDGF-BB. Taken together, these data highlight the importance of biophysical cues during corneal wound healing and suggest that keratocytes respond differently to changes in ECM stiffness in the presence of different growth factors.

Insertion mechanics of amorphous SiC ultra-micro scale neural probes.

Objective. Trauma induced by the insertion of microelectrodes into cortical neural tissue is a significant problem. Further, micromotion and mechanical mismatch between microelectrode probes and neural tissue is implicated in an adverse foreign body response (FBR). Hence, intracortical ultra-microelectrode probes have been proposed as alternatives that minimize this FBR. However, significant challenges in implanting these flexible probes remain. We investigated the insertion mechanics of amorphous silicon carbide (a-SiC) probes with a view to defining probe geometries that can be inserted into cortex without buckling.Approach. We determined the critical buckling force of a-SiC probes as a function of probe geometry and then characterized the buckling behavior of these probes by measuring force-displacement responses during insertion into agarose gel and rat cortex.Main results.Insertion forces for a range of probe geometries were determined and compared with critical buckling forces to establish geometries that should avoid buckling during implantation into brain. The studies show that slower insertion speeds reduce the maximum insertion force for single-shank probes but increase cortical dimpling during insertion of multi-shank probes.Significance.Our results provide a guide for selecting probe geometries and insertion speeds that allow unaided implantation of probes into rat cortex. The design approach is applicable to other animal models where insertion of intracortical probes to a depth of 2 mm is required.

Three-dimensional visualization and improved quantification with super-resolution ultrasound imaging - validation framework for analysis of microvascular morphology using a chicken embryo model.

The purpose of this study was to improve the morphological analysis of microvascular networks depicted in three-dimensional (3D) super-resolution ultrasound (SR-US) images. This was supported by qualitative and quantitative validation by comparison to matched brightfield microscopy and traditional B-mode ultrasound (US) images. Contrast-enhanced US (CEUS) images were collected using a preclinical US scanner (Vevo 3100, FUJIFILM VisualSonics Inc.) equipped with an MX250 linear array transducer. CEUS imaging was performed after administration of a microbubble (MB) contrast agent into the vitelline network of a developing chicken embryo. Volume data was collected by mechanically scanning the US transducer throughout a tissue volume-of-interest in 90µm step increments. CEUS images were collected at each increment and stored as in-phase/quadrature data (2000 frames at 152 frames per sec). SR-US images were created for each cross-sectional plane using established data processing methods. All SR-US images were then used to reconstruct a final 3D volume for vessel diameter (VD) quantification and for surface rendering. VD quantification from the 3D SR-US data exhibited an average error of 6.1% ± 6.0% when compared with matched brightfield microscopy images, whereas measurements from B-mode US images had an average error of 77.1% ± 68.9%. Volume and surface renderings in 3D space enabled qualitative validation and improved visualization of small vessels below the axial resolution of the US system. Overall, 3D SR-US image reconstructions depicted the microvascular network of the developing chicken embryos. Improved visualization of isolated vessels and quantification of microvascular morphology from SR-US images achieved a considerably greater accuracy compared to B-mode US measurements.

ECM Stiffness Controls the Activation and Contractility of Corneal Keratocytes in Response to TGF-ß1.

After surgery or traumatic injury, corneal wound healing can cause a scarring response that stiffens the tissue and impairs ocular function. This fibrosis is caused in part by the activation of corneal keratocytes from a native mechanically quiescent state to an activated myofibroblastic state. This transformation is tied to signaling downstream of transforming growth factor-ß1 (TGF-ß1). Here, to better understand how biochemical and biophysical cues interact to regulate keratocyte activation and contractility, we cultured primary rabbit corneal keratocytes on flexible substrata of varying stiffness in the presence (or absence) of TGF-ß1. Time-lapse fluorescence microscopy was used to assess changes in keratocyte morphology, as well as to quantify the dynamic traction stresses exerted by cells under different experimental conditions. In other experiments, keratocytes were fixed after 5 days of culture and stained for markers of both contractility and myofibroblastic activation. Treatment with TGF-ß1 elicited distinct phenotypes on substrata of different stiffnesses. Cells on soft (1 kPa) gels formed fewer stress fibers and retained a more dendritic morphology, indicative of a quiescent keratocyte phenotype. Keratocytes cultured on stiff (10 kPa) gels or collagen-coated glass coverslips, however, had broad morphologies, formed abundant stress fibers, exhibited greater levels of α-smooth muscle actin (α-SMA) expression, and exerted larger traction forces. Confocal images of phospho-myosin light chain (pMLC) immunofluorescence, moreover, revealed stiffness-dependent differences in the subcellular distribution of actomyosin contractility, with pMLC localized at the tips of thin cellular processes in mechanically quiescent cells. Importantly, keratocytes cultured in the absence of TGF-ß1 showed no stiffness-dependent differences in α-SMA immunofluorescence, suggesting that a stiff microenvironment alone is insufficient to induce myofibroblastic activation. Taken together, these data suggest that changes in ECM stiffness can modulate the morphology, cytoskeletal organization, and subcellular pattern of force generation in corneal keratocytes treated with TGF-ß1.

Keratocyte mechanobiology.

In vivo, corneal keratocytes reside within a complex 3D extracellular matrix (ECM) consisting of highly aligned collagen lamellae, growth factors, and other extracellular matrix components, and are subjected to various mechanical stimuli during developmental morphogenesis, fluctuations in intraocular pressure, and wound healing. The process by which keratocytes convert changes in mechanical stimuli (e.g. local topography, applied force, ECM stiffness) into biochemical signaling is known as mechanotransduction. Activation of the various mechanotransductive pathways can produce changes in cell migration, proliferation, and differentiation. Here we review how corneal keratocytes respond to and integrate different biochemical and biophysical factors. We first highlight how growth factors and other cytokines regulate the activity of Rho GTPases, cytoskeletal remodeling, and ultimately the mechanical phenotype of keratocytes. We then discuss how changes in the mechanical properties of the ECM have been shown to regulate keratocyte behavior in sophisticated 2D and 3D experimental models of the corneal microenvironment. Finally, we discuss how ECM topography and protein composition can modulate cell phenotypes, and review the different methods of fabricating in vitro mimics of corneal ECM topography, novel approaches for examining topographical effects in vivo, and the impact of different ECM glycoproteins and proteoglycans on keratocyte behavior.

Anisotropic, porous hydrogels templated by lyotropic chromonic liquid crystals.

Approaches to control the microstructure of hydrogels enable the control of cell-material interactions and the design of stimuli-responsive materials. We report a versatile approach for the synthesis of anisotropic polyacrylamide hydrogels using lyotropic chromonic liquid crystal (LCLC) templating. The orientational order of LCLCs in a mold can be patterned by controlling surface anchoring conditions, which in turn patterns the polymer network. The resulting hydrogels have tunable pore size and mechanical anisotropy. For example, the elastic moduli measured parallel and perpendicular to the LCLC order are 124.9 ± 6.4 kPa and 17.4 ± 1.1 kPa for a single composition. The resulting anisotropic hydrogels also have 30% larger swelling normal to the LCLC orientation than along the LCLC orientation. By patterning the LCLC order, this anisotropic swelling can be used to create 3D hydrogel structures. These anisotropic gels can also be functionalized with extracellular matrix (ECM) proteins and used as compliant substrata for cell culture. As an illustrative example, we show that the patterned hydrogel microstructure can be used to direct the orientation of cultured human corneal fibroblasts. This strategy to make anisotropic hydrogels has potential for enabling patternable tissue scaffolds, soft robotics, or microfluidic devices.

Deployable, liquid crystal elastomer-based intracortical probes.

Intracortical microelectrode arrays (MEAs) are currently limited in their chronic functionality due partially to the foreign body response (FBR) that develops in regions immediately surrounding the implant (typically within 50-100 µm). Mechanically flexible, polymer-based substrates have recently been explored for MEAs as a way of minimizing the FBR caused by the chronic implantation. Nonetheless, the FBR degrades the ability of the device to record neural activity. We are motivated to develop approaches to deploy multiple recording sites away from the initial site of implantation into regions of tissue outside the FBR zone. Liquid Crystal Elastomers (LCEs) are responsive materials capable of programmable and reversible shape change. These hydrophobic materials are also non-cytotoxic and compatible with photolithography. As such, these responsive materials may be well suited to serve as substrates for smart, implantable electronics. This study explores the feasibility of LCE-based deployable intracortical MEAs. LCE intracortical probes are fabricated on a planar substrate and adopt a 3D shape after being released from the substrate. The LCE probes are then fixed in a planar configuration using polyethylene glycol (PEG). The PEG layer dissolves in physiological conditions, allowing the LCE probe to deploy post-implantation. Critically, we show that LCE intracortical probes will deploy within a brain-like agarose tissue phantom. We also show that deployment distance increases with MEA width. A finite element model was then developed to predict the deformed shape of the deployed probe when embedded in an elastic medium. Finally, LCE-based deployable intracortical MEAs were capable of maintaining electrochemical stability, recording extracellular signals from cortical neurons in vivo, and deploying recording sites greater than 100 µm from the insertion site in vivo. Taken together, these results suggest the feasibility of using LCEs to develop deployable intracortical MEAs. STATEMENT OF SIGNIFICANCE: Deployable MEAs are a recently developed class of neural interfaces that aim to shift the recording sites away from the region of insertion to minimize the negative effects of FBR on the recording performance of MEAs. In this study, we explore LCEs as a potential substrate for deployable MEAs. The novelty of this study lies in the systematic and programmable deployment offered by LCE-based intracortical MEAs. These results illustrate the feasibility and potential application of LCEs as a substrate for deployable intracortical MEAs.

Three-dimensional super-resolution ultrasound imaging of chicken embryos - A validation framework for analysis of microvascular morphology.

The purpose of this present study was to improve the quantification of microvascular networks depicted in three-dimensional (3D) super-resolution ultrasound (SR-US) images and compare results with matched brightfield microscopy and B-mode ultrasound (US) images. Standard contrast-enhanced US (CEUS) images were collected using a high-frequency US scanner (Vevo 3100, FUJIFILM VisualSonics Inc) equipped with an MX250 linear array transducer. Using a developing chicken embryo as our model system, US imaging was performed after administration of a custom microbubble (MB) contrast agent. Guided by stereo microscopy, MBs were introduced into a perfused blood vessel by microinjection with a glass capillary needle. Volume data was collected by mechanically scanning the US transducer throughout a tissue volume-of-interest (VOI) in 90 µm step increments. CEUS images were collected at each increment and stored as in-phase/quadrature (IQ) data (N = 2000 at 152 frames per sec). SR-US images were created for each cross-sectional plane using established data processing methods, and all were then used to form a final 3D volume for subsequent quantification of morphological features. Vessel diameter quantifications from 3D SR-US data exhibited an average error of 1.9% when compared with microscopy images, whereas measures from B-mode US images had an average error of 75.3%. Overall, 3D SR-US images clearly depicted the microvascular network of the developing chicken embryo and measurements of microvascular morphology achieved better accuracy compared to traditional B-mode US.

A high-throughput microfluidic method for fabricating aligned collagen fibrils to study Keratocyte behavior.

In vivo, keratocytes are surrounded by aligned type I collagen fibrils that are organized into lamellae. A growing body of literature suggests that the unique topography of the corneal stroma is an important regulator of keratocyte behavior. In this study we describe a microfluidic method to deposit aligned fibrils of type I collagen onto glass coverslips. This high-throughput method allowed for the simultaneous coating of up to eight substrates with aligned collagen fibrils. When these substrates were integrated into a PDMS microwell culture system they provided a platform for high-resolution imaging of keratocyte behavior. Through the use of wide-field fluorescence and differential interference contrast microscopy, we observed that the density of collagen fibrils deposited was dependent upon both the perfusion shear rate of collagen and the time of perfusion. In contrast, a similar degree of fibril alignment was observed over a range of shear rates. When primary normal rabbit keratocytes (NRK) were seeded on substrates with a high density of aligned collagen fibrils and cultured in the presence of platelet derived growth factor (PDGF) the keratocytes displayed an elongated cell body that was co-aligned with the underlying collagen fibrils. In contrast, when NRK were cultured on substrates with a low density of aligned collagen fibrils, the cells showed no preferential orientation. These results suggest that this simple and inexpensive method can provide a general platform to study how simultaneous exposure to topographical and soluble cues influence cell behavior.

A Meta-Analysis of Intracortical Device Stiffness and Its Correlation with Histological Outcomes.

Neural implants offer solutions for a variety of clinical issues. While commercially available devices can record neural signals for short time periods, they fail to do so chronically, partially due to the sustained tissue response around the device. Our objective was to assess the correlation between device stiffness, a function of both material modulus and cross-sectional area, and the severity of immune response. Meta-analysis data were derived from nine previously published studies which reported device material and geometric properties, as well as histological outcomes. Device bending stiffness was calculated by treating the device shank as a cantilevered beam. Immune response was quantified through analysis of immunohistological images from each study, specifically looking at fluorescent markers for neuronal nuclei and astrocytes, to assess neuronal dieback and gliosis. Results demonstrate that the severity of the immune response, within the first 50 µm of the device, is highly correlated with device stiffness, as opposed to device modulus or cross-sectional area independently. In general, commercially available devices are around two to three orders of magnitude higher in stiffness than devices which induced a minimal tissue response. These results have implications for future device designs aiming to decrease chronic tissue response and achieve increased long-term functionality.

An In Vitro Model for Assessing Corneal Keratocyte Spreading and Migration on Aligned Fibrillar Collagen.

BACKGROUND: Corneal stromal cells (keratocytes) are responsible for developing and maintaining normal corneal structure and transparency, and for repairing the tissue after injury. Corneal keratocytes reside between highly aligned collagen lamellae in vivo. In addition to growth factors and other soluble biochemical factors, feedback from the extracellular matrix (ECM) itself has been shown to modulate corneal keratocyte behavior. METHODS: In this study, we fabricate aligned collagen substrates using a microfluidics approach and assess their impact on corneal keratocyte morphology, cytoskeletal organization, and patterning after stimulation with platelet derived growth factor (PDGF) or transforming growth factor beta 1 (TGFß). We also use time-lapse imaging to visualize the dynamic interactions between cells and fibrillar collagen during wound repopulation following an in vitro freeze injury. RESULTS: Significant co-alignment between keratocytes and aligned collagen fibrils was detected, and the degree of cell/ECM co-alignment further increased in the presence of PDGF or TGFß. Freeze injury produced an area of cell death without disrupting the collagen. High magnification, time-lapse differential interference contrast (DIC) imaging allowed cell movement and subcellular interactions with the underlying collagen fibrils to be directly visualized. CONCLUSIONS: With continued development, this experimental model could be an important tool for accessing how the integration of multiple biophysical and biochemical signals regulate corneal keratocyte differentiation.