A Protein-Adsorbent Hydrogel with Tunable Stiffness for Tissue Culture Demonstrates Matrix-Dependent Stiffness Responses.

Although tissue culture plastic has been widely employed for cell culture, the rigidity of plastic is not physiologic. Softer hydrogels used to culture cells have not been widely adopted in part because coupling chemistries are required to covalently capture extracellular matrix (ECM) proteins and support cell adhesion. To create an in vitro system with tunable stiffnesses that readily adsorbs ECM proteins for cell culture, we present a novel hydrophobic hydrogel system via chemically converting hydroxyl residues on the dextran backbone to methacrylate groups, thereby transforming non-protein adhesive, hydrophilic dextran to highly protein adsorbent substrates. Increasing methacrylate functionality increases the hydrophobicity in the resulting hydrogels and enhances ECM protein adsorption without additional chemical reactions. These hydrophobic hydrogels permit facile and tunable modulation of substrate stiffness independent of hydrophobicity or ECM coatings. Using this approach, we show that substrate stiffness and ECM adsorption work together to affect cell morphology and proliferation, but the strengths of these effects vary in different cell types. Furthermore, we reveal that stiffness mediated differentiation of dermal fibroblasts into myofibroblasts is modulated by the substrate ECM. Our material system demonstrates remarkable simplicity and flexibility to tune ECM coatings and substrate stiffness and study their effects on cell function.

A 3D biomimetic model of lymphatics reveals cell-cell junction tightening and lymphedema via a cytokine-induced ROCK2/JAM-A complex.

Impaired lymphatic drainage and lymphedema are major morbidities whose mechanisms have remained obscure. To study lymphatic drainage and its impairment, we engineered a microfluidic culture model of lymphatic vessels draining interstitial fluid. This lymphatic drainage-on-chip revealed that inflammatory cytokines that are known to disrupt blood vessel junctions instead tightened lymphatic cell-cell junctions and impeded lymphatic drainage. This opposing response was further demonstrated when inhibition of rho-associated protein kinase (ROCK) was found to normalize fluid drainage under cytokine challenge by simultaneously loosening lymphatic junctions and tightening blood vessel junctions. Studies also revealed a previously undescribed shift in ROCK isoforms in lymphatic endothelial cells, wherein a ROCK2/junctional adhesion molecule-A (JAM-A) complex emerges that is responsible for the cytokine-induced lymphatic junction zippering. To validate these in vitro findings, we further demonstrated in a genetic mouse model that lymphatic-specific knockout of ROCK2 reversed lymphedema in vivo. These studies provide a unique platform to generate interstitial fluid pressure and measure the drainage of interstitial fluid into lymphatics and reveal a previously unappreciated ROCK2-mediated mechanism in regulating lymphatic drainage.

Fibroblast clearance of damaged tissue following laser ablation in engineered microtissues.

Although the mechanisms underlying wound healing are largely preserved across wound types, the method of injury can affect the healing process. For example, burn wounds are more likely to undergo hypertrophic scarring than are lacerations, perhaps due to the increased underlying damage that needs to be cleared. This tissue clearance is thought to be mainly managed by immune cells, but it is unclear if fibroblasts contribute to this process. Herein, we utilize a 3D in vitro model of stromal wound healing to investigate the differences between two modes of injury: laceration and laser ablation. We demonstrate that laser ablation creates a ring of damaged tissue around the wound that is cleared by fibroblasts prior to wound closure. This process is dependent on ROCK and dynamin activity, suggesting a phagocytic or endocytic process. Transmission electron microscopy of fibroblasts that have entered the wound area reveals large intracellular vacuoles containing fibrillar extracellular matrix. These results demonstrate a new model to study matrix clearance by fibroblasts in a 3D soft tissue. Because aberrant wound healing is thought to be caused by an imbalance between matrix degradation and production, this model, which captures both aspects, will be a valuable addition to the study of wound healing.

Mechanical response of cardiac microtissues to acute localized injury.

After a myocardial infarction (MI), the heart undergoes changes including local remodeling that can lead to regional abnormalities in mechanical and electrical properties, ultimately increasing the risk of arrhythmias and heart failure. Although these responses have been successfully recapitulated in animal models of MI, local changes in tissue and cell-level mechanics caused by MI remain difficult to study in vivo. Here, we developed an in vitro cardiac microtissue (CMT) injury system that through acute focal injury recapitulates aspects of the regional responses seen following an MI. With a pulsed laser, cell death was induced in the center of the microtissue causing a loss of calcium signaling and a complete loss of contractile function in the injured region and resulting in a 39% reduction in the CMT's overall force production. After 7 days, the injured area remained void of cardiomyocytes (CMs) and showed increased expression of vimentin and fibronectin, two markers for fibrotic remodeling. Interestingly, although the injured region showed minimal recovery, calcium amplitudes in uninjured regions returned to levels comparable with control. Furthermore, overall force production returned to preinjury levels despite the lack of contractile function in the injured region. Instead, uninjured regions exhibited elevated contractile function, compensating for the loss of function in the injured region, drawing parallels to changes in tissue-level mechanics seen in vivo. Overall, this work presents a new in vitro model to study cardiac tissue remodeling and electromechanical changes after injury.NEW & NOTEWORTHY We report an in vitro cardiac injury model that uses a high-powered laser to induce regional cell death and a focal fibrotic response within a human-engineered cardiac microtissue. The model captures the effects of acute injury on tissue response, remodeling, and electromechanical recovery in both the damaged region and surrounding healthy tissue, modeling similar changes to contractile function observed in vivo following myocardial infarction.

Engineering a living cardiac pump on a chip using high-precision fabrication.

Biomimetic on-chip tissue models serve as a powerful tool for studying human physiology and developing therapeutics; however, their modeling power is hindered by our inability to develop highly ordered functional structures in small length scales. Here, we demonstrate how high-precision fabrication can enable scaled-down modeling of organ-level cardiac mechanical function. We use two-photon direct laser writing (TPDLW) to fabricate a nanoscale-resolution metamaterial scaffold with fine-tuned mechanical properties to support the formation and cyclic contraction of a miniaturized, induced pluripotent stem cell-derived ventricular chamber. Furthermore, we fabricate microfluidic valves with extreme sensitivity to rectify the flow generated by the ventricular chamber. The integrated microfluidic system recapitulates the ventricular fluidic function and exhibits a complete pressure-volume loop with isovolumetric phases. Together, our results demonstrate a previously unexplored application of high-precision fabrication that can be generalized to expand the accessible spectrum of organ-on-a-chip models toward structurally and biomechanically sophisticated tissue systems.

Notch1 and Notch3 coordinate for pericyte-induced stabilization of vasculature.

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires the cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for the stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases the accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.

Plakophilin-2 truncating variants impair cardiac contractility by disrupting sarcomere stability and organization.

Progressive loss of cardiac systolic function in arrhythmogenic cardiomyopathy (ACM) has recently gained attention as an important clinical consideration in managing the disease. However, the mechanisms leading to reduction in cardiac contractility are poorly defined. Here, we use CRISPR gene editing to generate human induced pluripotent stem cells (iPSCs) that harbor plakophilin-2 truncating variants (PKP2tv), the most prevalent ACM-linked mutations. The PKP2tv iPSC­derived cardiomyocytes are shown to have aberrant action potentials and reduced systolic function in cardiac microtissues, recapitulating both the electrical and mechanical pathologies reported in ACM. By combining cell micropatterning with traction force microscopy and live imaging, we found that PKP2tvs impair cardiac tissue contractility by destabilizing cell-cell junctions and in turn disrupting sarcomere stability and organization. These findings highlight the interplay between cell-cell adhesions and sarcomeres required for stabilizing cardiomyocyte structure and function and suggest fundamental pathogenic mechanisms that may be shared among different types of cardiomyopathies.

Extracellular Matrix Alignment Directs Provisional Matrix Assembly and Three Dimensional Fibrous Tissue Closure.

Gap closure is a dynamic process in wound healing, in which a wound contracts and a provisional matrix is laid down, to restore structural integrity to injured tissues. The efficiency of wound closure has been found to depend on the shape of a wound, and this shape dependence has been echoed in various in vitro studies. While wound shape itself appears to contribute to this effect, it remains unclear whether the alignment of the surrounding extracellular matrix (ECM) may also contribute. In this study, we investigate the role both wound curvature and ECM alignment have on gap closure in a 3D culture model of fibrous tissue. Using microfabricated flexible micropillars positioned in rectangular and octagonal arrangements, seeded 3T3 fibroblasts embedded in a collagen matrix formed microtissues with different ECM alignments. Wounding these microtissues with a microsurgical knife resulted in wounds with different shapes and curvatures that closed at different rates. Observing different regions around the wounds, we noted local wound curvature did not impact the rate of production of provisional fibronectin matrix assembled by the fibroblasts. Instead, the rate of provisional matrix assembly was lowest emerging from regions of high fibronectin alignment and highest in the areas of low matrix alignment. Our data suggest that the underlying ECM structure affects the shape of the wound as well as the ability of fibroblasts to build provisional matrix, an important step in the process of tissue closure and restoration of tissue architecture. The study highlights an important interplay between ECM alignment, wound shape, and tissue healing that has not been previously recognized and may inform approaches to engineer tissues. Impact statement Current models of tissue growth have identified a role for curvature in driving provisional matrix assembly. However, most tissue repair occurs in fibrous tissues with different levels of extracellular matrix (ECM) alignment. Here, we show how this underlying ECM alignment may affect the ability of fibroblasts to build new provisional matrix, with implications for in vivo wound healing and providing insight for engineering of new tissues.

Force-FAK signaling coupling at individual focal adhesions coordinates mechanosensing and microtissue repair.

How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Using cells adhering to deformable micropillar arrays, we demonstrate that traction force and FAK localization as well as traction force and Y397-FAK phosphorylation are linearly coupled at individual FAs on stiff, but not soft, substrates. Similarly, FAK phosphorylation increases linearly with external forces applied to FAs using magnetic beads. This mechanosignaling coupling requires actomyosin contractility, talin-FAK binding, and full-length vinculin that binds talin and actin. Using an in vitro 3D biomimetic wound healing model, we show that force-FAK signaling coupling coordinates cell migration and tissue-scale forces to promote microtissue repair. A simple kinetic binding model of talin-FAK interactions under force can recapitulate the experimental observations. This study provides insights on how talin and vinculin convert forces into FAK signaling events regulating cell migration and tissue repair.

Reconstituting the dynamics of endothelial cells and fibroblasts in wound closure.

The formation of healthy vascularized granulation tissue is essential for rapid wound closure and the prevention of chronic wounds in humans, yet how endothelial cells and fibroblasts coordinate during this process has been difficult to study. Here, we have developed an in vitro system that reveals how human endothelial and stromal cells in a 3D matrix respond during wound healing and granulation tissue formation. By creating incisions in engineered cultures composed of human umbilical vein endothelial cells and human lung fibroblasts embedded within a 3D matrix, we observed that these tissues are able to close the wound within approximately 4 days. Live tracking of cells during wound closure revealed that the process is mediated primarily by fibroblasts. The fibroblasts migrate circumferentially around the wound edge during early phases of healing, while contracting the wound. The fibroblast-derived matrix is, then, deposited into the void, facilitating fibroblast migration toward the wound center and filling of the void. Interestingly, the endothelial cells remain at the periphery of the wound rather than actively sprouting into the healing region to restore the vascular network. This study captures the dynamics of endothelial and fibroblast-mediated closure of three-dimensional wounds, which results in the repopulation of the wound with the cell-derived extracellular matrix representative of early granulation tissue, thus presenting a model for future studies to investigate factors regulating vascularized granulation tissue formation.

Transient Support from Fibroblasts is Sufficient to Drive Functional Vascularization in Engineered Tissues.

Formation of capillary blood vasculature is a critical requirement for native as well as engineered organs and can be induced in vitro by co-culturing endothelial cells with fibroblasts. However, whether these fibroblasts are required only in the initial morphogenesis of endothelial cells or needed throughout is unknown, and the ability to remove these stromal cells after assembly could be useful for clinical translation. In this study, we introduce a technique termed CAMEO (Controlled Apoptosis in Multicellular Tissues for Engineered Organogenesis), whereby fibroblasts are selectively ablated on demand, and utilize it to probe the dispensability of fibroblasts in vascular morphogenesis. The presence of fibroblasts is shown to be necessary only during the first few days of endothelial cell morphogenesis, after which they can be ablated without significantly affecting the structural and functional features of the developed vasculature. Furthermore, we demonstrate the use of CAMEO to vascularize a construct containing primary human hepatocytes that improved tissue function. In conclusion, this study suggests that transient, initial support from fibroblasts is sufficient to drive vascular morphogenesis in engineered tissues, and this strategy of engineering-via-elimination may provide a new general approach for achieving desired functions and cell compositions in engineered organs.

A biomimetic pancreatic cancer on-chip reveals endothelial ablation via ALK7 signaling.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, lethal malignancy that invades adjacent vasculatures and spreads to distant sites before clinical detection. Although invasion into the peripancreatic vasculature is one of the hallmarks of PDAC, paradoxically, PDAC tumors also exhibit hypovascularity. How PDAC tumors become hypovascular is poorly understood. We describe an organotypic PDAC-on-a-chip culture model that emulates vascular invasion and tumor-blood vessel interactions to better understand PDAC-vascular interactions. The model features a 3D matrix containing juxtaposed PDAC and perfusable endothelial lumens. PDAC cells invaded through intervening matrix, into vessel lumen, and ablated the endothelial cells, leaving behind tumor-filled luminal structures. Endothelial ablation was also observed in in vivo PDAC models. We also identified the activin-ALK7 pathway as a mediator of endothelial ablation by PDAC. This tumor-on-a-chip model provides an important in vitro platform for investigating the process of PDAC-driven endothelial ablation and may provide a mechanism for tumor hypovascularity.

Surface and Bulk Stresses Drive Morphological Changes in Fibrous Microtissues.

Engineered fibrous tissues consisting of cells encapsulated within collagen gels are widely used three-dimensional in vitro models of morphogenesis and wound healing. Although cell-mediated matrix remodeling that occurs within these scaffolds has been extensively studied, less is known about the mesoscale physical principles governing the dynamics of tissue shape. Here, we show both experimentally and by using computer simulations how surface contraction through the development of surface stresses (analogous to surface tension in fluids) coordinates with bulk contraction to drive shape evolution in constrained three-dimensional microtissues. We used microelectromechanical systems technology to generate arrays of fibrous microtissues and robot-assisted microsurgery to perform local incisions and implantation. We introduce a technique based on phototoxic activation of a small molecule to selectively kill cells in a spatially controlled manner. The model simulations, which reproduced the experimentally observed shape changes after surgical and photochemical operations, indicate that fitting of only bulk and surface contractile moduli is sufficient for the prediction of the equilibrium shape of the microtissues. The computational and experimental methods we have developed provide a general framework to study and predict the morphogenic states of contractile fibrous tissues under external loading at multiple length scales.

Myosin IIA-mediated forces regulate multicellular integrity during vascular sprouting.

Angiogenic sprouting is a critical process involved in vascular network formation within tissues. During sprouting, tip cells and ensuing stalk cells migrate collectively into the extracellular matrix while preserving cell-cell junctions, forming patent structures that support blood flow. Although several signaling pathways have been identified as controlling sprouting, it remains unclear to what extent this process is mechanoregulated. To address this question, we investigated the role of cellular contractility in sprout morphogenesis, using a biomimetic model of angiogenesis. Three-dimensional maps of mechanical deformations generated by sprouts revealed that mainly leader cells, not stalk cells, exert contractile forces on the surrounding matrix. Surprisingly, inhibiting cellular contractility with blebbistatin did not affect the extent of cellular invasion but resulted in cell-cell dissociation primarily between tip and stalk cells. Closer examination of cell-cell junctions revealed that blebbistatin impaired adherens-junction organization, particularly between tip and stalk cells. Using CRISPR/Cas9-mediated gene editing, we further identified NMIIA as the major isoform responsible for regulating multicellularity and cell contractility during sprouting. Together, these studies reveal a critical role for NMIIA-mediated contractile forces in maintaining multicellularity during sprouting and highlight the central role of forces in regulating cell-cell adhesions during collective motility.

A BMP/activin A chimera is superior to native BMPs and induces bone repair in nonhuman primates when delivered in a composite matrix.

Bone morphogenetic protein (BMP)/carriers approved for orthopedic procedures achieve efficacy superior or equivalent to autograft bone. However, required supraphysiological BMP concentrations have been associated with potential local and systemic adverse events. Suboptimal BMP/receptor binding and rapid BMP release from approved carriers may contribute to these outcomes. To address these issues and improve efficacy, we engineered chimeras with increased receptor binding by substituting BMP-6 and activin A receptor binding domains into BMP-2 and optimized a carrier for chimera retention and tissue ingrowth. BV-265, a BMP-2/BMP-6/activin A chimera, demonstrated increased binding affinity to BMP receptors, including activin-like kinase-2 (ALK2) critical for bone formation in people. BV-265 increased BMP intracellular signaling, osteogenic activity, and expression of bone-related genes in murine and human cells to a greater extent than BMP-2 and was not inhibited by BMP antagonist noggin or gremlin. BV-265 induced larger ectopic bone nodules in rats compared to BMP-2 and was superior to BMP-2, BMP-2/6, and other chimeras in nonhuman primate bone repair models. A composite matrix (CM) containing calcium-deficient hydroxyapatite granules suspended in a macroporous, fenestrated, polymer mesh-reinforced recombinant human type I collagen matrix demonstrated improved BV-265 retention, minimal inflammation, and enhanced handling. BV-265/CM was efficacious in nonhuman primate bone repair models at concentrations ranging from 1/10 to 1/30 of the BMP-2/absorbable collagen sponge (ACS) concentration approved for clinical use. Initial toxicology studies were negative. These results support evaluations of BV-265/CM as an alternative to BMP-2/ACS in clinical trials for orthopedic conditions requiring augmented healing.

Studies of 3D directed cell migration enabled by direct laser writing of curved wave topography.

Cell migration, critical to numerous biological processes, can be guided by surface topography. Studying the effects of topography on cell migration is valuable for enhancing our understanding of directional cell migration and for functionally engineering cell behavior. However, fabrication limitations constrain topography studies to geometries that may not adequately mimic physiological environments. Direct Laser Writing (DLW) provides the necessary 3D flexibility and control to create well-defined waveforms with curvature and length scales that are similar to those found in physiological settings, such as the luminal walls of blood vessels that endothelial cells migrate along. We find that endothelial cells migrate fastest along square waves, intermediate along triangular waves, and slowest along sine waves and that directional cell migration on sine waves decreases as sinusoid wavelength increases. Interestingly, inhibition of Rac1 decreases directional migration on sine wave topographies but not on flat surfaces with micropatterned lines, suggesting that cells may utilize different molecular pathways to sense curved topographies. Our study demonstrates that DLW can be employed to investigate the effects and mechanisms of topography on cell migration by fabricating a wide array of physiologically-relevant surfaces with curvatures that are challenging to fabricate using conventional manufacturing techniques.

Effects of Geometry on the Mechanics and Alignment of Three-Dimensional Engineered Microtissues.

The structure and stiffness of the extracellular matrix (ECM) in living tissues play a significant role in facilitating cellular functions and maintaining tissue homeostasis. However, the wide variation and complexity in tissue composition across different tissue types make comparative study of the impact of matrix architecture and alignment on tissue mechanics difficult. Here we present a microtissue-based system capable of controlling the degree of ECM alignment in 3D self-assembled fibroblast-populated collagen matrix, anchored around multiple elastic micropillars. The pillars provide structural constraints, control matrix alignment, enable measurement of the microtissues' contractile forces, and provide the ability to apply tensile strain using magnetic particles. Utilizing finite element models (FEMs) to parametrize results of mechanical measurements, spatial variations in the microtissues' Young's moduli across different regions were shown to be correlated with the degree of ECM fiber alignment. The aligned regions were up to six times stiffer than the unaligned regions. The results were not affected by suppression of cellular contractile forces in matured microtissues. However, comparison to a distributed fiber anisotropic model shows that variations in fiber alignment alone cannot account for the variations in the observed moduli, indicating that fiber density and tissue geometry also play important roles in the microtissues' properties. These results suggest a complex interplay between mechanical boundary constraints, ECM alignment, density, and mechanics and offer an approach combining engineered microtissues and computational modeling to elucidate these relationships.

Force Generation via ß-Cardiac Myosin, Titin, and α-Actinin Drives Cardiac Sarcomere Assembly from Cell-Matrix Adhesions.

Truncating mutations in the sarcomere protein titin cause dilated cardiomyopathy due to sarcomere insufficiency. However, it remains mechanistically unclear how these mutations decrease sarcomere content in cardiomyocytes. Utilizing human induced pluripotent stem cell-derived cardiomyocytes, CRISPR/Cas9, and live microscopy, we characterize the fundamental mechanisms of human cardiac sarcomere formation. We observe that sarcomerogenesis initiates at protocostameres, sites of cell-extracellular matrix adhesion, where nucleation and centripetal assembly of α-actinin-2-containing fibers provide a template for the fusion of Z-disk precursors, Z bodies, and subsequent striation. We identify that ß-cardiac myosin-titin-protocostamere form an essential mechanical connection that transmits forces required to direct α-actinin-2 centripetal fiber assembly and sarcomere formation. Titin propagates diastolic traction stresses from ß-cardiac myosin, but not α-cardiac myosin or non-muscle myosin II, to protocostameres during sarcomerogenesis. Ablating protocostameres or decoupling titin from protocostameres abolishes sarcomere assembly. Together these results identify the mechanical and molecular components critical for human cardiac sarcomerogenesis.