Publications by authors named "Jeroen Eyckmans"

33 Publications

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

Sci Adv 2022 Apr 22;8(16):eabm3791. Epub 2022 Apr 22.

Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.

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.
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http://dx.doi.org/10.1126/sciadv.abm3791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9032966PMC
April 2022

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

Am J Physiol Cell Physiol 2022 02 8;322(2):C185-C196. Epub 2021 Dec 8.

The Biological Design Center and Department of Biomedical Engineering, Boston University, Boston, Massachusetts.

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.
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http://dx.doi.org/10.1152/ajpcell.00320.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8791789PMC
February 2022

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

Sci Adv 2021 Oct 15;7(42):eabh3995. Epub 2021 Oct 15.

Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.

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 (tv), the most prevalent ACM-linked mutations. The tv iPSC–derived 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 tvs 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.
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http://dx.doi.org/10.1126/sciadv.abh3995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8519574PMC
October 2021

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

Tissue Eng Part A 2021 12 12;27(23-24):1447-1457. Epub 2021 May 12.

Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA.

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 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 wound healing and providing insight for engineering of new tissues.
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http://dx.doi.org/10.1089/ten.tea.2020.0332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8742281PMC
December 2021

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

Nat Commun 2021 04 21;12(1):2359. Epub 2021 Apr 21.

Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA.

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.
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http://dx.doi.org/10.1038/s41467-021-22602-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8060400PMC
April 2021

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

Adv Funct Mater 2020 Nov 25;30(48). Epub 2020 Jun 25.

Biological Design Center, Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.

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.
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http://dx.doi.org/10.1002/adfm.202003777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891457PMC
November 2020

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

APL Bioeng 2021 Mar 19;5(1):016102. Epub 2021 Jan 19.

The Biological Design Center and Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA.

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 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.
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http://dx.doi.org/10.1063/5.0028651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817247PMC
March 2021

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

Sci Adv 2019 08 28;5(8):eaav6789. Epub 2019 Aug 28.

Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.

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.
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http://dx.doi.org/10.1126/sciadv.aav6789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713506PMC
August 2019

Surface and Bulk Stresses Drive Morphological Changes in Fibrous Microtissues.

Biophys J 2019 09 31;117(5):975-986. Epub 2019 Jul 31.

Institute of Mechanical Engineering and Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bpj.2019.07.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731460PMC
September 2019

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

ACS Biomater Sci Eng 2019 Aug 20;5(8):3843-3855. Epub 2018 Dec 20.

Department of Physics and Astronomy, Johns Hopkins University, Baltimore, Maryland 21218, United States.

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.
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http://dx.doi.org/10.1021/acsbiomaterials.8b01183DOI Listing
August 2019

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

Mol Biol Cell 2019 07 18;30(16):1974-1984. Epub 2019 Jul 18.

Biological Design Center, Department of Biomedical Engineering, Boston University, Boston, MA 02215.

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.
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http://dx.doi.org/10.1091/mbc.E19-02-0076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727772PMC
July 2019

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

Sci Transl Med 2019 04;11(489)

Bioventus Surgical, Bioventus LLC, Boston, MA 02215, USA.

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 / to / 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.
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http://dx.doi.org/10.1126/scitranslmed.aar4953DOI Listing
April 2019

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

Biofabrication 2019 02 25;11(2):021001. Epub 2019 Feb 25.

Department of Biomedical Engineering, Boston University, Boston, MA 02215, United States of America.

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.
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http://dx.doi.org/10.1088/1758-5090/ab047fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8117727PMC
February 2019

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

Dev Cell 2018 01 8;44(1):87-96.e5. Epub 2018 Jan 8.

Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA; The Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA 02115, USA. Electronic address:

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 β-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 β-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.
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http://dx.doi.org/10.1016/j.devcel.2017.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6421364PMC
January 2018

Fabrication and Mechanical Properties Measurements of 3D Microtissues for the Study of Cell-Matrix Interactions.

Methods Mol Biol 2018 ;1722:303-328

Department of Physics and Astronomy, Johns Hopkins University, Baltimore, MD, USA.

Cell interactions with the extracellular matrix (ECM) are critical to cell and tissue functions involving adhesion, communication, and differentiation. Three-dimensional (3D) in vitro culture systems are an important approach to mimic in vivo cell-matrix interactions for mechanobiology studies and tissue engineering applications. This chapter describes the use of engineered microtissues as 3D constructs in combination with a magnetic tissue gauge (μTUG) system to analyze tissue mechanical properties. The μTUG system is composed of poly(dimethylsiloxane) (PDMS) microwells with vertical pillars in the wells. Self-assembled microtissues containing cells and ECM gel can form between the pillars, and generate mechanical forces that deform the pillars, which provides a readout of those forces. Herein, detailed procedures for microfabrication of the PDMS μTUG system, seeding and growth of cells with ECM gels in the microwells, and measurements of the mechanical properties of the resulting microtissues via magnetic actuation of magnetic sphere-tagged μTUGs are described.
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http://dx.doi.org/10.1007/978-1-4939-7553-2_18DOI Listing
August 2018

Designer biomaterials for mechanobiology.

Nat Mater 2017 11;16(12):1164-1168

Biological Design Center and in the Department of Biomedical Engineering, Boston University, Boston, Massachusetts, 02215, USA, and at the Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, 02115, USA.

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http://dx.doi.org/10.1038/nmat5049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001850PMC
November 2017

A non-canonical Notch complex regulates adherens junctions and vascular barrier function.

Nature 2017 12 13;552(7684):258-262. Epub 2017 Nov 13.

The Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA.

The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and haemostasis. Haemodynamic shear stress plays a critical role in maintaining endothelial barrier function, but how this occurs remains unknown. Here we use an engineered organotypic model of perfused microvessels to show that activation of the transmembrane receptor NOTCH1 directly regulates vascular barrier function through a non-canonical, transcription-independent signalling mechanism that drives assembly of adherens junctions, and confirm these findings in mouse models. Shear stress triggers DLL4-dependent proteolytic activation of NOTCH1 to expose the transmembrane domain of NOTCH1. This domain mediates establishment of the endothelial barrier; expression of the transmembrane domain of NOTCH1 is sufficient to rescue defects in barrier function induced by knockout of NOTCH1. The transmembrane domain restores barrier function by catalysing the formation of a receptor complex in the plasma membrane consisting of vascular endothelial cadherin, the transmembrane protein tyrosine phosphatase LAR, and the RAC1 guanidine-exchange factor TRIO. This complex activates RAC1 to drive assembly of adherens junctions and establish barrier function. Canonical transcriptional signalling via Notch is highly conserved in metazoans and is required for many processes in vascular development, including arterial-venous differentiation, angiogenesis and remodelling. We establish the existence of a non-canonical cortical NOTCH1 signalling pathway that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodelling.
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http://dx.doi.org/10.1038/nature24998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730479PMC
December 2017

Making bone via nanoscale kicks.

Nat Biomed Eng 2017 Sep;1(9):689-690

Biological Design Center, Department of Biomedical Engineering, Boston University, 610 Commonwealth Avenue, Boston, MA, 02215, USA.

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http://dx.doi.org/10.1038/s41551-017-0133-6DOI Listing
September 2017

Three-dimensional biomimetic vascular model reveals a RhoA, Rac1, and -cadherin balance in mural cell-endothelial cell-regulated barrier function.

Proc Natl Acad Sci U S A 2017 08 1;114(33):8758-8763. Epub 2017 Aug 1.

Department of Biomedical Engineering, Boston University, Boston, MA 02215;

The integrity of the endothelial barrier between circulating blood and tissue is important for blood vessel function and, ultimately, for organ homeostasis. Here, we developed a vessel-on-a-chip with perfused endothelialized channels lined with human bone marrow stromal cells, which adopt a mural cell-like phenotype that recapitulates barrier function of the vasculature. In this model, barrier function is compromised upon exposure to inflammatory factors such as LPS, thrombin, and TNFα, as has been observed in vivo. Interestingly, we observed a rapid physical withdrawal of mural cells from the endothelium that was accompanied by an inhibition of endogenous Rac1 activity and increase in RhoA activity in the mural cells themselves upon inflammation. Using a system to chemically induce activity in exogenously expressed Rac1 or RhoA within minutes of stimulation, we demonstrated RhoA activation induced loss of mural cell coverage on the endothelium and reduced endothelial barrier function, and this effect was abrogated when Rac1 was simultaneously activated. We further showed that -cadherin expression in mural cells plays a key role in barrier function, as CRISPR-mediated knockout of -cadherin in the mural cells led to loss of barrier function, and overexpression of -cadherin in CHO cells promoted barrier function. In summary, this bicellular model demonstrates the continuous and rapid modulation of adhesive interactions between endothelial and mural cells and its impact on vascular barrier function and highlights an in vitro platform to study the biology of perivascular-endothelial interactions.
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http://dx.doi.org/10.1073/pnas.1618333114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5565405PMC
August 2017

3D culture models of tissues under tension.

J Cell Sci 2017 01 1;130(1):63-70. Epub 2016 Dec 1.

Department of Biomedical Engineering and the Biological Design Center, Boston University, Boston, MA 02215, USA

Cells dynamically assemble and organize into complex tissues during development, and the resulting three-dimensional (3D) arrangement of cells and their surrounding extracellular matrix in turn feeds back to regulate cell and tissue function. Recent advances in engineered cultures of cells to model 3D tissues or organoids have begun to capture this dynamic reciprocity between form and function. Here, we describe the underlying principles that have advanced the field, focusing in particular on recent progress in using mechanical constraints to recapitulate the structure and function of musculoskeletal tissues.
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http://dx.doi.org/10.1242/jcs.198630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5394782PMC
January 2017

Cellular forces and matrix assembly coordinate fibrous tissue repair.

Nat Commun 2016 Mar 16;7:11036. Epub 2016 Mar 16.

Department of Biomedical Engineering, Boston University, 36 Cummington Mall, Boston, Massachusetts 02215, USA.

Planar in vitro models have been invaluable tools to identify the mechanical basis of wound closure. Although these models may recapitulate closure dynamics of epithelial cell sheets, they fail to capture how a wounded fibrous tissue rebuilds its 3D architecture. Here we develop a 3D biomimetic model for soft tissue repair and demonstrate that fibroblasts ensconced in a collagen matrix rapidly close microsurgically induced defects within 24 h. Traction force microscopy and time-lapse imaging reveal that closure of gaps begins with contractility-mediated whole-tissue deformations. Subsequently, tangentially migrating fibroblasts along the wound edge tow and assemble a progressively thickening fibronectin template inside the gap that provide the substrate for cells to complete closure. Unlike previously reported mechanisms based on lamellipodial protrusions and purse-string contraction, our data reveal a mode of stromal closure in which coordination of tissue-scale deformations, matrix assembly and cell migration act together to restore 3D tissue architecture.
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http://dx.doi.org/10.1038/ncomms11036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4799373PMC
March 2016

Patterning vascular networks in vivo for tissue engineering applications.

Tissue Eng Part C Methods 2015 May 18;21(5):509-17. Epub 2015 Feb 18.

1 Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania , Philadelphia, Pennsylvania.

The ultimate design of functionally therapeutic engineered tissues and organs will rely on our ability to engineer vasculature that can meet tissue-specific metabolic needs. We recently introduced an approach for patterning the formation of functional spatially organized vascular architectures within engineered tissues in vivo. Here, we now explore the design parameters of this approach and how they impact the vascularization of an engineered tissue construct after implantation. We used micropatterning techniques to organize endothelial cells (ECs) into geometrically defined "cords," which in turn acted as a template after implantation for the guided formation of patterned capillaries integrated with the host tissue. We demonstrated that the diameter of the cords before implantation impacts the location and density of the resultant capillary network. Inclusion of mural cells to the vascularization response appears primarily to impact the dynamics of vascularization. We established that clinically relevant endothelial sources such as induced pluripotent stem cell-derived ECs and human microvascular endothelial cells can drive vascularization within this system. Finally, we demonstrated the ability to control the juxtaposition of parenchyma with perfused vasculature by implanting cords containing a mixture of both a parenchymal cell type (hepatocytes) and ECs. These findings define important characteristics that will ultimately impact the design of vasculature structures that meet tissue-specific needs.
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http://dx.doi.org/10.1089/ten.TEC.2014.0258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4410304PMC
May 2015

Stem cell differentiation: sticky mechanical memory.

Nat Mater 2014 Jun;13(6):542-3

Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02115, USA, and the Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts 02115, USA.

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http://dx.doi.org/10.1038/nmat3989DOI Listing
June 2014

Geometric control of vascular networks to enhance engineered tissue integration and function.

Proc Natl Acad Sci U S A 2013 May 22;110(19):7586-91. Epub 2013 Apr 22.

Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA 19104, USA.

Tissue vascularization and integration with host circulation remains a key barrier to the translation of engineered tissues into clinically relevant therapies. Here, we used a microtissue molding approach to demonstrate that constructs containing highly aligned "cords" of endothelial cells triggered the formation of new capillaries along the length of the patterned cords. These vessels became perfused with host blood as early as 3 d post implantation and became progressively more mature through 28 d. Immunohistochemical analysis showed that the neovessels were composed of human and mouse endothelial cells and exhibited a mature phenotype, as indicated by the presence of alpha-smooth muscle actin-positive pericytes. Implantation of cords with a prescribed geometry demonstrated that they provided a template that defined the neovascular architecture in vivo. To explore the utility of this geometric control, we implanted primary rat and human hepatocyte constructs containing randomly organized endothelial networks vs. ordered cords. We found substantially enhanced hepatic survival and function in the constructs containing ordered cords following transplantation in mice. These findings demonstrate the importance of multicellular architecture in tissue integration and function, and our approach provides a unique strategy to engineer vascular architecture.
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http://dx.doi.org/10.1073/pnas.1217796110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651499PMC
May 2013

Mapping calcium phosphate activated gene networks as a strategy for targeted osteoinduction of human progenitors.

Biomaterials 2013 Jun 26;34(19):4612-21. Epub 2013 Mar 26.

Skeletal Biology and Engineering Research Center, Department of Development and Regeneration, KU Leuven, O&N1, Herestraat 49, PB 813, 3000 Leuven, Belgium.

Although calcium phosphate-containing biomaterials are promising scaffolds for bone regenerative strategies, the osteoinductive capacity of such materials is poorly understood. In this study, we investigated whether endogenous mechanisms of in vivo calcium phosphate-driven, ectopic bone formation could be identified and used to induce enhanced differentiation in vitro of the same progenitor population. To accomplish this, human periosteum derived cells (hPDCs) were seeded on hydroxyapatite/collagen scaffolds (calcium phosphate rich matrix or CPRM), or on decalcified scaffolds (calcium phosphate depleted matrix or CPDM), followed by subcutaneous implantation in nude mice to trigger ectopic bone formation. In this system, osteoblast differentiation occurred in CPRM scaffolds, but not in CPDM scaffolds. Gene expression was assessed by human full-genome microarray at 20 h after seeding, and 2, 8 and 18 days after implantation. In both matrices, implantation of the cell constructs triggered a similar gene expression cascade, however, gene expression dynamics progressed faster in CPRM scaffolds than in CPDM scaffolds. The difference in gene expression dynamics was associated with differential activation of hub genes and molecular signaling pathways related to calcium signaling (CREB), inflammation (TNFα, NFkB, and IL6) and bone development (TGFβ, β-catenin, BMP, EGF, and ERK signaling). Starting from this set of pathways, a growth factor cocktail was developed that robustly enhanced osteogenesis in vitro and in vivo. Taken together, our data demonstrate that through the identification and subsequent stimulation of genes, proteins and signaling pathways associated with calcium phosphate mediated osteoinduction, a focused approach to develop targeted differentiation protocols in adult progenitor cells can be achieved.
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http://dx.doi.org/10.1016/j.biomaterials.2013.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4037407PMC
June 2013

Adhesive and mechanical regulation of mesenchymal stem cell differentiation in human bone marrow and periosteum-derived progenitor cells.

Biol Open 2012 Nov 27;1(11):1058-68. Epub 2012 Aug 27.

Department of Bioengineering, University of Pennsylvania , 510 Skirkanich Hall, 210 South 33rd Street, Philadelphia, PA 19104 , USA ; Laboratory for Skeletal Development and Joint Disorders, Katholieke Universiteit Leuven , Herestraat 49, Box 813, B-3000 Leuven , Belgium.

It has previously been demonstrated that cell shape can influence commitment of human bone marrow-derived mesenchymal stem cells (hBMCs) to adipogenic, osteogenic, chondrogenic, and other lineages. Human periosteum-derived cells (hPDCs) exhibit multipotency similar to hBMCs, but hPDCs may offer enhanced potential for osteogenesis and chondrogenesis given their apparent endogenous role in bone and cartilage repair in vivo. Here, we examined whether hPDC differentiation is regulated by adhesive and mechanical cues comparable to that reported for hBMC differentiation. When cultured in the appropriate induction media, hPDCs at high cell seeding density demonstrated enhanced levels of adipogenic or chondrogenic markers as compared with hPDCs at low cell seeding density. Cell seeding density correlated inversely with projected area of cell spreading, and directly limiting cell spreading with micropatterned substrates promoted adipogenesis or chondrogenesis while substrates promoting cell spreading supported osteogenesis. Interestingly, cell seeding density influenced differentiation through both changes in cell shape and non-shape-mediated effects: density-dependent adipogenesis and chondrogenesis were regulated primarily by cell shape whereas non-shape effects strongly influenced osteogenic potential. Inhibition of cytoskeletal contractility by adding the Rho kinase inhibitor Y27632 further enhanced adipogenic differentiation and discouraged osteogenic differentiation of hPDCs. Together, our results suggest that multipotent lineage decisions of hPDCs are impacted by cell adhesive and mechanical cues, though to different extents than hBMCs. Thus, future studies of hPDCs and other primary stem cell populations with clinical potential should consider varying biophysical metrics for more thorough optimization of stem cell differentiation.
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http://dx.doi.org/10.1242/bio.20122162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507189PMC
November 2012

Bone morphogenetic protein-2-induced signaling and osteogenesis is regulated by cell shape, RhoA/ROCK, and cytoskeletal tension.

Stem Cells Dev 2012 May 3;21(7):1176-86. Epub 2011 Oct 3.

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.

Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is classically thought to be mediated by different cytokines such as the bone morphogenetic proteins (BMPs). Here, we report that cell adhesion to extracellular matrix (ECM), and its effects on cell shape and cytoskeletal mechanics, regulates BMP-induced signaling and osteogenic differentiation of hMSCs. Using micropatterned substrates to progressively restrict cell spreading and flattening against ECM, we demonstrated that BMP-induced osteogenesis is progressively antagonized with decreased cell spreading. BMP triggered rapid and sustained RhoA/Rho-associated protein kinase (ROCK) activity and contractile tension only in spread cells, and this signaling was required for BMP-induced osteogenesis. Exploring the molecular basis for this effect, we found that restricting cell spreading, reducing ROCK signaling, or inhibiting cytoskeletal tension prevented BMP-induced SMA/mothers against decapentaplegic (SMAD)1 c-terminal phosphorylation, SMAD1 dimerization with SMAD4, and SMAD1 translocation into the nucleus. Together, these findings demonstrate the direct involvement of cell spreading and RhoA/ROCK-mediated cytoskeletal tension generation in BMP-induced signaling and early stages of in vitro osteogenesis, and highlight the essential interplay between biochemical and mechanical cues in stem cell differentiation.
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http://dx.doi.org/10.1089/scd.2011.0293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3328763PMC
May 2012

A hitchhiker's guide to mechanobiology.

Dev Cell 2011 Jul;21(1):35-47

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.

More than a century ago, it was proposed that mechanical forces could drive tissue formation. However, only recently with the advent of enabling biophysical and molecular technologies are we beginning to understand how individual cells transduce mechanical force into biochemical signals. In turn, this knowledge of mechanotransduction at the cellular level is beginning to clarify the role of mechanics in patterning processes during embryonic development. In this perspective, we will discuss current mechanotransduction paradigms, along with the technologies that have shaped the field of mechanobiology.
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http://dx.doi.org/10.1016/j.devcel.2011.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3155761PMC
July 2011

Effects of MRI contrast agents on the stem cell phenotype.

Cell Transplant 2010 26;19(8):919-36. Epub 2010 Mar 26.

Stem Cell Institute, K.U. Leuven, Leuven, Belgium.

The ultimate therapy for ischemic stroke is restoration of blood supply in the ischemic region and regeneration of lost neural cells. This might be achieved by transplanting cells that differentiate into vascular or neuronal cell types, or secrete trophic factors that enhance self-renewal, recruitment, long-term survival, and functional integration of endogenous stem/progenitor cells. Experimental stroke models have been developed to determine potential beneficial effect of stem/progenitor cell-based therapies. To follow the fate of grafted cells in vivo, a number of noninvasive imaging approaches have been developed. Magnetic resonance imaging (MRI) is a high-resolution, clinically relevant method allowing in vivo monitoring of cells labeled with contrast agents. In this study, labeling efficiency of three different stem cell populations [mouse embryonic stem cells (mESC), rat multipotent adult progenitor cells (rMAPC), and mouse mesenchymal stem cells (mMSC)] with three different (ultra)small superparamagnetic iron oxide [(U)SPIO] particles (Resovist, Endorem, Sinerem) was compared. Labeling efficiency with Resovist and Endorem differed significantly between the different stem cells. Labeling with (U)SPIOs in the range that allows detection of cells by in vivo MRI did not affect differentiation of stem cells when labeled with concentrations of particles needed for MRI-based visualization. Finally, we demonstrated that labeled rMAPC could be detected in vivo and that labeling did not interfere with their migration. We conclude that successful use of (U)SPIOs for MRI-based visualization will require assessment of the optimal (U)SPIO for each individual (stem) cell population to ensure the most sensitive detection without associated toxicity.
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http://dx.doi.org/10.3727/096368910X494623DOI Listing
March 2011

Bone augmentation with autologous periosteal cells and two different calcium phosphate scaffolds under an occlusive titanium barrier: an experimental study in rabbits.

J Periodontol 2008 May;79(5):896-904

Department of Periodontology, Catholic University of Leuven, Leuven, Belgium.

Background: This study used a tissue-engineering approach, which combined autologous periosteal cells with a scaffold material, to promote bone augmentation under an occlusive titanium barrier that was placed on the skull of rabbits. Because the cell-matrix interaction is of key importance in tissue engineering, two different calcium phosphate-based scaffolds were seeded with autologous periosteal cells. One scaffold contained hydroxyapatite, tricalcium phosphate, and collagen; the other scaffold was a beta-tricalcium phosphate structure.

Methods: The experiment involved 38 rabbits divided into five groups: the two different scaffolds with and without cells and a blood clot only. Prior to implantation, autologous periosteal cells were harvested from the tibia by stripping the periosteum. Cells were cultured, and 1 day before the implantation approximately 20 million cells were collected and seeded onto the scaffolds. Two preformed dome-shaped full titanium barriers were placed subperiosteally onto the frontal and parietal bones of each rabbit. Before placement of the barriers, the different scaffolds, seeded with or without cells, were put on top of the skull. As a negative control, autologous blood was injected into the barriers. Histologic evaluation and histomorphometric analysis were performed after 12 weeks of undisturbed bone growth. Measurements involved the amounts of newly formed tissue and of new bone distinguishing between trabecular bone and osteoid.

Results: No significant differences were found between the four treatment groups (scaffolds with or without cells). However, the amount of new bone tissue found underneath the titanium barriers with scaffolds was significantly higher (P <0.04) than with a blood clot only.

Conclusion: A better understanding of the mode of action is required to optimize tissue-engineering procedures before entering clinical applications.
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http://dx.doi.org/10.1902/jop.2008.070043DOI Listing
May 2008
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