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Fibrin sealants have long been used as carrier for osteogenic cells in bone regeneration. However, it has not been demonstrated whether fibrin’s role is limited to delivering cells to the bone defect or whether fibrin enhances osteogenesis. This study investigated fibrin’s influence on the behaviour of human periosteum-derived cells (hPDCs) when cultured in vitro under osteogenic conditions in two-dimensional (fibrin substrate) and three-dimensional (fibrin carrier) environments. Tranexamic acid (TEA) was used to reduce fibrin degradation after investigating its effect on hPDCs in monolayer culture on plastic.TEA did not affect proliferation nor calcium deposition of hPDCs under these conditions. Expression profiles of specific osteogenic markers were also maintained within the presence of TEA, apart from reduced alkaline phosphatase (ALP) expression (day 14). Compared to plastic, proliferation was upregulated on 2D fibrin substrates with a 220% higher DNA content by day 21. Gene expression was also altered, with significantly (p

The health and function of microvascular beds are dramatically impacted by the mechanical forces that they experience due to fluid flow. These fluid flow-generated forces are challenging to measure directly and are typically calculated from experimental flow data. However, current computational fluid dynamics (CFD) models either employ truncated 2D models or overlook the presence of extraluminal flows within the interstitial space between vessels that result from the permeability of the endothelium lining the vessels, which are crucial components affecting flow dynamics. To address this, we present a bottom-up modeling approach that assesses fluid flow in 3D-engineered vessel networks featuring an endothelial lining and interstitial space. Using image processing algorithms to segment 3D confocal image stacks from engineered capillary networks, we reconstructed a 3D computational model of the networks. We incorporated vascular permeability and matrix porosity values to model the contributions of the endothelial lining and interstitial spaces to the flow dynamics in the networks. Simulations suggest that including the endothelial monolayer and the interstitium significantly affects the predicted flow magnitude in the vessels and flow profiles in the interstitium. To demonstrate the importance of these factors, we showed experimentally and computationally that while cytokine (IL-1β) treatment did not affect the network architecture, it significantly increased vessel permeability and resulted in a dramatic decrease in wall shear stresses and flow velocities intraluminally within the networks. In conclusion, this framework offers a robust methodology for studying flow dynamics in 3D in vitro vessel networks, enhancing our understanding of vascular physiology and pathology.

A longstanding challenge in microfluidics has been the efficient delivery of fluids from macro-scale pumping systems into microfluidic devices, known as the "world-to-chip" problem. Thus far, the entire industry has accepted the use of imperfect, rigid tubing and connectors as the ecosystem within which to operate, which, while functional, are often cumbersome, labor-intensive, prone to errors, and ill-suited for high-throughput experimentation. In this paper, we introduce TapeTech microfluidics, a flexible and scalable solution designed to address the persistent "world-to-chip" problem in microfluidics, particularly in organ-on-a-chip (OoC) applications. TapeTech offers a streamlined alternative, utilizing adhesive tape and thin-film polymers to create adaptable, integrated multi-channel ribbon connectors that simplify fluidic integration with pumps and reservoirs. Key features of TapeTech include reduced pressure surges, easy priming, rapid setup, easy multiplexing, and broad compatibility with existing devices and components, which are essential for maintaining stable fluid dynamics and protecting sensitive cell cultures. Furthermore, TapeTech is designed to flex around the lids of Petri dishes, enhancing sterility and transportability by enabling easy transfer between incubators, biosafety cabinets (BSCs), and microscopes. The rapid design-to-prototype iteration enabled by TapeTech allows users to quickly develop connectors for a wide range of microfluidic devices. Importantly, we showcase the utility of TapeTech in OoC cultures requiring fluid flow. We also highlight other utilities, such as real-time microscopy and a well-plate medium exchanger. The accessibility of this technology should enable more laboratories to simplify design and setup of microfluidic experiments, and increase technology adoption.

Natural tissues are composed of diverse cells and extracellular materials whose arrangements across several length scales-from subcellular lengths 1 (micrometre) to the organ scale 2 (centimetre)-regulate biological functions. Tissue-fabrication methods have progressed to large constructs, for example, through stereolithography 3 and nozzle-based bioprinting 4,5 , and subcellular resolution through subtractive photoablation 6-8 . However, additive bioprinting struggles with sub-nozzle/voxel features 9 and photoablation is restricted to small volumes by prohibitive heat generation and time 10 . Building across several length scales with temperature-sensitive, water-based soft biological matter has emerged as a critical challenge, leaving large classes of biological motifs-such as multiscalar vascular trees with varying calibres-inaccessible with present technologies 11,12 . Here we use gallium-based engineered sacrificial capillary pumps for evacuation (ESCAPE) during moulding to generate multiscalar structures in soft natural hydrogels, achieving both cellular-scale (<10 µm) and millimetre-scale features. Decoupling the biomaterial of interest from the process of constructing the geometry allows non-biocompatible tools to create the initial geometry. As an exemplar, we fabricated branched, cell-laden vascular trees in collagen, spanning approximately 300-µm arterioles down to the microvasculature (roughly ten times smaller). The same approach can micropattern the inner surface of vascular walls with topographical cues to orient cells in 3D and engineer fine structures such as vascular malformations. ESCAPE moulding enables the fabrication of multiscalar forms in soft biomaterials, paving the way for a wide range of tissue architectures that were previously inaccessible in vitro., Competing Interests: Competing interests: A patent application (U.S. application no. 18/422,963) has been filed by Boston University based on this work. C.S.C. is a founder and owns shares in Innolign Biomedical, a company that is developing engineered organ models for pharmaceutical research and development, and Satellite Biosciences, a company that is developing cell-based therapies. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

In brain metastasis, cancer cells remain in close contact with the existing vasculature and can use vessels as migratory paths-a process known as vessel co-option. However, the mechanisms regulating this form of migration are poorly understood. Here we use ex vivo brain slices and an organotypic in vitro model for vessel co-option to show that cancer cell invasion along brain vasculature is driven by the difference in stiffness between vessels and the brain parenchyma. Imaging analysis indicated that cells move along the basal surface of vessels by adhering to the basement membrane extracellular matrix. We further show that vessel co-option is enhanced by both the stiffness of brain vasculature, which reinforces focal adhesions through a talin-dependent mechanism, and the softness of the surrounding environment that permits cellular movement. Our work reveals a mechanosensing mechanism that guides cell migration in response to the tissue's intrinsic mechanical heterogeneity, with implications in cancer invasion and metastasis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the left ventricular wall, diastolic dysfunction, and fibrosis, and is associated with mutations in genes encoding sarcomere proteins. While in vitro studies have used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study HCM, these models have not examined the multicellular interactions involved in fibrosis. Using engineered cardiac microtissues (CMTs) composed of HCM-causing MYH7 -variant hiPSC-CMs and wild-type fibroblasts, we observed cell-cell cross-talk leading to increased collagen deposition, tissue stiffening, and decreased contractility dependent on fibroblast proliferation. hiPSC-CM conditioned media and single-nucleus RNA sequencing data suggested that fibroblast proliferation is mediated by paracrine signals from MYH7 -variant cardiomyocytes. Furthermore, inhibiting epidermal growth factor receptor tyrosine kinase with erlotinib hydrochloride attenuated stromal activation. Last, HCM-causing MYBPC3 -variant CMTs also demonstrated increased stromal activation and reduced contractility, but with distinct characteristics. Together, these findings establish a paracrine-mediated cross-talk potentially responsible for fibrotic changes observed in HCM.

Generating stable and customizable topography on hydrogel surfaces with contact guidance potential is critical as it can direct/influence cell growth. This necessitates the development of new techniques for surface patterning of the hydrogels. We report on the design of a square grid template for surface patterning hydrogels. The template was 3-D printed and has the diameter of a well in a 24-well plate. Hyaluronic acid methacrylate (HA) hydrogel precursor solutions were cast on the 3D printed template's surface, which generated 3D square shape topographies on the HA hydrogel surface upon demolding. The 3D Laser Microscopy has shown the formation of a periodic array of 3D topographies on hydrogel surfaces. 3D Laser and Electron Microscopy Imaging have revealed that this new method has increased the surface area and exposed the underlying pore structure of the HA hydrogels. To demonstrate the method's versatility, we have successfully applied this technique to generate 3D topography on two more acrylate hydrogel formulations, gelatin Methacrylate and polyethylene glycol dimethacrylate. Human neonatal dermal fibroblast cells were used as a model cell line to evaluate the cell guidance potential of patterned HA hydrogel. Confocal fluorescence microscopy imaging has revealed that the 3D surface topographies on HA hydrogels can guide and align the actin filaments of the fibroblasts presumably due to the contact guidance mechanism. The newly developed methodology of 3D topography generation in acrylate hydrogels may influence the cell responses on hydrogel surfaces which can impact biomedical applications such as tissue engineering, wound healing, and disease modeling., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Previous lung-on-chip devices have facilitated significant advances in our understanding of lung biology and pathology. Here, we describe a novel lung-on-a-chip model in which human induced pluripotent stem cell-derived alveolar epithelial type II cells (iAT2s) form polarized duct-like lumens alongside engineered perfused vessels lined with human umbilical vein endothelium, all within a 3D, physiologically relevant microenvironment. Using this model, we investigated the morphologic and signaling consequences of the KRAS G12D mutation, a commonly identified oncogene in human lung adenocarcinoma (LUAD). We show that expression of the mutant KRAS G12D isoform in iAT2s leads to a hyperproliferative response and morphologic dysregulation in the epithelial monolayer. Interestingly, the mutant epithelia also drive an angiogenic response in the adjacent vasculature that is mediated by enhanced secretion of the pro-angiogenic factor soluble uPAR. These results demonstrate the functionality of a multi-cellular in vitro platform capable of modeling mutation-specific behavioral and signaling changes associated with lung adenocarcinoma., Competing Interests: C.S.C. is a founder and owns shares of Innolign Biomedical, a company that is developing engineered organ models for pharmaceutical research and development, and Satellite Biosciences, a company that is developing cell-based therapies. The other authors declare that they have no competing interest., (© 2024 Author(s).)

Tissue engineering has long sought to rapidly generate perfusable vascularized tissues with vessel sizes spanning those seen in humans. Current techniques such as biological 3D printing (top-down) and cellular self-assembly (bottom-up) are resource intensive and have not overcome the inherent tradeoff between vessel resolution and assembly time, limiting their utility and scalability for engineering tissues. We present a flexible and scalable technique termed SPAN - S acrificial P ercolation of A nisotropic N etworks, where a network of perfusable channels is created throughout a tissue in minutes, irrespective of its size. Conduits with length scales spanning arterioles to capillaries are generated using pipettable alginate fibers that interconnect above a percolation density threshold and are then degraded within constructs of arbitrary size and shape. SPAN is readily used within common tissue engineering processes, can be used to generate endothelial cell-lined vasculature in a multi-cell type construct, and paves the way for rapid assembly of perfusable tissues., Competing Interests: DECLARATION OF INTERESTS A provisional patent application has been filed by Boston University based on this work. C.S.C. is a founder and owns shares of Innolign Biomedical, a company that is developing engineered organ models for pharmaceutical research and development; Satellite Biosciences, a company that is developing cell-based therapies; and Ropirio Therapeutics, a company developing molecular therapeutics for vascular disease. The other authors declare that they have no competing interests.

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., Competing Interests: Conflict of Interest C.S.C. is a founder and own shares of Innolign Biomedical, a company that is developing engineered organ models for pharmaceutical research and development, and Satellite Biosciences, a company that is developing cell-based therapies. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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.

Competing Interests: Declaration of interests The author declares no competing interests.

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., (© 2023 Author(s).)

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.

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.

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.

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.

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 ( PKP2 tv), the most prevalent ACM-linked mutations. The PKP2 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 PKP2 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.

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.

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., (© 2021 Author(s).)

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., Competing Interests: Conflict of Interest S.N.B. is a director at Vertex, co-founder and consultant at Glympse Bio, consultant for Cristal, Maverick, and Moderna, and receives sponsored research funding from Johnson & Johnson.

The COVID-19 pandemic induces the worst economic downturn since the Second World War, requiring governments to design large-scale recovery plans to overcome this crisis. This paper quantitatively assesses the potential of government investments in eco-friendly construction projects to boost the economy and simultaneously realise environmental gains through reduced energy consumption and related greenhouse gas emissions. The analysis uses a Computable General Equilibrium model that examines the macroeconomic impact of the COVID-19 crisis in a small open economy (Belgium). Subsequently, the impact of the proposed policy is assessed through comparative analysis for macroeconomic parameters as well as CO 2 equivalent emissions for four scenarios. Our findings demonstrate that the COVID-19 pandemic damages economies considerably, however, the reduction in emissions is less than proportionate. Still, well-designed public policies can reverse this trend, achieving both economic growth and a disproportionally large decrease in emissions. Moreover, the positive effect of such a decoupling policy on GDP is even stronger during the pandemic than compared to the pre-COVID-19 period. This is the result of a targeted, investment-induced green transition towards low energy-intensive economic activities. Finally, this paper describes how the net effect on the government budget is positive through the indirect gains of the economic uptake., (© Springer Nature B.V. 2020.)

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., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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.

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.

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.

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., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

Urgent solutions are needed in Europe to deal with construction and demolition waste (CDW). EU policy has contributed to significantly reducing the amount of CDW going to landfill, but most of the effort has been put in downcycling practices. Therefore, further policies are needed to stimulate high-quality recycling of CDW. The present paper presents a combined life cycle assessment (LCA) and life cycle costing (LCC) methodologies to analyse the environmental and the economic drivers in four alternative CDW end-of-life scenarios in the region of Flanders, in Belgium. The four analysed alternatives are (i) landfilling, (ii) downcycling, (iii) advanced recycling and (iv) recycling after selective demolition. LCA results show that landiflling is the scenario having the highest environmental impacts in terms of person equivalent (PE), followed by downcycling and recycling (-36%) and recycling after selective demolition (-59%). The decrease in environmental impacts is mostly due to the avoided landfilling of CDW and the recovery of materials from selective demolition. LCC results indicate that landfilling is the scenario bearing the highest total economic costs. This is due to the high landfill tax in Flanders. The recycling after selective demolition bears the second highest cost. The increase of high-quality CDW recycling can significantly reduce the overall environmental impact of the system. Implementing a high landfill tax, increasing the gate fee to the recycling plant, and boosting the sales price of recycled aggregates are the most effective drivers to facilitate a transition towards a more sustainable CDW management system. The paper demonstrates that the combined LCA and LCC results can highlight the environmental and economic drivers in CDW management. The results of the combined analysis can help policymakers to promote the aspects contributing to sustainability and to limit the ones creating a barrier., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

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.

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 N -cadherin expression in mural cells plays a key role in barrier function, as CRISPR-mediated knockout of N -cadherin in the mural cells led to loss of barrier function, and overexpression of N -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., Competing Interests: The authors declare no conflict of interest.

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., (© 2017. Published by The Company of Biologists Ltd.)

Both landfill taxes and Enhanced Waste Management (EWM) practices can mitigate the scarcity issue of landfill capacity by respectively reducing landfilled waste volumes and valorising future waste streams. However, high landfill taxes might erode incentives for EWM, even though EWM creates value by valorising waste. Concentrating on Flanders (Belgium), the paper applies dynamic optimisation modelling techniques to analyse how landfill taxation and EWM can reinforce each other and how taxation schemes can be adjusted in order to foster sustainable and welfare maximising ways of processing future waste streams. Based on the Flemish simulation results, insights are offered that are generally applicable in international waste and resource management policy. As shown, the optimal Flemish landfill tax that optimises welfare in the no EWM scenario is higher than the one in the EWM scenario (93 against €50/ton). This difference should create incentives for applying EWM and is driven by the positive external effects that are generated by EWM practices. In Flanders, as the current landfill tax is slightly lower than these optimal levels, the choice that can be made is to further increase taxation levels or show complete commitment to EWM. A first generally applicable insight that was found points to the fact that it is not necessarily the case that the higher the landfill tax, the more effective waste management improvements can be realised. Other insights are about providing sufficient incentives for applying EMW practices and formulating appropriate pleas in support of technological development. By these insights, this paper should provide relevant information that can assist in triggering the transition towards a resource-efficient, circular economy in Europe., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

Weight-based pricing systems for municipal solid waste collection and processing are increasingly popular in many European countries and regions. However, the impact on waste generation of such pricing schedules remains debated and depends strongly on the practical details of the system used. This article assesses the impact of a voluntary transition by Flemish municipalities from the default price-per-bag pricing systems to a more sophisticated weight-based pricing schedule. By (1) exploiting alternative statistical techniques, (2) using more recent data and (3) focusing on the introduction effect of the new pricing schedule, we are able to complement and refine prior research on this topic. Our results indicate that introducing weight-based pricing has initially a significant and substantial downward impact on the amount of residual municipal solid waste per capita. This result is robust under different methodologies that control for selection bias. There are however, indications that this initial effect does not persist in the years after introduction., (© The Author(s) 2015.)

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.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We study the impact of some local policies aimed at municipal solid waste (MSW) reduction on the cost efficiency of MSW collection and disposal. We explicitly account for differences between municipalities in background conditions by using a bootstrapped version of the Data Envelopment Analysis methodology in combination with a matching technique. Using data on 299 municipalities in Flanders, Belgium, for the year 2003, our results indicate that municipalities that are member of a waste collection joint venture, or that subscribe to a voluntary agreement to reduce MSW at the highest ambition level, collect and process MSW more efficiently than other municipalities. Weekly instead of two-weekly waste collection, or using a weight-based pricing system appears to have no impact on efficiency. Our results show that aiming at MSW reduction does not lead to lower efficiency of public service provision, even on the contrary., (Copyright © 2011 Elsevier Ltd. All rights reserved.)