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Cells select from a diverse repertoire of migration strategies. Recent developments in tunable biomaterials have helped identify how extracellular matrix properties influence migration, however, many settings lack the fibrous architecture characteristic of native tissues. To investigate migration in fibrous contexts, we independently varied the alignment and stiffness of synthetic 3D fiber matrices and identified two phenotypically distinct migration modes. In contrast to stiff matrices where cells migrated continuously in a traditional mesenchymal fashion, cells in deformable matrices stretched matrix fibers to store elastic energy; subsequent adhesion failure triggered sudden matrix recoil and rapid cell translocation. Across a variety of cell types, traction force measurements revealed a relationship between cell contractility and the matrix stiffness where this migration mode occurred optimally. Given the prevalence of fibrous tissues, an understanding of how matrix structure and mechanics influences migration could improve strategies to recruit repair cells to wound sites or inhibit cancer metastasis. How cells migrate in fibrous tissues is still poorly understood. Here, with synthetic 3D fibre matrices of controlled alignment and stiffness, the authors report that cells in stiff matrices move slowly and continuously, but in softer, deformable matrices cells can rapidly slingshot forward via stretch and recoil of matrix fibres.

Vasculogenesis is the de novo formation of a vascular network from individual endothelial progenitor cells occurring during embryonic development, organogenesis, and adult neovascularization. Vasculogenesis can be mimicked and studied in vitro using network formation assays, in which endothelial cells (ECs) spontaneously form capillary-like structures when seeded in the appropriate microenvironment. While the biochemical regulators of network formation have been well studied using these assays, the role of mechanical and topographical properties of the extracellular matrix (ECM) is less understood. Here, we utilized both natural and synthetic fibrous materials to better understand how physical attributes of the ECM influence the assembly of EC networks. Our results reveal that active cell-mediated matrix recruitment through actomyosin force generation occurs concurrently with network formation on Matrigel, a reconstituted basement membrane matrix regularly used to promote EC networks, and on synthetic matrices composed of electrospun dextran methacrylate (DexMA) fibers. Furthermore, modulating physical attributes of DexMA matrices that impair matrix recruitment consequently inhibited the formation of cellular networks. These results suggest an iterative process in which dynamic cell-induced changes to the physical microenvironment reciprocally modulate cell behavior to guide the formation and stabilization of multicellular networks.

Intercellular communication is critical to the formation and homeostatic function of all tissues. Previous work has shown that cells can communicate mechanically via the transmission of cell‐generated forces through their surrounding extracellular matrix, but this process is not well understood. Here, mechanically defined, synthetic electrospun fibrous matrices are utilized in conjunction with a microfabrication‐based cell patterning approach to examine mechanical intercellular communication (MIC) between endothelial cells (ECs) during their assembly into interconnected multicellular networks. It is found that cell force‐mediated matrix displacements in deformable fibrous matrices underly directional extension and migration of neighboring ECs toward each other prior to the formation of stable cell‐cell connections enriched with vascular endothelial cadherin (VE‐cadherin). A critical role is also identified for calcium signaling mediated by focal adhesion kinase and mechanosensitive ion channels in MIC that extends to multicellular assembly of 3D vessel‐like networks when ECs are embedded within fibrin hydrogels. These results illustrate a role for cell‐generated forces and ECM mechanical properties in multicellular assembly of capillary‐like EC networks and motivates the design of biomaterials that promote MIC for vascular tissue engineering. This work describes how endothelial cells (ECs) utilize mechanical intercellular communication (MIC) during vasculogenic assembly into capillary‐like, multicellular networks. Mechanically permissive matrices and EC actomyosin activity enable long‐range force transmission to direct cell migration and subsequent cell‐cell contact formation. In particular, focal adhesion signaling and stretch‐induced activation of ion channels (Piezo1/TRPV4) are critical to EC MIC and vasculogenic assembly.

The mechanical function of the myocardium is defined by cardiomyocyte contractility and the biomechanics of the extracellular matrix (ECM). Understanding this relationship remains an important unmet challenge due to limitations in existing approaches for engineering myocardial tissue. Here, they established arrays of cardiac microtissues with tunable mechanics and architecture by integrating ECM‐mimetic synthetic, fiber matrices, and induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs), enabling real‐time contractility readouts, in‐depth structural assessment, and tissue‐specific computational modeling. They found that the stiffness and alignment of matrix fibers distinctly affect the structural development and contractile function of pure iPSC‐CM tissues. Further examination into the impact of fibrous matrix stiffness enabled by computational models and quantitative immunofluorescence implicates cell‐ECM interactions in myofibril assembly, myofibril maturation, and notably costamere assembly, which correlates with improved contractile function of tissues. These results highlight how iPSC‐CM tissue models with controllable architecture and mechanics can elucidate mechanisms of tissue maturation and disease. Arrays of cardiac microtissues with tunable mechanics and architecture are created by integrating extracellular matrix‐mimetic, synthetic fiber matrices, and induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs). Real‐time contractility readouts, in‐depth structural assessment, and tissue‐specific computational modeling reveal that stiffness and alignment of matrix fibers distinctly affect tissue structural development and contractile function, thus informing the design of translatable regenerative cardiac therapies.

Vasculogenic assembly of 3D capillary networks remains a promising approach to vascularizing tissue-engineered grafts, a significant outstanding challenge in tissue engineering and regenerative medicine. Current approaches for vasculogenic assembly rely on the inclusion of supporting mesenchymal cells alongside endothelial cells, co-encapsulated within vasculo-conducive materials such as low-density fibrin hydrogels. Here, we established a material-based approach to circumvent the need for supporting mesenchymal cells and report that the inclusion of synthetic matrix fibers in dense (>3 mg mL-1) 3D fibrin hydrogels can enhance vasculogenic assembly in endothelial cell monocultures. Surprisingly, we found that the addition of non-cell-adhesive synthetic matrix fibers compared to cell-adhesive synthetic fibers best encouraged vasculogenic assembly, proliferation, lumenogenesis, a vasculogenic transcriptional program, and additionally promoted cell-matrix interactions and intercellular force transmission. Implanting fiber-reinforced prevascularized constructs to assess graft-host vascular integration, we demonstrate additive effects of enhanced vascular network assembly during in vitro pre-culture, fiber-mediated improvements in endothelial cell survival and vascular maintenance post-implantation, and enhanced host cell infiltration that collectively enabled graft vessel integration with host circulation. This work establishes synthetic matrix fibers as an inexpensive alternative to sourcing and expanding secondary supporting cell types for the prevascularization of tissue constructs. [Display omitted] •Tunable semi-synthetic hydrogel composites of electrospun fibers interspersed in fibrin were developed and characterized.•The role of fibrous topography and fiber RGD functionalization on vasculogenic assembly and vessel lumenization was assessed.•FHCs with non-adhesive fibers best encouraged vasculogenic assembly, cell proliferation, lumenogenesis, and vessel stability.•FHCs with non-adhesive fibers also promoted cell-matrix interactions and cell-cell communication via force transmission.•Implanted prevascularized FHCs enhanced host cell infiltration and graft vessel integration with systemic circulation.

Fibrosis is central to numerous fatal conditions including solid cancers, pulmonary fibrosis, cirrhosis and post-infarct cardiac fibrosis amongst many others, thereby collectively contributing to 45% of all deaths in developed nations. The potential for fibrosis across most organ systems may stem from its connections to wound healing and the ubiquitous presence of vascular endothelium. Endothelial cells (ECs) and angiogenesis, cells and associated biological program central to wound healing, have been heavily implicated in many organ-specific fibroses, but the relationship between angiogenesis and fibrogenesis remains debated and little has been established in terms of how the EC phenotype governs tissue healing vs. fibrosis. Here, we examine a murine lung injury model enabling EC lineage tracing and observe the invasion of aberrant ECs from the bronchial microvasculature following lung injury along with concurrent densification of matrix fibers surrounding these vessels. To investigate the underlying mechanisms governing their appearance, we established a microphysiological system (MPS) of arteriole/venule-scale microvessels embedded within a tunable stromal mimetic matrix and find that heightened extracellular matrix fiber density activates ECs, drives endothelial to mesenchymal transition, and promotes aberrant tip EC (ATEC) invasion into the matrix. ATECs remain adherent to fibrotic matrix and possess a pro-inflammatory phenotype that secretes TGF-β2. Notably, our studies establish that the formation of ATECs is gated by destabilization of endothelial adherens junction upon EC adhesion to fibrous matrix, and associated regulation of TGF-β signaling that is mediated by a novel VE-cadherin - TGF-βR2 axis. The current lack of effective anti-fibrotic therapies suggests potential critical involvement of other cell types such as ECs, and our findings suggest new contributions of ECs to fibrotic progression that may better inform future targets for novel anti-fibrotic therapeutics.

The mechanical function of the myocardium is defined by cardiomyocyte contractility and the biomechanics of the extracellular matrix (ECM). Understanding this relationship remains an important unmet challenge due to limitations in existing approaches for engineering myocardial tissue. Here, we established arrays of cardiac microtissues with tunable mechanics and architecture by integrating ECM-mimetic synthetic, fiber matrices and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), enabling real-time contractility readouts, in-depth structural assessment, and tissue-specific computational modeling. We find that the stiffness and alignment of matrix fibers distinctly affect the structural development and contractile function of pure iPSC-CM tissues. Further examination into the impact of fibrous matrix stiffness enabled by computational models and quantitative immunofluorescence implicates cell-ECM interactions in myofibril assembly and notably costamere assembly, which correlates with improved contractile function of tissues. These results highlight how iPSC-CM tissue models with controllable architecture and mechanics can inform the design of translatable regenerative cardiac therapies.

The ability of cells to communicate and coordinate their activity is crucial to the development and homeostatic function of all tissues. In addition to the well-established means of biochemically mediated signaling, a more recent body of evidence has indicated that cells can also communicate via cell-generated forces transmitted to neighboring cells through the extracellular matrix (ECM). One setting in which a deeper understanding of mechanical intercellular communication (MIC) would be extremely valuable is in vasculogenesis, or the de novo formation of a microvascular network. This dynamic process involves the assembly and organization of individual endothelial progenitor cells into an interconnected network of capillaries, thus requiring cellular communication and coordination over large spatial scales. If fully understood and harnessed, vasculogenic assembly presents a promising approach to vascularizing engineered tissue constructs for regenerative medicine applications. We hypothesize physical properties of the ECM are critical to MIC as the matrix context defines not only the generation of cell forces but also force transmission through the matrix to nearby cells. Thus, the focus of this dissertation is to study cell force propagation and MIC between endothelial cells (ECs) in controllable synthetic ECMs towards the informed design of biomaterials that drive rapid self-assembly of functional microvascular networks.First, this thesis explores how physical attributes of the ECM regulate the assembly of ECs into interconnected multicellular networks. To mimic the fibrous microenvironments where neovascularization typically occurs in the body, we developed a novel model of the EC network formation assay utilizing 2.5D matrices of electrospun synthetic dextran methacrylate (DexMA) polymeric fibers. Our results revealed that active cell-mediated matrix deformations and fiber recruitment through actomyosin force generation occurs concurrently with the formation and stabilization of multicellular EC networks.Next, this thesis describes the development and characterization of a new material system composed of electrospun dextran vinyl sulfone (DexVS) polymeric fibers that possess longer-term mechanical stability in culture as compared to DexMA matrices. These matrices were utilized for two major objectives: 1) investigating the role of matrix mechanics on the activation of fibroblasts into myofibroblasts, a key component of wound healing and the fibrotic progression, and 2) exploring the impact of nonlinear matrix mechanical properties on vasculogenic assembly by imbuing fibers with crimped microstructure.Lastly, this thesis describes the mechanism of MIC between individual ECs during vasculogenic assembly. By combining electrospun DexMA fiber matrices with a microfabrication-based cell-patterning method, we investigated EC force-mediated matrix displacements and MIC as a function of matrix stiffness and identified the critical cellular machinery required for ECs to sense and respond to mechanical signals emanating from neighboring cells. We then sought to harness these observations in more translatable 3D hydrogel constructs by using a composite approach where fibrin hydrogels were reinforced with electrospun DexVS fiber segments. While traditional approaches to prevascularize 3D hydrogels require long-term cocultures of ECs and support stromal cells, our work demonstrated that mechanical cues from synthetic fibers enable ECs alone to rapidly self-assemble into networks of lumenized capillary-like structures.Overall, the work presented in this dissertation integrates biomaterials, tissue engineering, and microfabrication approaches to investigate the mechanobiology of how cell forces regulate intercellular communication during vasculogenic assembly. The results presented here are critical to the design of biomaterials that promote robust capillary network assembly for applications in tissue engineering and regenerative medicine.

Intercellular communication is critical to the development and homeostatic function of all tissues. Previous work has shown that cells can communicate mechanically via transmission of cell-generated forces through their surrounding extracellular matrix, but this process is not well understood. Here, we utilized synthetic, electrospun fibrous matrices in conjunction with a microfabrication-based cell patterning approach to examine mechanical intercellular communication (MIC) between endothelial cells (ECs) during the assembly of microvascular networks. We found that cell force-mediated matrix displacements in deformable fibrous matrices underly directional migration of neighboring ECs towards each other prior to the formation of stable cell-cell connections. We also identified a critical role for intracellular calcium signaling mediated by focal adhesion kinase and TRPV4 during MIC that extends to multicellular assembly of vessel-like networks in 3D fibrin hydrogels. The results presented here are critical to the design of biomaterials that support cellular self-assembly for tissue engineering applications.

Fibrosis is often untreatable and is characterized by aberrant tissue scarring from activated myofibroblasts. Although the extracellular matrix becomes increasingly stiff and fibrous during disease progression, how these physical cues impact myofibroblast differentiation in 3D is poorly understood. Here we describe a multicomponent hydrogel that recapitulates the 3D fibrous structure hallmark to the interstitial tissue regions where idiopathic pulmonary fibrosis (IPF) initiates. In contrast to findings on 2D hydrogels, myofibroblast differentiation in 3D was inversely correlated with hydrogel stiffness, but positively correlated with matrix fiber density. Employing a multi-step bioinformatics analysis of IPF patient transcriptomes and in vitro pharmacologic screening, we identify matrix-metalloprotease activity to be essential for 3D but not 2D myofibroblast differentiation. Given our observation that compliant degradable 3D matrices amply support fibrogenesis, these studies demonstrate a departure from the established relationship between stiffness and myofibroblast differentiation in 2D, and provide a new 3D model for studying fibrosis.