Synthetic hydrogels represent an exciting avenue in the field of regenerative biomaterials given their injectability, orthogonally tunable mechanical properties, and potential for modular inclusion ...of cellular cues. Separately, recent advances in soluble factor release technology have facilitated control over the soluble milieu in cell microenvironments via tunable microparticles. A composite hydrogel incorporating both of these components can robustly mediate tendon healing following a single injection. Here, a synthetic hydrogel system with encapsulated electrospun fiber segments and a novel microgel‐based soluble factor delivery system achieves precise control over topographical and soluble features of an engineered microenvironment, respectively. It is demonstrated that three‐dimensional migration of tendon progenitor cells can be enhanced via combined mechanical, topographical, and microparticle‐delivered soluble cues in both a tendon progenitor cell spheroid model and an ex vivo murine Achilles tendon model. These results indicate that fiber reinforced hydrogels can drive the recruitment of endogenous progenitor cells relevant to the regeneration of tendon and, likely, a broad range of connective tissues.
A composite hydrogel‐based scaffold providing control over soluble and physical microenvironmental cues is designed to direct the 3D migration of tendon progenitor cells. Microgel‐mediated chemokine delivery, along with contact guidance afforded by RGD‐functionalized fiber segments, promoted tendon progenitor cell invasion from ex vivo tendons into an injectable composite hydrogel. This material strategy offers a promising route toward augmenting tendon repair and, likely, that of a range of other connective tissues.
Acoustically-responsive scaffolds (ARSs), which are composite fibrin hydrogels, have been used to deliver regenerative molecules. ARSs respond to ultrasound in an on-demand, ...spatiotemporally-controlled manner via a mechanism termed acoustic droplet vaporization (ADV). Here, we study the ADV-induced, time-dependent micromechanical and microstructural changes to the fibrin matrix in ARSs using confocal fluorescence microscopy as well as atomic force microscopy. ARSs, containing phase-shift double emulsion (PSDE, mean diameter: 6.3 μm), were exposed to focused ultrasound to generate ADV - the phase transitioning of the PSDE into gas bubbles. As a result of ADV-induced mechanical strain, localized restructuring of fibrin occurred at the bubble-fibrin interface, leading to formation of locally denser regions. ADV-generated bubbles significantly reduced fibrin pore size and quantity within the ARS. Two types of ADV-generated bubble responses were observed in ARSs: super-shelled spherical bubbles, with a growth rate of 31 μm per day in diameter, as well as fluid-filled macropores, possibly as a result of acoustically-driven microjetting. Due to the strain stiffening behavior of fibrin, ADV induced a 4-fold increase in stiffness in regions of the ARS proximal to the ADV-generated bubble versus distal regions. These results highlight that the mechanical and structural microenvironment within an ARS can be spatiotemporally modulated using ultrasound, which could be used to control cellular processes and further the understanding of ADV-triggered drug delivery for regenerative applications.
Stem cells transit along a variety of lineage-specific routes towards differentiated phenotypes. These fate decisions are dependent not just on the soluble chemical cues that are encountered or ...enforced in vivo and in vitro, but also on physical cues from the cellular microenvironment. These physical cues can consist of both nano- and micro-scale topographical features, as well as mechanical inputs provided passively (from the base properties of the materials to which they adhere) or actively (from extrinsic applied mechanical deformations). A suitable tool to investigate the coordination of these cues lies in nanofibrous scaffolds, which can both dictate cellular and cytoskeletal orientation and facilitate mechanical perturbation of seeded cells. Here, we demonstrate a coordinated influence of scaffold architecture (aligned vs. randomly organized fibers) and tensile deformation on nuclear shape and orientation. Sensitivity of nuclear morphology to scaffold architecture was more pronounced in stem cell populations than in terminally differentiated fibrochondrocytes. Tension applied to the scaffold elicited further alterations in nuclear morphology, greatest in stem cells, that were mediated by the filamentous actin cytoskeleton, but not the microtubule or intermediate filament network. Nuclear perturbations were time and direction dependent, suggesting that the modality and direction of loading influenced nuclear architecture. The present work may provide additional insight into the mechanisms by which the physical microenvironment influences cell fate decisions, and has specific application to the design of new materials for regenerative medicine applications with adult stem cells.
Collective cell migration is critical for proper embryonic development, wound healing, and cancer cell invasion. However, much of our knowledge of cell migration has been performed using flat ...surfaces that lack topographical features and do not recapitulate the complex fibrous architecture of the extracellular matrix (ECM). The recent availability of synthetic fibrous networks designed to mimic
ECM has been key to identify the topological features that dictate cell migration patterns as well as to determine the underlying mechanisms that regulate topography-sensing. Recent studies have underscored the prevalence of collective cell migration during cancer invasion, and these observations present a compelling need to understand the mechanisms controlling contact guidance within migratory, multicellular groups. Therefore, we designed an integrated migration analysis platform combining tunable electrospun fibers that recapitulate aspects of the biophysical properties of the ECM, and computational approaches to investigate collective cell migration. To quantitatively assess migration as a function of matrix topography, we developed an automated MATLAB code that quantifies cell migration dynamics, including speed, directionality, and the number of detached cells. This platform enables live cell imaging while providing enough cells for biochemical, proteomic, and genomic analyses, making our system highly adaptable to multiple experimental investigations.
Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) show great potential for engineering myocardium to study cardiac disease and create regenerative therapies. However, iPSC-CMs ...typically possess a late embryonic stage phenotype, with cells failing to exhibit markers of mature adult tissue. This is due in part to insufficient knowledge and control of microenvironmental cues required to facilitate the organization and maturation of iPSC-CMs. Here, we employed a cell-adhesive, mechanically tunable synthetic fibrous extracellular matrix (ECM) consisting of electrospun dextran vinyl sulfone (DVS) fibers and examined how biochemical, architectural, and mechanical properties of the ECM impact iPSC-CM tissue assembly and subsequent function. Exploring a multidimensional parameter space spanning cell-adhesive ligand, seeding density, fiber alignment, and stiffness, we found that fibronectin-functionalized DVS matrices composed of highly aligned fibers with low stiffness optimally promoted the organization of functional iPSC-CM tissues. Tissues generated on these matrices demonstrated improved calcium handling and increased end-to-end localization of N-cadherin as compared to micropatterned fibronectin lines or fibronectin-coated glass. Furthermore, DVS matrices supported long-term culture (45 days) of iPSC-CMs; N-cadherin end-to-end localization and connexin43 expression both increased as a function of time in culture. In sum, these findings demonstrate the importance of recapitulating the fibrous myocardial ECM in engineering structurally organized and functional iPSC-CM tissues.
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.
Emerging cell-based therapies such as stem cell therapy and immunotherapy have attracted broad attention in both biological research and clinical practice. However, a long-standing technical gap of ...cell-based therapies is the difficulty of directly assessing treatment efficacy via tracking therapeutically administered cells. Therefore, imaging techniques to follow the
distribution and migration of cells are greatly needed. Optical coherence tomography (OCT) is a clinically available imaging technology with ultrahigh-resolution and excellent imaging depth. It also shows great potential for
cellular imaging. However, due to the homogeneity of current OCT cell labeling contrast agents (such as gold and polymer nanoparticles), only the distribution of entire cell populations can be observed. Precise tracking of the trajectory of individual single cells is not possible with such conventional contrast agents. Microlasers may provide a route to track unique cell identifiers given their small size, high emission intensities, rich emission spectra, and narrow linewidths. Here, we demonstrate that nanowire lasers internalized by cells provide both OCT and fluorescence signal. In addition, cells can be individually identified by the unique lasing emission spectra of the nanowires that they carry. Furthermore, single cell migration trajectories can be monitored both
and
with OCT and fluorescence microscopy dual-modality imaging system. Our study demonstrates the feasibility of nanowire lasers combined with the dual-modality imaging system for
single cell tracking with a high spatial resolution and identity verification, an approach with great utility for stem cell and immunomodulatory therapies.
Over the past 3 decades, there has been a vast expansion of research in both tissue engineering and organic electronics. Although the two fields have interacted little, the materials and fabrication ...technologies which have accompanied the rise of organic electronics offer the potential for innovation and translation if appropriately adapted to pattern biological materials for tissue engineering. In this work, we use two organic electronic materials as adhesion points on a biocompatible poly(p-xylylene) surface. The organic electronic materials are precisely deposited via vacuum thermal evaporation and organic vapor jet printing, the proven, scalable processes used in the manufacture of organic electronic devices. The small molecular-weight organics prevent the subsequent growth of antifouling polyethylene glycol methacrylate polymer brushes that grow within the interstices between the molecular patches, rendering these background areas both protein and cell resistant. Last, fibronectin attaches to the molecular patches, allowing for the selective adhesion of fibroblasts. The process is simple, reproducible, and promotes a high yield of cell attachment to the targeted sites, demonstrating that biocompatible organic small-molecule materials can pattern cells at the microscale, utilizing techniques widely used in electronic device fabrication.
Mechanical forces provide critical biological signals to cells during healthy and aberrant organ development as well as during disease processes in adults. Within the cardiopulmonary system, ...mechanical forces, such as shear, compressive, and tensile forces, act across various length scales, and dysregulated forces are often a leading cause of disease initiation and progression such as in bronchopulmonary dysplasia and cardiomyopathies. Engineered in vitro models have supported studies of mechanical forces in a number of tissue and disease-specific contexts, thus enabling new mechanistic insights into cardiopulmonary development and disease. This review first provides fundamental examples where mechanical forces operate at multiple length scales to ensure precise lung and heart function. Next, we survey recent engineering platforms and tools that have provided new means to probe and modulate mechanical forces across in vitro and in vivo settings. Finally, the potential for interdisciplinary collaborations to inform novel therapeutic approaches for a number of cardiopulmonary diseases are discussed.
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Bioengineering; Biomaterials; Biomechanics
Tissue fibrosis is a leading cause of mortality and is characterized by excessive protein deposition and altered tissue mechanical properties. In pathological fibrosis, as well as cancer related ...fibrosis, tissue pericytes and fibroblasts transition from a quiescent to a myofibroblastic phenotype. In vitro models are needed to better understand how these cells are influenced by their local microenvironment. Here, we developed a fibrous network platform to mimic the structure of the extracellular matrix, where fibers consist of cross-linked hyaluronic acid hydrogels with controlled cross-link density and mechanical properties. As a model myofibroblast precursor, primary hepatic stellate cells were seeded onto fibers with either low (soft) or high (stiff) cross-link density, either directly after isolation (quiescent) or following preculture on tissue culture plates (activated). In general, both quiescent and activated cells showed an increase in spreading, alpha smooth muscle actin expression, and the formation of multicellular clusters on soft fibers when compared to stiff fibers. Further, inhibition of alpha smooth muscle actin decreased activation of cells on soft fibers. This is likely due to fiber recruitment in soft fibers that increased local fiber density, whereas stiff fibers resisted recruitment. This work emphasizes the importance of substrate topography on cell–material interactions and shows that tunable fibrous hydrogels are a relevant culture platform for studying fibrosis and mechanotransduction in disease.
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