Biophysical forces mediated by respiration maintain lung alveolar epithelial cell fate.

Lungs undergo mechanical strain during breathing, but how these biophysical forces affect cell fate and tissue homeostasis are unclear. We show that biophysical forces through normal respiratory motion actively maintain alveolar type 1 (AT1) cell identity and restrict these cells from reprogramming into AT2 cells in the adult lung. AT1 cell fate is maintained at homeostasis by Cdc42- and Ptk2-mediated actin remodeling and cytoskeletal strain, and inactivation of these pathways causes a rapid reprogramming into the AT2 cell fate. This plasticity induces chromatin reorganization and changes in nuclear lamina-chromatin interactions, which can discriminate AT1 and AT2 cell identity. Unloading the biophysical forces of breathing movements leads to AT1-AT2 cell reprogramming, revealing that normal respiration is essential to maintain alveolar epithelial cell fate. These data demonstrate the integral function of mechanotransduction in maintaining lung cell fate and identifies the AT1 cell as an important mechanosensor in the alveolar niche.

Bioprinting for the Biologist.

Building tissues from scratch to explore entirely new cell configurations could revolutionize fundamental understanding in biology. Bioprinting is an emerging technology to do this. Although typically applied to engineer tissues for therapeutic tissue repair or drug screening, there are many opportunities for bioprinting within biology, such as for exploring cellular crosstalk or cellular morphogenesis. The overall goals of this Primer are to provide an overview of bioprinting with the biologist in mind, outline the steps in extrusion bioprinting (the most widely used and accessible technology), and discuss alternative bioprinting technologies and future opportunities for bioprinting in biology.

Nuclear-Import Receptors Reverse Aberrant Phase Transitions of RNA-Binding Proteins with Prion-like Domains.

RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-ß2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-ß1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-ß2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. In vivo, Karyopherin-ß2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.

Modeling development using hydrogels.

The development of multicellular complex organisms relies on coordinated signaling from the microenvironment, including both biochemical and mechanical interactions. To better understand developmental biology, increasingly sophisticated in vitro systems are needed to mimic these complex extracellular features. In this Primer, we explore how engineered hydrogels can serve as in vitro culture platforms to present such signals in a controlled manner and include examples of how they have been used to advance our understanding of developmental biology.

A fetal wound healing program after intrauterine bile duct injury may contribute to biliary atresia.

BACKGROUND & AIMS: Biliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause. Fetal tissues have increased levels of hyaluronic acid (HA), which plays an integral role in fetal wound healing. The objective of this study was to determine whether a program of fetal wound healing is part of the response to fetal EHBD injury. METHODS: Mouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes. RESULTS: A wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device. CONCLUSION: The fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with prolonged high levels of HA typical of fetal wound healing. The expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD. IMPACT AND IMPLICATIONS: Biliary atresia is a pediatric cholangiopathy associated with high morbidity and mortality rates; although multiple etiologies have been proposed, the fetal response to bile duct damage is largely unknown. This study explores the fetal pathogenesis after extrahepatic bile duct damage, thereby opening a completely new avenue to study therapeutic targets in the context of biliary atresia.

Synthesis, Characterization, and Digital Light Processing of a Hydrolytically Degradable Hyaluronic Acid Hydrogel.

Numerous chemical modifications of hyaluronic acid (HA) have been explored for the formation of degradable hydrogels that are suitable for a variety of biomedical applications, including biofabrication and drug delivery. Thiol-ene step-growth chemistry is of particular interest due to its lower oxygen sensitivity and ability to precisely tune mechanical properties. Here, we utilize an aqueous esterification route via reaction with carbic anhydride to synthesize norbornene-modified HA (NorHACA) that is amenable to thiol-ene crosslinking to form hydrolytically unstable networks. NorHACA is first synthesized with varying degrees of modification (∼15-100%) by adjusting the ratio of reactive carbic anhydride to HA. Thereafter, NorHACA is reacted with dithiol crosslinker in the presence of visible light and photoinitiator to form hydrogels within tens of seconds. Unlike conventional NorHA, NorHACA hydrogels are highly susceptible to hydrolytic degradation through enhanced ester hydrolysis. Both the mechanical properties and the degradation timescales of NorHACA hydrogels are tuned via macromer concentration and/or the degree of modification. Moreover, the degradation behavior of NorHACA hydrogels is validated through a statistical-co-kinetic model of ester hydrolysis. The rapid degradation of NorHACA hydrogels can be adjusted by incorporating small amounts of slowly degrading NorHA macromer into the network. Further, NorHACA hydrogels are implemented as digital light processing (DLP) resins to fabricate hydrolytically degradable scaffolds with complex, macroporous structures that can incorporate cell-adhesive sites for cell attachment and proliferation after fabrication. Additionally, DLP bioprinting of NorHACA hydrogels to form cell-laden constructs with high viability is demonstrated, making them useful for applications in tissue engineering and regenerative medicine.

Chemically Modified Biopolymers for the Formation of Biomedical Hydrogels.

Biopolymers are natural polymers sourced from plants and animals, which include a variety of polysaccharides and polypeptides. The inclusion of biopolymers into biomedical hydrogels is of great interest because of their inherent biochemical and biophysical properties, such as cellular adhesion, degradation, and viscoelasticity. The objective of this Review is to provide a detailed overview of the design and development of biopolymer hydrogels for biomedical applications, with an emphasis on biopolymer chemical modifications and cross-linking methods. First, the fundamentals of biopolymers and chemical conjugation methods to introduce cross-linking groups are described. Cross-linking methods to form biopolymer networks are then discussed in detail, including (i) covalent cross-linking (e.g., free radical chain polymerization, click cross-linking, cross-linking due to oxidation of phenolic groups), (ii) dynamic covalent cross-linking (e.g., Schiff base formation, disulfide formation, reversible Diels-Alder reactions), and (iii) physical cross-linking (e.g., guest-host interactions, hydrogen bonding, metal-ligand coordination, grafted biopolymers). Finally, recent advances in the use of chemically modified biopolymer hydrogels for the biofabrication of tissue scaffolds, therapeutic delivery, tissue adhesives and sealants, as well as the formation of interpenetrating network biopolymer hydrogels, are highlighted.

Sticking Together: Injectable Granular Hydrogels with Increased Functionality via Dynamic Covalent Inter-Particle Crosslinking.

Granular hydrogels are an exciting class of microporous and injectable biomaterials that are being explored for many biomedical applications, including regenerative medicine, 3D printing, and drug delivery. Granular hydrogels often possess low mechanical moduli and lack structural integrity due to weak physical interactions between microgels. This has been addressed through covalent inter-particle crosslinking; however, covalent crosslinking often occurs through temporal enzymatic methods or photoinitiated reactions, which may limit injectability and material processing. To address this, a hyaluronic acid (HA) granular hydrogel is developed with dynamic covalent (hydrazone) inter-particle crosslinks. Extrusion fragmentation is used to fabricate microgels from photocrosslinkable norbornene-modified HA, additionally modified with either aldehyde or hydrazide groups. Aldehyde and hydrazide-containing microgels are mixed and jammed to form adhesive granular hydrogels. These granular hydrogels possess enhanced mechanical integrity and shape stability over controls due to the covalent inter-particle bonds, while maintaining injectability due to the dynamic hydrazone bonds. The adhesive granular hydrogels are applied to 3D printing, which allows the printing of structures that are stable without any further post-processing. Additionally, the authors demonstrate that adhesive granular hydrogels allow for cell invasion in vitro. Overall, this work demonstrates the use of dynamic covalent inter-particle crosslinking to enhance injectable granular hydrogels.

Fundamentals and Applications of Photo-Cross-Linking in Bioprinting.

This review provides a detailed overview of the rapidly advancing field of biofabrication, particularly with regards to the use of photo-cross-linking (i.e., light-based) techniques. The major emphasis of this review is on the fundamentals of photo-cross-linking and key criteria identified for the successful design and implementation of photo-cross-linked bioinks and bioresins in extrusion-based and lithography-based bioprinting. The general mechanisms associated with photo-cross-linking (e.g., free-radical chain polymerization, thiol-ene, photomediated redox) of natural and synthetic materials are described to inform bioink and bioresin design, which includes the selection of polymers, functional group modifications, photoinitiators, and light sources that enable facile and cytocompatible photo-cross-linking. Depending on material selection and the bioprinting technique of interest, we describe the specific bioink or bioresin properties and criteria that must be achieved to ensure optimal printability and utility. Finally, examples of current state-of-the-art applications of light-based bioprinting for in vitro tissue models, tissue engineering, and regenerative medicine are provided to further motivate future opportunities within the bioprinting landscape that are facilitated with light.

Extracellular vesicles mediate improved functional outcomes in engineered cartilage produced from MSC/chondrocyte cocultures.

Several recent studies have demonstrated that coculture of chondrocytes (CHs) with bone marrow-derived mesenchymal stem cells (MSCs) improves their chondrogenesis. This implies that intercellular communication dictates fate decisions in recipient cells and/or reprograms their metabolic state to support a differentiated function. While this coculture phenomenon is compelling, the differential chondroinductivity of zonal CHs on MSC cocultures, the nature of the molecular cargo, and their transport mechanisms remains undetermined. Here, we demonstrate that juvenile CHs in coculture with adult MSCs promote functional differentiation and improved matrix production. We further demonstrate that close proximity between the two cell types is a prerequisite for this response and that the outcome of this interaction improves viability, chondrogenesis, matrix formation, and homeostasis in the recipient MSCs. Furthermore, we visualized the transfer of intracellular contents from CHs to nearby MSCs and showed that inhibition of extracellular vesicle (EV) transfer blocks the synergistic effect of coculture, identifying EVs as the primary mode of communication in these cocultures. These findings will forward the development of therapeutic agents and more effective delivery systems to promote cartilage repair.

Matching material and cellular timescales maximizes cell spreading on viscoelastic substrates.

Recent evidence has shown that, in addition to rigidity, the viscous response of the extracellular matrix (ECM) significantly affects the behavior and function of cells. However, the mechanism behind such mechanosensitivity toward viscoelasticity remains unclear. In this study, we systematically examined the dynamics of motor clutches (i.e., focal adhesions) formed between the cell and a viscoelastic substrate using analytical methods and direct Monte Carlo simulation. Interestingly, we observe that, for low ECM rigidity, maximum cell spreading is achieved at an optimal level of viscosity in which the substrate relaxation time falls between the timescale for clutch binding and its characteristic binding lifetime. That is, viscosity serves to stiffen soft substrates on a timescale faster than the clutch off-rate, which enhances cell-ECM adhesion and cell spreading. On the other hand, for substrates that are stiff, our model predicts that viscosity will not influence cell spreading, since the bound clutches are saturated by the elevated stiffness. The model was tested and validated using experimental measurements on three different material systems and explained the different observed effects of viscosity on each substrate. By capturing the mechanism by which substrate viscoelasticity affects cell spreading across a wide range of material parameters, our analytical model provides a useful tool for designing biomaterials that optimize cellular adhesion and mechanosensing.

Therapeutic Efficacy of Cryopreserved, Allogeneic Extracellular Vesicles for Treatment of Acute Myocardial Infarction.

Extracellular vesicles (EV) that are derived from endothelial progenitor cells (EPC) have been determined to be a novel therapy for acute myocardial infarction, with a promise for immediate "off-the-shelf" delivery. Early experience suggests delivery of EVs from allogeneic sources is safe. Yet, clinical translation of this therapy requires assurances of both EV stability following cryopreservation and absence of an adverse immunologic response to EVs from allogeneic donors. Thus, more bioactivity studies on allogeneic EVs after cold storage are necessary to establish quality standards for its widespread clinical use. Thus, in this study, we aimed to demonstrate the safety and efficacy in delivering cryopreserved EVs in allogeneic recipients as a therapy for acute myocardial infarction.In this present study, we have analyzed the cardioprotective effects of allogeneic EPC-derived EVs after storage at -80°C for 2 months, using a shear-thinning gel (STG) as an in vivo delivery vehicle. EV size, proteome, and nucleic acid cargo were observed to remain steady through extended cryopreservation via nanoparticle tracking analysis, mass spectrometry, and nanodrop analysis, respectively. Fresh and previously frozen EVs in STG were delivered intramyocardially in a rat model of myocardial infarction (MI), with both showing improvements in contractility, angiogenesis, and scar thickness in comparison to phosphate-buffered saline (PBS) and STG controls at 4 weeks post-MI. Pathologic analyses and flow cytometry revealed minimal inflammatory and immune upregulation upon exposure of tissue to EVs pooled from allogeneic donor cells.Allogeneic EPC-EVs have been known to elicit minimal immune activity and retain therapeutic efficacy after at least 2 months of cryopreservation in a post-MI model.

Metabolic Labeling to Probe the Spatiotemporal Accumulation of Matrix at the Chondrocyte-Hydrogel Interface.

Hydrogels are engineered with biochemical and biophysical signals to recreate aspects of the native microenvironment and to control cellular functions such as differentiation and matrix deposition. This deposited matrix accumulates within the pericellular space and likely affects the interactions between encapsulated cells and the engineered hydrogel; however, there has been little work to study the spatiotemporal evolution of matrix at this interface. To address this, metabolic labeling is employed to visualize the temporal and spatial positioning of nascent proteins and proteoglycans deposited by chondrocytes. Within covalently crosslinked hyaluronic acid hydrogels, chondrocytes deposit nascent proteins and proteoglycans in the pericellular space within 1 d after encapsulation. The accumulation of this matrix, as measured by an increase in matrix thickness during culture, depends on the initial hydrogel crosslink density with decreased thicknesses for more crosslinked hydrogels. Encapsulated fluorescent beads are used to monitor the hydrogel location and indicate that the emerging nascent matrix physically displaces the hydrogel from the cell membrane with extended culture. These findings suggest that secreted matrix increasingly masks the presentation of engineered hydrogel cues and may have implications for the design of hydrogels in tissue engineering and regenerative medicine.

Local nascent protein deposition and remodelling guide mesenchymal stromal cell mechanosensing and fate in three-dimensional hydrogels.

Hydrogels serve as valuable tools for studying cell-extracellular matrix interactions in three-dimensional environments that recapitulate aspects of native extracellular matrix. However, the impact of early protein deposition on cell behaviour within hydrogels has largely been overlooked. Using a bio-orthogonal labelling technique, we visualized nascent proteins within a day of culture across a range of hydrogels. In two engineered hydrogels of interest in three-dimensional mechanobiology studies-proteolytically degradable covalently crosslinked hyaluronic acid and dynamic viscoelastic hyaluronic acid hydrogels-mesenchymal stromal cell spreading, YAP/TAZ nuclear translocation and osteogenic differentiation were observed with culture. However, inhibition of cellular adhesion to nascent proteins or reduction in nascent protein remodelling reduced mesenchymal stromal cell spreading and nuclear translocation of YAP/TAZ, resulting in a shift towards adipogenic differentiation. Our findings emphasize the role of nascent proteins in the cellular perception of engineered materials and have implications for in vitro cell signalling studies and application to tissue repair.

Multiscale model predicts increasing focal adhesion size with decreasing stiffness in fibrous matrices.

We describe a multiscale model that incorporates force-dependent mechanical plasticity induced by interfiber cross-link breakage and stiffness-dependent cellular contractility to predict focal adhesion (FA) growth and mechanosensing in fibrous extracellular matrices (ECMs). The model predicts that FA size depends on both the stiffness of ECM and the density of ligands available to form adhesions. Although these two quantities are independent in commonly used hydrogels, contractile cells break cross-links in soft fibrous matrices leading to recruitment of fibers, which increases the ligand density in the vicinity of cells. Consequently, although the size of focal adhesions increases with ECM stiffness in nonfibrous and elastic hydrogels, plasticity of fibrous networks leads to a departure from the well-described positive correlation between stiffness and FA size. We predict a phase diagram that describes nonmonotonic behavior of FA in the space spanned by ECM stiffness and recruitment index, which describes the ability of cells to break cross-links and recruit fibers. The predicted decrease in FA size with increasing ECM stiffness is in excellent agreement with recent observations of cell spreading on electrospun fiber networks with tunable cross-link strengths and mechanics. Our model provides a framework to analyze cell mechanosensing in nonlinear and inelastic ECMs.

A practical guide to hydrogels for cell culture.

There is growing appreciation of the role that the extracellular environment plays in regulating cell behavior. Mechanical, structural, and compositional cues, either alone or in concert, can drastically alter cell function. Biomaterials, and particularly hydrogels, have been developed and implemented to present defined subsets of these cues for investigating countless cellular processes as a means of understanding morphogenesis, aging, and disease. Although most scientists concede that standard cell culture materials (tissue culture plastic and glass) do a poor job of recapitulating native cellular milieus, there is currently a knowledge barrier for many researchers in regard to the application of hydrogels for cell culture. Here, we introduce hydrogels to those who may be unfamiliar with procedures to culture and study cells with these systems, with a particular focus on commercially available hydrogels.

Delivery of a matrix metalloproteinase-responsive hydrogel releasing TIMP-3 after myocardial infarction: effects on left ventricular remodeling.

Although improvements in timing and approach for early reperfusion with acute coronary syndromes have occurred, myocardial injury culminating in a myocardial infarction (MI) remains a common event. Although a multifactorial process, an imbalance between the induction of proteolytic pathways, such as matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of metalloproteinase (TIMPs), has been shown to contribute to this process. In the present study, a full-length TIMP-3 recombinant protein (rTIMP-3) was encapsulated in a specifically formulated hyaluronic acid (HA)-based hydrogel that contained MMP-cleavable peptide cross-links, which influenced the rate of rTIMP-3 release from the HA gel. The effects of localized delivery of this MMP-sensitive HA gel (HAMMPS) alone and containing rTIMP-3 (HAMMPS/rTIMP-3) were examined in terms of the natural history of post-MI remodeling. Pigs were randomized to one of the following three different groups: MI and saline injection (MI/saline group, 100-µl injection at nine injection sites, n = 7), MI and HAMMPS injection (MI/HAMMPS group; 100-µl injection at nine injection sites, n = 7), and MI and HAMMPS/rTIMP-3 injection (MI/HAMMPS/rTIMP-3 group; 20-µg/100-µl injection at nine injection sites, n = 7). Left ventricular (LV) echocardiography was serially performed up to 28 days post-MI. LV dilation, as measured by end-diastolic volume, and the degree of MI wall thinning were reduced by ~50% in the HAMMPS/rTIMP-3 group ( P < 0.05). Furthermore, indexes of heart failure progression post-MI, such as LV filling pressures and left atrial size, were also attenuated to the greatest degree in the HAMMPS/rTIMP-3 group. At 28 days post-MI, HAMMPS/rTIMP-3 caused a relative reduction in the transcriptional profile for myofibroblasts as well as profibrotic pathways, which was confirmed by subsequent histochemistry. In conclusion, these findings suggest that localized delivery of a MMP-sensitive biomaterial that releases a recombinant TIMP holds promise as a means to interrupt adverse post-MI remodeling. NEW & NOTEWORTHY The present study targeted a myocardial matrix proteolytic system, matrix metalloproteinases (MMPs), through the use of a recombinant tissue inhibitor of MMPs incorporated into a MMP-sensitive hydrogel, which was regionally injected using a large animal model of myocardial infarction. Left ventricular geometry and function and indexes of myocardial remodeling were improved with this approach and support the advancement of localized therapeutic strategies that specifically target the myocardial matrix.

Reversible Control of Network Properties in Azobenzene-Containing Hyaluronic Acid-Based Hydrogels.

Biomimetic hydrogels fabricated from biologically derived polymers, such as hyaluronic acid (HA), are useful for numerous biomedical applications. Due to the dynamic nature of biological processes, it is of great interest to synthesize hydrogels with dynamically tunable network properties where various functions (e.g., cargo delivery, mechanical signaling) can be changed over time. Among the various stimuli developed to control hydrogel properties, light stands out for its exquisite spatiotemporal control; however, most light-based chemistries are unidirectional in their ability to manipulate network changes. Here, we report a strategy to reversibly modulate HA hydrogel properties with light, using supramolecular cross-links formed via azobenzene bound to ß-cyclodextrin. Upon isomerization with 365 nm or 400-500 nm light, the binding affinity between azobenzene and ß-cyclodextrin changed and altered the network connectivity. The hydrogel mechanical properties depended on both the azobenzene modification and isomeric state (lower for cis state), with up to a 60% change in storage modulus with light exposure. Furthermore, the release of a fluorescently labeled protein was accelerated with light exposure under conditions that were cytocompatible to encapsulated cells. These results indicate that the developed hydrogels may be suitable for applications in which temporal regulation of material properties is important, such as drug delivery or mechanobiology studies.

Ruthenium-Crosslinked Hydrogels with Rapid, Visible-Light Degradation.

Incorporation of photoresponsive molecules within soft materials can provide spatiotemporal control over bulk properties and address challenges in targeted delivery and mechanical variability. However, the kinetics of in situ photochemical reactions are often slow and typically employ ultraviolet wavelengths. Here, we present a novel photoactive crosslinker Ru(bipyridine)2 (3-pyridinaldehyde)2 (RuAldehyde), which was reacted with hydrazide-functionalized hyaluronic acid to form hydrogels capable of encapsulating protein cargo. Visible light irradiation (400-500 nm) initiated rapid ligand exchange on the ruthenium center, which degraded the hydrogel within seconds to minutes, depending on gel thickness. An exemplar enzyme cargo, TEM1 ß-lactamase, was loaded into and photoreleased from the Ru-hydrogel. To expand their applications, Ru-hydrogels were also processed into microgels using a microfluidic platform.