Chandipura viral glycoprotein (CNV-G) promotes Gectosome generation and enables delivery of intracellular therapeutics.

Overexpression of vesicular stomatitis virus G protein (VSV-G) elevates the secretion of EVs known as gectosomes, which contain VSV-G. Such vesicles can be engineered to deliver therapeutic macromolecules. We investigated viral glycoproteins from several viruses for their potential in gectosome production and intracellular cargo delivery. Expression of the viral glycoprotein (viral glycoprotein from the Chandipura virus [CNV-G]) from the human neurotropic pathogen Chandipura virus in 293T cells significantly augments the production of CNV-G-containing gectosomes. In comparison with VSV-G gectosomes, CNV-G gectosomes exhibit heightened selectivity toward specific cell types, including primary cells and tumor cell lines. Consistent with the differential tropism between CNV-G and VSV-G gectosomes, cellular entry of CNV-G gectosome is independent of the Low-density lipoprotein receptor, which is essential for VSV-G entry, and shows varying sensitivity to pharmacological modulators. CNV-G gectosomes efficiently deliver diverse intracellular cargos for genomic modification or responses to stimuli in vitro and in the brain of mice in vivo utilizing a split GFP and chemical-induced dimerization system. Pharmacokinetics and biodistribution analyses support CNV-G gectosomes as a versatile platform for delivering macromolecular therapeutics intracellularly.

Network modeling predicts personalized gene expression and drug responses in valve myofibroblasts cultured with patient sera.

Aortic valve stenosis (AVS) patients experience pathogenic valve leaflet stiffening due to excessive extracellular matrix (ECM) remodeling. Numerous microenvironmental cues influence pathogenic expression of ECM remodeling genes in tissue-resident valvular myofibroblasts, and the regulation of complex myofibroblast signaling networks depends on patient-specific extracellular factors. Here, we combined a manually curated myofibroblast signaling network with a data-driven transcription factor network to predict patient-specific myofibroblast gene expression signatures and drug responses. Using transcriptomic data from myofibroblasts cultured with AVS patient sera, we produced a large-scale, logic-gated differential equation model in which 11 biochemical and biomechanical signals were transduced via a network of 334 signaling and transcription reactions to accurately predict the expression of 27 fibrosis-related genes. Correlations were found between personalized model-predicted gene expression and AVS patient echocardiography data, suggesting links between fibrosis-related signaling and patient-specific AVS severity. Further, global network perturbation analyses revealed signaling molecules with the most influence over network-wide activity, including endothelin 1 (ET1), interleukin 6 (IL6), and transforming growth factor ß (TGFß), along with downstream mediators c-Jun N-terminal kinase (JNK), signal transducer and activator of transcription (STAT), and reactive oxygen species (ROS). Lastly, we performed virtual drug screening to identify patient-specific drug responses, which were experimentally validated via fibrotic gene expression measurements in valvular interstitial cells cultured with AVS patient sera and treated with or without bosentan-a clinically approved ET1 receptor inhibitor. In sum, our work advances the ability of computational approaches to provide a mechanistic basis for clinical decisions including patient stratification and personalized drug screening.

Substrate stiffness modulates cardiac fibroblast activation, senescence, and proinflammatory secretory phenotype.

In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These phenotypic switches confound the interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we used poly(ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic hearts, respectively. Low hydrogel stiffness converted activated CFs into a quiescent state with a reduced abundance of α-smooth muscle actin (α-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated ß-galactosidase (SA-ß-Gal) activity and increased expression of p16 and p21, which are antiproliferative markers of senescence. Using dynamically stiffening hydrogels with phototunable cross-linking capabilities, we demonstrate that premature, substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low-stiffness hydrogels, with a reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-κB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment in which they reside.NEW & NOTEWORTHY Our findings highlight the advantages and pitfalls associated with culturing cardiac fibroblasts on hydrogels of varying stiffness. The findings also define stiffness-dependent signaling and transcriptional networks in cardiac fibroblasts.

Photo-expansion microscopy enables super-resolution imaging of cells embedded in 3D hydrogels.

Hydrogels are extensively used as tunable, biomimetic three-dimensional cell culture matrices, but optically deep, high-resolution images are often difficult to obtain, limiting nanoscale quantification of cell-matrix interactions and outside-in signalling. Here we present photopolymerized hydrogels for expansion microscopy that enable optical clearance and tunable ×4.6-6.7 homogeneous expansion of not only monolayer cell cultures and tissue sections, but cells embedded within hydrogels. The photopolymerized hydrogels for expansion microscopy formulation relies on a rapid photoinitiated thiol/acrylate mixed-mode polymerization that is not inhibited by oxygen and decouples monomer diffusion from polymerization, which is particularly beneficial when expanding cells embedded within hydrogels. Using this technology, we visualize human mesenchymal stem cells and their interactions with nascently deposited proteins at <120 nm resolution when cultured in proteolytically degradable synthetic polyethylene glycol hydrogels. Results support the notion that focal adhesion maturation requires cellular fibronectin deposition; nuclear deformation precedes cellular spreading; and human mesenchymal stem cells display cell-surface metalloproteinases for matrix remodelling.

Genes That Escape X Chromosome Inactivation Modulate Sex Differences in Valve Myofibroblasts.

BACKGROUND: Aortic valve stenosis is a sexually dimorphic disease, with women often presenting with sustained fibrosis and men with more extensive calcification. However, the intracellular molecular mechanisms that drive these clinically important sex differences remain underexplored. METHODS: Hydrogel biomaterials were designed to recapitulate key aspects of the valve tissue microenvironment and to serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to profibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA sequencing was used to define pathways involved in driving sex-dependent activation. Interventions with small molecule inhibitors and siRNA transfections were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors. RESULTS: In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker α-smooth muscle actin compared with male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-smooth muscle actin stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than in male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase signaling as a potential driver of this sex-dependent myofibroblast activation. Furthermore, we found that genes that escape X-chromosome inactivation such as BMX and STS (encoding for Bmx nonreceptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation through Rho-associated protein kinase signaling. This finding was confirmed by treating male and female VICs with endothelin-1 and plasminogen activator inhibitor-1, factors that are secreted by endothelial cells and known to drive myofibroblast activation through Rho-associated protein kinase signaling. CONCLUSIONS: Together, in vivo and in vitro results confirm sex dependencies in myofibroblast activation pathways and implicate genes that escape X-chromosome inactivation in regulating sex differences in myofibroblast activation and subsequent aortic valve stenosis progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of aortic valve stenosis and to help guide sex-based precision therapies.

Hydrogel cultures reveal Transient Receptor Potential Vanilloid 4 regulation of myofibroblast activation and proliferation in valvular interstitial cells.

As aortic valve stenosis develops, valve tissue becomes stiffer. In response to this change in environmental mechanical stiffness, valvular interstitial cells (VICs) activate into myofibroblasts. We aimed to investigate the role of mechanosensitive calcium channel Transient Receptor Potential Vanilloid type 4 (TRPV4) in stiffness induced myofibroblast activation. We verified TRPV4 functionality in VICs using live calcium imaging during application of small molecule modulators of TRPV4 activity. We designed hydrogel biomaterials that mimic mechanical features of healthy or diseased valve tissue microenvironments, respectively, to investigate the role of TRPV4 in myofibroblast activation and proliferation. Our results show that TRPV4 regulates VIC proliferation in a microenvironment stiffness-independent manner. While there was a trend toward inhibiting myofibroblast activation on soft microenvironments during TRPV4 inhibition, we observed near complete deactivation of myofibroblasts on stiff microenvironments. We further identified Yes-activated protein (YAP) as a downstream target for TRPV4 activity on stiff microenvironments. Mechanosensitive TRPV4 channels regulate VIC myofibroblast activation, whereas proliferation regulation is independent of the microenvironmental stiffness. Collectively, the data suggests differential regulation of stiffness-induced proliferation and myofibroblast activation. Our data further suggest a regulatory role for TRPV4 regarding YAP nuclear localization. TRPV4 is an important regulator for VIC myofibroblast activation, which is linked to the initiation of valve fibrosis. Although more validation studies are necessary, we suggest TRPV4 as a promising pharmaceutical target to slow aortic valve stenosis progression.

Characterizing rheological properties and microstructure of thioester networks during degradation.

Covalent adaptable networks are designed for applications including cell and drug delivery and tissue regeneration. These applications require network degradation at physiological conditions and on a physiological timescale with microstructures that can: (1) support, protect and deliver encapsulated cells or molecules and (2) provide structure to surrounding tissue. Due to this, the evolving microstructure and rheological properties during scaffold degradation must be characterized. In this work, we characterize degradation of covalent adaptable poly(ethylene glycol) (PEG)-thioester networks with different amounts of excess thiol. Networks are formed between PEG-thiol and PEG-thioester norbornene using photopolymerization. These networks are adaptable because of a thioester exchange reaction that takes place in the presence of excess thiol. We measure degradation of PEG-thioester networks with L-cysteine using multiple particle tracking microrheology (MPT). MPT measures the Brownian motion of fluorescent probe particles embedded in a material and relates this motion to rheological properties. Using time-cure superposition (TCS), we characterize the microstructure of these networks at the gel-sol phase transition by calculating the critical relaxation exponent, n, for each network with different amounts of excess thiol. Based on the measured n values, networks formed with 0% and 50% excess thiol are tightly cross-linked and elastic in nature. While networks formed with 100% excess are similar to ideal, percolated networks, which have equal viscous and elastic components. MPT measurements during degradation of these networks also measure a non-monotonic increase in probe motility. We hypothesize that this is network rearrangement near the phase transition. We then measure macroscopic material properties including the equilibrium modulus and stress relaxation. We measure a trend in bulk network properties that agrees with the values of n. Elastic modulus and stress relaxation measurements show that networks with 50% excess thiol are more elastic compared to the other two networks. As the amount of excess thiol is increased from 0% to 50%, the networks become more elastic. Further increasing excess thiol to 100% reduces the elastically effective cross-links. We hypothesize that these properties are due to network non-idealities, resulting in networks with 50% excess thiol that are more elastic. This work characterizes dynamic rheological properties during degradation, which mimics processes that could occur during implantation. This work provides information that can be used in the future design of implantable materials enabling both the rheological properties and timescale of degradation to be specified.

Photoclick Chemistry: A Bright Idea.

At its basic conceptualization, photoclick chemistry embodies a collection of click reactions that are performed via the application of light. The emergence of this concept has had diverse impact over a broad range of chemical and biological research due to the spatiotemporal control, high selectivity, and excellent product yields afforded by the combination of light and click chemistry. While the reactions designated as "photoclick" have many important features in common, each has its own particular combination of advantages and shortcomings. A more extensive realization of the potential of this chemistry requires a broader understanding of the physical and chemical characteristics of the specific reactions. This review discusses the features of the most frequently employed photoclick reactions reported in the literature: photomediated azide-alkyne cycloadditions, other 1,3-dipolarcycloadditions, Diels-Alder and inverse electron demand Diels-Alder additions, radical alternating addition chain transfer additions, and nucleophilic additions. Applications of these reactions in a variety of chemical syntheses, materials chemistry, and biological contexts are surveyed, with particular attention paid to the respective strengths and limitations of each reaction and how that reaction benefits from its combination with light. Finally, challenges to broader employment of these reactions are discussed, along with strategies and opportunities to mitigate such obstacles.

Nuclear mechanosensing controls MSC osteogenic potential through HDAC epigenetic remodeling.

Cells sense mechanical cues from the extracellular matrix to regulate cellular behavior and maintain tissue homeostasis. The nucleus has been implicated as a key mechanosensor and can directly influence chromatin organization, epigenetic modifications, and gene expression. Dysregulation of nuclear mechanosensing has been implicated in several diseases, including bone degeneration. Here, we exploit photostiffening hydrogels to manipulate nuclear mechanosensing in human mesenchymal stem cells (hMSCs) in vitro. Results show that hMSCs respond to matrix stiffening by increasing nuclear tension and causing an increase in histone acetylation via deactivation of histone deacetylases (HDACs). This ultimately induces osteogenic fate commitment. Disrupting nuclear mechanosensing by disconnecting the nucleus from the cytoskeleton up-regulates HDACs and prevents osteogenesis. Resetting HDAC activity back to healthy levels rescues the epigenetic and osteogenic response in hMSCs with pathological nuclear mechanosensing. Notably, bone from patients with osteoarthritis displays similar defective nuclear mechanosensing. Collectively, our results reveal that nuclear mechanosensing controls hMSC osteogenic potential mediated by HDAC epigenetic remodeling and that this cellular mechanism is likely relevant to bone-related diseases.

4D Printing of Extrudable and Degradable Poly(Ethylene Glycol) Microgel Scaffolds for Multidimensional Cell Culture.

Granular synthetic hydrogels are useful bioinks for their compatibility with a variety of chemistries, affording printable, stimuli-responsive scaffolds with programmable structure and function. Additive manufacturing of microscale hydrogels, or microgels, allows for the fabrication of large cellularized constructs with percolating interstitial space, providing a platform for tissue engineering at length scales that are inaccessible by bulk encapsulation where transport of media and other biological factors are limited by scaffold density. Herein, synthetic microgels with varying degrees of degradability are prepared with diameters on the order of hundreds of microns by submerged electrospray and UV photopolymerization. Porous microgel scaffolds are assembled by particle jamming and extrusion printing, and semi-orthogonal chemical cues are utilized to tune the void fraction in printed scaffolds in a logic-gated manner. Scaffolds with different void fractions are easily cellularized post printing and microgels can be directly annealed into cell-laden structures. Finally, high-throughput direct encapsulation of cells within printable microgels is demonstrated, enabling large-scale 3D culture in a macroporous biomaterial. This approach provides unprecedented spatiotemporal control over the properties of printed microporous annealed particle scaffolds for 2.5D and 3D tissue culture.

Tumor necrosis factor-α promotes and exacerbates calcification in heart valve myofibroblast populations.

Pro-inflammatory cytokines play critical roles in regulating valvular interstitial cell (VIC) phenotypic changes that can cause heart valve fibrosis and calcification. Tumor necrosis factor alpha (TNF-α) is a cytokine known to influence VIC behavior and has been reported at high levels in calcified valves ex vivo. We sought to understand the specific effects of TNF-α on VIC phenotypes (eg, fibroblast, profibrotic activated myofibroblasts) and its link with heart valve disorders. We characterize human aortic valve tissue from patients with valve disorders and identify a high variability of fibrotic and calcific markers between tissues. These results motivated in vitro studies to explore the effects of TNF-α on defined VIC fibroblasts and profibrotic activated myofibroblasts, induced via FGF-2 and TGF-ß1 treatment. Using 3D hydrogels to culture VICs, we measure the effect of TNF-α (0.1-10 ng/mL) on key markers of fibrosis (eg, αSMA, COL1A1) and calcification (eg, RUNX2, BMP2, and calcium deposits). We observe calcification in TNF-α-treated VIC activated myofibroblasts and identify the MAPK/ERK signaling cascade as a potential pathway for TNF-α mediated calcification. Conversely, VIC fibroblasts respond to TNF-α with decreased calcification. Treatment of VIC profibrotic activated myofibroblast populations with TNF-α leads to increased calcification. Our in vitro findings correlate with findings in diseased human valves and highlight the importance of understanding the effect of cytokines and signaling pathways on specific VIC phenotypes. Finally, we reveal MAPK/ERK as a potential pathway involved in VIC-mediated matrix calcification with TNF-α treatment, suggesting this pathway as a potential pharmaceutical target for aortic valve disease.

Matters of the heart: Cellular sex differences.

Nearly all cardiovascular diseases show sexual dimorphisms in prevalence, presentation, and outcomes. Until recently, most clinical trials were carried out in males, and many animal studies either failed to identify the sex of the animals or combined data obtained from males and females. Cellular sex in the heart is relatively understudied and many studies fail to report the sex of the cells used for in vitro experiments. Moreover, in the small number of studies in which sex is reported, most of those studies use male cells. The observation that cells from males and females are inherently different is becoming increasingly clear - either due to acquired differences from hormones and other factors or due to intrinsic differences in genotype (XX or XY). Because of the likely contribution of cellular sex differences in cardiac health and disease, here, we explore differences in mammalian male and female cells in the heart, including the less-studied non-myocyte cell populations. We discuss how the heart's microenvironment impacts male and female cellular phenotypes and vice versa, including how secretory profiles are dependent on cellular sex, and how hormones contribute to sexually dimorphic phenotypes and cellular functions. Intracellular mechanisms that contribute to sex differences, including gene expression and epigenetic remodeling, are also described. Recent single-cell sequencing studies have revealed unexpected sex differences in the composition of cell types in the heart which we discuss. Finally, future recommendations for considering cellular sex differences in the design of bioengineered in vitro disease models of the heart are provided.

Secreted Factors From Proinflammatory Macrophages Promote an Osteoblast-Like Phenotype in Valvular Interstitial Cells.

OBJECTIVE: Resident valvular interstitial cells (VICs) activate to myofibroblasts during aortic valve stenosis progression, which further promotes fibrosis or even differentiate into osteoblast-like cells that can lead to calcification of valve tissue. Inflammation is a hallmark of aortic valve stenosis, so we aimed to determine proinflammatory cytokines secreted from M1 macrophages that give rise to a transient VIC phenotype that leads to calcification of valve tissue. Approach and Results: We designed hydrogel biomaterials as valve extracellular matrix mimics enabling the culture of VICs in either their quiescent fibroblast or activated myofibroblast phenotype in response to the local matrix stiffness. When VIC fibroblasts and myofibroblasts were treated with conditioned media from THP-1-derived M1 macrophages, we observed robust reduction of αSMA (alpha smooth muscle actin) expression, reduced stress fiber formation, and increased proliferation, suggesting a potent antifibrotic effect. We further identified TNF (tumor necrosis factor)-α and IL (interleukin)-1ß as 2 cytokines in M1 media that cause the observed antifibrotic effect. After 7 days of culture in M1 conditioned media, VICs began differentiating into osteoblast-like cells, as measured by increased expression of RUNX2 (runt-related transcription factor 2) and osteopontin. We also identified and validated IL-6 as a critical mediator of the observed pro-osteogenic effect. CONCLUSIONS: Proinflammatory cytokines in M1 conditioned media inhibit myofibroblast activation in VICs (eg, TNF-α and IL-1ß) and promote their osteogenic differentiation (eg, IL-6). Together, our work suggests inflammatory M1 macrophages may drive a myofibroblast-to-osteogenic intermediate VIC phenotype, which may mediate the switch from fibrosis to calcification during aortic valve stenosis progression.

Designing Microgels for Cell Culture and Controlled Assembly of Tissue Microenvironments.

Micron-sized hydrogels, termed microgels, are emerging as multifunctional platforms that can recapitulate tissue heterogeneity in engineered cell microenvironments. The microgels can function as either individual cell culture units or can be assembled into larger scaffolds. In this manner, individual microgels can be customized for single or multi-cell co-culture applications, or heterogeneous populations can be used as building blocks to create microporous assembled scaffolds that more closely mimic tissue heterogeneities. The inherent versatility of these materials allows user-defined control of the microenvironments, from the order of singly encapsulated cells to entire three-dimensional cell scaffolds. These hydrogel scaffolds are promising for moving towards personalized medicine approaches and recapitulating the multifaceted microenvironments that exist in vivo.

Spatially patterned matrix elasticity directs stem cell fate.

There is a growing appreciation for the functional role of matrix mechanics in regulating stem cell self-renewal and differentiation processes. However, it is largely unknown how subcellular, spatial mechanical variations in the local extracellular environment mediate intracellular signal transduction and direct cell fate. Here, the effect of spatial distribution, magnitude, and organization of subcellular matrix mechanical properties on human mesenchymal stem cell (hMSCs) function was investigated. Exploiting a photodegradation reaction, a hydrogel cell culture substrate was fabricated with regions of spatially varied and distinct mechanical properties, which were subsequently mapped and quantified by atomic force microscopy (AFM). The variations in the underlying matrix mechanics were found to regulate cellular adhesion and transcriptional events. Highly spread, elongated morphologies and higher Yes-associated protein (YAP) activation were observed in hMSCs seeded on hydrogels with higher concentrations of stiff regions in a dose-dependent manner. However, when the spatial organization of the mechanically stiff regions was altered from a regular to randomized pattern, lower levels of YAP activation with smaller and more rounded cell morphologies were induced in hMSCs. We infer from these results that irregular, disorganized variations in matrix mechanics, compared with regular patterns, appear to disrupt actin organization, and lead to different cell fates; this was verified by observations of lower alkaline phosphatase (ALP) activity and higher expression of CD105, a stem cell marker, in hMSCs in random versus regular patterns of mechanical properties. Collectively, this material platform has allowed innovative experiments to elucidate a novel spatial mechanical dosing mechanism that correlates to both the magnitude and organization of spatial stiffness.

PEG-Anthracene Hydrogels as an On-Demand Stiffening Matrix To Study Mechanobiology.

There is a growing interest in materials that can dynamically change their properties in the presence of cells to study mechanobiology. Herein, we exploit the 365 nm light mediated [4+4] photodimerization of anthracene groups to develop cytocompatible PEG-based hydrogels with tailorable initial moduli that can be further stiffened. A hydrogel formulation that can stiffen from 10 to 50 kPa, corresponding to the stiffness of a healthy and fibrotic heart, respectively, was prepared. This system was used to monitor the stiffness-dependent localization of NFAT, a downstream target of intracellular calcium signaling using a reporter in live cardiac fibroblasts (CFbs). NFAT translocates to the nucleus of CFbs on stiffening hydrogels within 6 h, whereas it remains cytoplasmic when the CFbs are cultured on either 10 or 50 kPa static hydrogels. This finding demonstrates how dynamic changes in the mechanical properties of a material can reveal the kinetics of mechanoresponsive cell signaling pathways that may otherwise be missed in cells cultured on static substrates.

Secondary Photocrosslinking of Click Hydrogels To Probe Myoblast Mechanotransduction in Three Dimensions.

Muscle cells sense the mechanical properties of their microenvironment, and these properties can change in response to injury or disease. Hydrogels with dynamic material properties can be used to study the effect of such varying mechanical signals. Here, we report the ability of azadibenzocyclooctyne to undergo a cytocompatible, photoinitiated crosslinking reaction. This reaction is exploited as a strategy for on-demand stiffening of three-dimensional cell scaffolds formed through an initial strain-promoted azide-alkyne cycloaddition. Myoblasts encapsulated in these networks respond to increased matrix stiffness through decreased cell spreading and nuclear localization of Yes-associated protein 1 (YAP). However, when the photocrosslinking reaction is delayed to allow cell spreading, elongated myoblasts display increased YAP nuclear localization.

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.

Thermal Stabilization of Biologics with Photoresponsive Hydrogels.

Modern medicine, biological research, and clinical diagnostics depend on the reliable supply and storage of complex biomolecules. However, biomolecules are inherently susceptible to thermal stress and the global distribution of value-added biologics, including vaccines, biotherapeutics, and Research Use Only (RUO) proteins, requires an integrated cold chain from point of manufacture to point of use. To mitigate reliance on the cold chain, formulations have been engineered to protect biologics from thermal stress, including materials-based strategies that impart thermal stability via direct encapsulation of the molecule. While direct encapsulation has demonstrated pronounced stabilization of proteins and complex biological fluids, no solution offers thermal stability while enabling facile and on-demand release from the encapsulating material, a critical feature for broad use. Here we show that direct encapsulation within synthetic, photoresponsive hydrogels protected biologics from thermal stress and afforded user-defined release at the point of use. The poly(ethylene glycol) (PEG)-based hydrogel was formed via a bioorthogonal, click reaction in the presence of biologics without impact on biologic activity. Cleavage of the installed photolabile moiety enabled subsequent dissolution of the network with light and release of the encapsulated biologic. Hydrogel encapsulation improved stability for encapsulated enzymes commonly used in molecular biology (ß-galactosidase, alkaline phosphatase, and T4 DNA ligase) following thermal stress. ß-galactosidase and alkaline phosphatase were stabilized for 4 weeks at temperatures up to 60 °C, and for 60 min at 85 °C for alkaline phosphatase. T4 DNA ligase, which loses activity rapidly at moderately elevated temperatures, was protected during thermal stress of 40 °C for 24 h and 60 °C for 30 min. These data demonstrate a general method to employ reversible polymer networks as robust excipients for thermal stability of complex biologics during storage and shipment that additionally enable on-demand release of active molecules at the point of use.

Measuring dynamic cell-material interactions and remodeling during 3D human mesenchymal stem cell migration in hydrogels.

Biomaterials that mimic aspects of the extracellular matrix by presenting a 3D microenvironment that cells can locally degrade and remodel are finding increased applications as wound-healing matrices, tissue engineering scaffolds, and even substrates for stem cell expansion. In vivo, cells do not simply reside in a static microenvironment, but instead, they dynamically reengineer their surroundings. For example, cells secrete proteases that degrade extracellular components, attach to the matrix through adhesive sites, and can exert traction forces on the local matrix, causing its spatial reorganization. Although biomaterials scaffolds provide initially well-defined microenvironments for 3D culture of cells, less is known about the changes that occur over time, especially local matrix remodeling that can play an integral role in directing cell behavior. Here, we use microrheology as a quantitative tool to characterize dynamic cellular remodeling of peptide-functionalized poly(ethylene glycol) (PEG) hydrogels that degrade in response to cell-secreted matrix metalloproteinases (MMPs). This technique allows measurement of spatial changes in material properties during migration of encapsulated cells and has a sensitivity that identifies regions where cells simply adhere to the matrix, as well as the extent of local cell remodeling of the material through MMP-mediated degradation. Collectively, these microrheological measurements provide insight into microscopic, cellular manipulation of the pericellular region that gives rise to macroscopic tracks created in scaffolds by migrating cells. This quantitative and predictable information should benefit the design of improved biomaterial scaffolds for medically relevant applications.