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.

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.

Quantifying stiffness and forces of tumor colonies and embryos using a magnetic microrobot.

Stiffness and forces are two fundamental quantities essential to living cells and tissues. However, it has been a challenge to quantify both 3D traction forces and stiffness (or modulus) using the same probe in vivo. Here, we describe an approach that overcomes this challenge by creating a magnetic microrobot probe with controllable functionality. Biocompatible ferromagnetic cobalt-platinum microcrosses were fabricated, and each microcross (about 30 micrometers) was trapped inside an arginine-glycine-apartic acid-conjugated stiff poly(ethylene glycol) (PEG) round microgel (about 50 micrometers) using a microfluidic device. The stiff magnetic microrobot was seeded inside a cell colony and acted as a stiffness probe by rigidly rotating in response to an oscillatory magnetic field. Then, brief episodes of ultraviolet light exposure were applied to dynamically photodegrade and soften the fluorescent nanoparticle-embedded PEG microgel, whose deformation and 3D traction forces were quantified. Using the microrobot probe, we show that malignant tumor-repopulating cell colonies altered their modulus but not traction forces in response to different 3D substrate elasticities. Stiffness and 3D traction forces were measured, and both normal and shear traction force oscillations were observed in zebrafish embryos from blastula to gastrula. Mouse embryos generated larger tensile and compressive traction force oscillations than shear traction force oscillations during blastocyst. The microrobot probe with controllable functionality via magnetic fields could potentially be useful for studying the mechanoregulation of cells, tissues, and embryos.

Inflammatory serum factors from aortic valve stenosis patients modulate sex differences in valvular myofibroblast activation and osteoblast-like differentiation.

Aortic valve stenosis (AVS) is a sexually dimorphic cardiovascular disease that is driven by fibrosis and calcification of the aortic valve leaflets. Circulating inflammatory factors present in serum from AVS patients contribute to sex differences in valve fibro-calcification by driving the activation of valvular interstitial cells (VICs) to myofibroblasts and/or osteoblast-like cells. However, the molecular mechanisms by which inflammatory factors contribute to sex-specific valve fibro-calcification remain largely unknown. In this study, we identified inflammatory factors present in serum samples from AVS patients that regulate sex-specific myofibroblast activation and osteoblast-like differentiation. After correlating serum proteomic datasets with clinical and in vitro myofibroblast datasets, we identified annexin A2 and cystatin C as candidate inflammatory factors that correlate with both AVS patient severity and myofibroblast activation measurements in vitro. Validation experiments utilizing hydrogel biomaterials as cell culture platforms that mimic the valve extracellular matrix confirmed that annexin A2 and cystatin C promote sex-specific VIC activation to myofibroblasts via p38 MAPK signaling. Additionally, annexin A2 and cystatin C increase osteoblast-like differentiation primarily in male VICs. Our results implicate serum inflammatory factors as potential AVS biomarkers that also contribute to sexually dimorphic AVS progression by driving VIC myofibroblast activation and/or osteoblast-like differentiation. Collectively, the results herein further our overall understanding as to how biological sex may impact inflammation-driven AVS and may lead to the development of sex-specific drug treatment strategies.

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.

Stress Relaxation and Composition of Hydrazone-Crosslinked Hybrid Biopolymer-Synthetic Hydrogels Determine Spreading and Secretory Properties of MSCs.

The extracellular matrix plays a critical role in mechanosensing and thereby influences the secretory properties of bone-marrow-derived mesenchymal stem/stromal cells (MSCs). As a result, interest has grown in the development of biomaterials with tunable properties for the expansion and delivery of MSCs that are used in cell-based therapies. Herein, stress-relaxing hydrogels are synthesized as hybrid networks containing both biopolymer and synthetic macromer components. Hyaluronic acid is functionalized with either aldehyde or hydrazide groups to form covalent adaptable hydrazone networks, which are stabilized by poly(ethylene glycol) functionalized with bicyclononyne and heterobifunctional small molecule crosslinkers containing azide and benzaldehyde moieties. Tuning the composition of these gels allows for controlled variation in the characteristic timescale for stress relaxation and the amount of stress relaxed. Over this compositional space, MSCs are observed to spread in formulations with higher degrees of adaptability, with aspect ratios of 1.60 ± 0.18, and YAP nuclear:cytoplasm ratios of 6.5 ± 1.3. Finally, a maximum MSC pericellular protein thickness of 1.45 ± 0.38 µm occurred in highly stress-relaxing gels, compared to 1.05 ± 0.25 µm in non-adaptable controls. Collectively, this study contributes a new understanding of the role of compositionally defined stress relaxation on MSCs mechanosensing and secretion.

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.

Granular PEG hydrogels mediate osteoporotic MSC clustering via N-cadherin influencing the pro-resorptive bias of their secretory profile.

Postmenopausal osteoporosis results from a pro-resorptive bone environment, which decreases bone mineral density causing increased fracture risk. Bone marrow derived mesenchymal stem/stromal cells (MSCs) secrete factors involved in bone homeostasis, but osteoporosis mediated changes to their secretions remain understudied. Herein, we examined the secretome of MSCs isolated from ovariectomized rats (OVX rMSCs), a model of post-menopausal osteoporosis, as a function of cell-cell interactions. Specifically, we controlled clustering of OVX and SHAM rMSCs by assembling them in granular hydrogels synthesized from poly(ethylene glycol) microgels with average diameters of ∼10, 100, and 200 µm. We directed both the sizes of rMSC clusters (single cells to ∼30 cells/cluster) and the percentages of cells within clusters (∼20-90%) by controlling the scaffold pore dimensions. Large clusters of OVX rMSCs had a pro-resorptive secretory profile, with increased concentrations of Activin A, CXCL1, CX3CL1, MCP-1, TIMP-1, and TNF-ɑ, compared to SHAM rMSCs. As this pro-resorptive bias was only observed in large cell clusters, we characterized the expression of several cadherins, mediators of cell-cell contacts. N-cadherin expression was elevated (∼4-fold) in OVX relative to SHAM rMSCs, in both cell clusters and single cells. Finally, TIMP-1 and MCP-1 secretion was only decreased in large cell clusters of OVX rMSCs when N-cadherin interactions were blocked, highlighting the dependence of OVX rMSC secretion of pro-resorptive cytokines on N-cadherin mediated cell-cell contacts. Further elucidation of the N-cadherin mediated osteoporotic MSC secretome may have implications for developing therapies for postmenopausal osteoporosis. STATEMENT OF SIGNIFICANCE: Postmenopausal osteoporosis is a prevalent bone disorder that affects tens of millions of women worldwide. This disease is characterized by severe bone loss resulting from a pro-resorptive bone marrow environment, where the rates of bone resorption outpace the rates of bone deposition. The paracrine factors secreted by bone marrow MSCs can influence cell types responsible for bone homeostasis, but the osteoporosis-mediated changes to MSC secretory properties remains understudied. In this study, we used PEG-based porous granular scaffolds to study the influence of cell clustering on the secretory properties of osteoporotic MSCs. We observed increased secretion of several pro-resorptive factors by osteoporotic MSCs in large clusters. Further, we explored the dependence of this altered secretion profile on N-cadherin mediated cell-cell contacts.

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.

Impact of Collagen Triple Helix Structure on Melanoma Cell Invadopodia Formation and Matrix Degradation upon BRAF Inhibitor Treatment.

A collagen-rich tumor microenvironment (TME) is associated with worse outcomes in cancer patients and contributes to drug resistance in many cancer types. In melanoma, stiff and fibrillar collagen-abundant tissue is observed after failure of therapeutic treatments with BRAF inhibitors. Increased collagen in the TME can affect properties of the extracellular matrix (ECM), including stiffness, adhesiveness, and interaction of integrins with triple helix forming nanostructures. Decoupling these biochemical and biophysical properties of the ECM can lead to a better understanding of how each of these individual properties affect melanoma cancer behavior and drug efficacy. In addition, as drug treatment can induce cancer cell phenotypic switch, cancer cell responsiveness to the TME can be dynamically changed during therapeutic treatments. To investigate cancer cell phenotype changes and the role of the cancer TME, poly(ethylene glycol) (PEG) hydrogels functionalized with collagen mimetic peptides (CMPs) is utilized, or an interpenetrating network (IPN) of type І collagen within the PEG system to culture various melanoma cell lines in the presence or absence of Vemurafenib (PLX4032) drug treatment is prepared. Additionally, the potential of using CMP functionalized PEG hydrogels, which can provide better tunability is explored, to replace the existing invadopodia assay platform based on fluorescent gelatin.

Recent advances in 3D models of tumor invasion.

This review presents recent advances in the design of in vitro cancer models to study tumor cell migration, metastasis, and invasion in three-dimensions (3D). These cancer models are divided into two categories based on the biophysiological processes and structures simulated, namely (i) spheroid invasion models or (ii) vascularization models. Some recent advances to spheroid invasion models include new methods to make them amenable to high-throughput settings. In vascularization models, cancer cell extravasation, intravasation, and angiogenesis have been emulated. Finally, 3D bioprinting and microfluidic technologies are allowing researchers to recapitulate some of the complex architectural and microenvironmental changes that can drive cancer cells migration from the extracellular matrix and basement membrane to blood vessels.

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.

Engineering the MSC Secretome: A Hydrogel Focused Approach.

The therapeutic benefits of exogenously delivered mesenchymal stromal/stem cells (MSCs) have been largely attributed to their secretory properties. However, clinical translation of MSC-based therapies is hindered due to loss of MSC regenerative properties during large-scale expansion and low survival/retention post-delivery. These limitations might be overcome by designing hydrogel culture platforms to modulate the MSC microenvironment. Hydrogel systems could be engineered to i) promote MSC proliferation and maintain regenerative properties (i.e., stemness and secretion) during ex vivo expansion, ii) improve MSC survival, retention, and engraftment in vivo, and/or iii) direct the MSC secretory profile using tailored biochemical and biophysical cues. Herein, it is reviewed how hydrogel material properties (i.e., matrix modulus, viscoelasticity, dimensionality, cell adhesion, and porosity) influence MSC secretion, mediated through cell-matrix and cell-cell interactions. In addition, it is highlighted how biochemical cues (i.e., small molecules, peptides, and proteins) can improve and direct the MSC secretory profile. Last, the authors' perspective is provided on future work toward the understanding of how microenvironmental cues influence the MSC secretome, and designing the next generation of biomaterials, with optimized biophysical and biochemical cues, to direct the MSC secretory profile for improved clinical translation outcomes.

Collagen networks within 3D PEG hydrogels support valvular interstitial cell matrix mineralization.

Enzymatically degradable hydrogels were designed for the 3D culture of valvular interstitial cells (VICs), and through the incorporation of various functionalities, we aimed to investigate the role of the tissue microenvironment in promoting the osteogenic properties of VICs and matrix mineralization. Specifically, porcine VICs were encapsulated in a poly(ethylene glycol) hydrogel crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinker (KCGPQG↓IWGQCK) and formed via a thiol-ene photoclick reaction in the presence or absence of collagen type I to promote matrix mineralization. VIC-laden hydrogels were treated with osteogenic medium for up to 15 days, and the osteogenic response was characterized by the expression of RUNX2 as an early marker of an osteoblast-like phenotype, osteocalcin (OCN) as a marker of a mature osteoblast-like phenotype, and vimentin (VIM) as a marker of the fibroblast phenotype. In addition, matrix mineralization was characterized histologically with Von Kossa stain for calcium phosphate. Osteogenic response was further characterized biochemically with calcium assays, and physically via optical density measurements. When the osteogenic medium was supplemented with calcium chloride, OCN expression was upregulated and mineralization was discernable at 12 days of culture. Finally, this platform was used to screen various drug therapeutics that were assessed for their efficacy in preventing mineralization using optical density as a higher throughput readout. Collectively, these results suggest that matrix composition has a key role in supporting mineralization deposition within diseased valve tissue.

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.

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.

Viscoelasticity of hydrazone crosslinked poly(ethylene glycol) hydrogels directs chondrocyte morphology during mechanical deformation.

Chondrocyte deformation influences disease progression and tissue regeneration in load-bearing joints. In this work, we found that viscoelasticity of dynamic covalent crosslinks temporally modulates the biophysical transmission of physiologically relevant compressive strains to encapsulated chondrocytes. Chondrocytes in viscoelastic alky-hydrazone hydrogels demonstrated (91.4 ± 4.5%) recovery of native rounded morphologies during mechanical deformation, whereas primarily elastic benzyl-hydrazone hydrogels significantly limited morphological recovery (21.2 ± 1.4%).

Bioorthogonal click chemistries enable simultaneous spatial patterning of multiple proteins to probe synergistic protein effects on fibroblast function.

Three biorthogonal click reactions, a photoinitiated thiol-yne reaction, an azide-alkyne cycloaddition, and a methyltetrazine-transcyclooctene Diels Alder, were used to independently control the presentation of several bioactive proteins to valvular interstitial cells (VICs) in hydrogel scaffolds. Tethered fibroblast growth factor (FGF-2) was found to suppress myofibroblast activation (from 48 ± 7% to 17 ± 6%) and promote proliferation (from 10 ± 2% to 54 ± 3%) at a concentration of 10 ng/mL. In the presence of the pro-fibrotic cytokine transforming growth factor-beta (TGF-ß1), FGF-2 could protect the VIC fibroblast phenotype, even at much higher concentrations of TGF-ß1 than that of FGF-2. With respect to the fibrocalcific VIC phenotype, TGF-ß1 and bone-morphogenic protein-2 (BMP-2) were found to synergistically promote calcific nodule formation (a five-fold increase in nodules compared to TGF-ß1 or BMP-2 alone). Exploiting the orthogonal click reactions, FGF-2, TGF-ß1 and BMP-2 combinations were patterned into distinct regions on a hydrogel to control VIC activation and nodule formation. Cellular crosstalk between separate regions of the same scaffold was affected by the size of each region as well as the interfacial area between different regions. Collectively, these results demonstrate the versatility and robustness of a photoinitiated thiol-yne reaction to template pendant functionalities that allow for the bioconjugation of multiple proteins. This approach maintains protein bioactivity, providing an in vitro platform capable of achieving a better understanding of the complex mechanisms involved in tissue fibrosis.

Phototunable Viscoelasticity in Hydrogels Through Thioester Exchange.

Mechanical cues are delivered to resident cells by the extracellular matrix and play an important role in directing cell processes, ranging from embryonic development and cancer metastasis to stem cell differentiation. Recently, cellular responses to viscoelastic and elastic mechanical cues have been studied; however, questions remain as to how cells identify and transduce these cues differently. We present a synthetic cell culture substrate with viscoelastic properties based on thioester exchange chemistry that can be modulated in situ with the photoinitiated thiol-ene 'click' reaction. With this method, stress relaxation in thioester hydrogels with an average relaxation time of 740,000 s can be switched off in the presence of cells without change to the elastic modulus. NIH 3T3 fibroblasts, cultured for 48 h on viscoelastic compared to elastic thioester substrates, displayed increased cell area (660-560 µm2) and increased nuclear to cytoplasmic YAP/TAZ ratios (2.4 to 2.2) when cultured on elastic compared to viscoelastic hydrogels, respectively. Next, when the viscoelasticity was switched off after 24 h, the fibroblasts responded to this change and exhibited an average cell area of 540 µm2, and nuclear to cytoplasmic YAP/TAZ ratio of 2.1, approaching that of the control elastic gels. Phototunable viscoelastic thioester hydrogels provide a tunable materials system to investigate time-dependent cellular responses to viscoelasticity and should prove useful for understanding the dynamics of mechanoresponsive cellular pathways.

Porous bio-click microgel scaffolds control hMSC interactions and promote their secretory properties.

Human mesenchymal stem/stromal cells (hMSCs) are known to secrete numerous cytokines that signal to endogenous cells and aid in tissue regeneration. However, the role that biomaterial scaffolds can play in controlling hMSC secretory properties has been less explored. Here, microgels were co-assembled with hMSCs using three different microgel populations, with large (190 ± 100 µm), medium (110 ± 60 µm), and small (13 ± 6 µm) diameters, to create distinct porous environments that influenced hMSC clustering. Cells embedded in large diameter microgel networks resided in large clusters (~40 cells), compared to small clusters (~6 cells) observed in networks using medium diameter microgels and primarily single cells in small diameter microgel networks. Using a cytokine microarray, an overall increase in secretion was observed in scaffolds that promoted hMSC clustering, with over 60% of the measured cytokines most elevated in the large diameter microgel networks. N-cadherin interactions were identified as partially mediating these differences, so the microgel formulations were modified with an N-cadherin epitope, HAVDI, to mimic cell-cell interactions. Results revealed increased secretory properties for hMSCs in HAVDI functionalized scaffolds, even the non-clustered cells in small diameter microgel networks. Together, these results demonstrate opportunities for microgel-based scaffold systems for hMSC delivery and tailoring of porous materials properties to promote their secretory potential.