Stiffness anisotropy coordinates supracellular contractility driving long-range myotube-ECM alignment.

The ability of cells to organize into tissues with proper structure and function requires the effective coordination of proliferation, migration, polarization, and differentiation across length scales. Skeletal muscle is innately anisotropic; however, few biomaterials can emulate mechanical anisotropy to determine its influence on tissue patterning without introducing confounding topography. Here, we demonstrate that substrate stiffness anisotropy coordinates contractility-driven collective cellular dynamics resulting in C2C12 myotube alignment over millimeter-scale distances. When cultured on mechanically anisotropic liquid crystalline polymer networks (LCNs) lacking topography, C2C12 myoblasts collectively polarize in the stiffest direction. Cellular coordination is amplified through reciprocal cell-ECM dynamics that emerge during fusion, driving global myotube-ECM ordering. Conversely, myotube alignment was restricted to small local domains with no directional preference on mechanically isotropic LCNs of the same chemical formulation. These findings provide valuable insights for designing biomaterials that mimic anisotropic microenvironments and underscore the importance of stiffness anisotropy in orchestrating tissue morphogenesis.

Engineering a Hydrazone and Triazole Crosslinked Hydrogel for Extrusion-Based Printing and Cell Delivery.

Covalent adaptable crosslinks, such as the alkyl-hydrazone, endow hydrogels with unique viscoelastic properties applicable to cell delivery and bioink systems. However, the alkyl-hydrazone crosslink lacks stability in biologically relevant environments. Furthermore, when formed with biopolymers such as hyaluronic acid (HA), low molecular weight polymers (<60 kDa), or low polymer content (<2 wt%) hydrogels are typically employed as entanglements reduce injectability. Here, a high molecular weight (>60 kDa) HA alkyl-hydrazone crosslinked hydrogel is modified with benzaldehyde-poly(ethylene glycol)3-azide to incorporate azide functional groups. By reacting azide-modified HA with a multi-arm poly(ethylene glycol) (PEG) functionalized with bicyclononyne, stabilizing triazole bonds are formed through strain-promoted azide-alkyne cycloaddition (SPAAC). Increasing the fraction of triazole bonds within the hydrogel network from 0% to 12% SPAAC substantially increases stability. The slow gelation kinetics of the SPAAC reaction in the 12% SPAAC hydrogel enables transient self-healing properties and a similar extrusion force as the 0% SPAAC hydrogel. Methyl-PEG4-hydrazide is then introduced to further slowdown network evolution, which temporarily lowers the extrusion force, improves printability, and increases post-extrusion mesenchymal stem cell viability and function in the 12% SPAAC hydrogel. This work demonstrates improved stability and temporal injectability of high molecular weight HA-PEG hydrogels for extrusion-based printing and cell delivery.

Photothermal Actuation of Thick 3D-Printed Liquid Crystalline Elastomer Nanocomposites.

Liquid crystalline elastomers (LCEs) are stimuli-responsive materials that transduce an input energy into a mechanical response. LCE composites prepared with photothermal agents, such as nanoinclusions, are a means to realize wireless, remote, and local control of deformation with light. Amongst photothermal agents, gold nanorods (AuNRs) are highly efficient converters when the irradiation wavelength matches the longitudinal surface plasmon resonance (LSPR) of the AuNRs. However, AuNR aggregation broadens the LSPR which also reduces photothermal efficiency. Here, the surface chemistry of AuNRs is engineered via a well-controlled two-step ligand exchange with a monofunctional poly(ethylene glycol) (PEG) thiol that greatly improves the dispersion of AuNRs in LCEs. Accordingly, LCE-AuNR nanocomposites with very low PEG-AuNR content (0.01 wt%) prepared by 3D printing are shown to be highly efficient photothermal actuators with rapid response (>60% strain s-1) upon irradiation with near-infrared (NIR; 808 nm) light. Because of the excellent dispersion of PEG-AuNR within the LCE, unabsorbed NIR light transmits through the nanocomposites and can actuate a series of samples. Further, the dispersion also allows for the optical deformation of millimeter-thick 3D printed structures without sacrificing actuation speed. The realization of well-dispersed nanoinclusions to maximize the stimulus-response of LCEs can benefit functional implementation in soft robotics or medical devices.

Facile Physicochemical Reprogramming of PEG-Dithiolane Microgels.

Granular biomaterials have found widespread applications in tissue engineering, in part because of their inherent porosity, tunable properties, injectability, and 3D printability. However, the assembly of granular hydrogels typically relies on spherical microparticles and more complex particle geometries have been limited in scope, often requiring templating of individual microgels by microfluidics or in-mold polymerization. Here, we use dithiolane-functionalized synthetic macromolecules to fabricate photopolymerized microgels via batch emulsion, and then harness the dynamic disulfide crosslinks to rearrange the network. Through unconfined compression between parallel plates in the presence of photoinitiated radicals, we transform the isotropic microgels are transformed into disks. Characterizing this process, we find that the areas of the microgel surface in contact with the compressive plates are flattened while the curvature of the uncompressed microgel boundaries increases. When cultured with C2C12 myoblasts, cells localize to regions of higher curvature on the disk-shaped microgel surfaces. This altered localization affects cell-driven construction of large supraparticle scaffold assemblies, with spherical particles assembling without specific junction structure while disk microgels assemble preferentially on their curved surfaces. These results represent a unique spatiotemporal process for rapid reprocessing of microgels into anisotropic shapes, providing new opportunities to study shape-driven mechanobiological cues during and after granular hydrogel assembly.

Stiffness anisotropy coordinates supracellular contractility driving long-range myotube-ECM alignment.

In skeletal muscle tissue, injury-related changes in stiffness activate muscle stem cells through mechanosensitive signaling pathways. Functional muscle tissue regeneration also requires the effective coordination of myoblast proliferation, migration, polarization, differentiation, and fusion across multiple length scales. Here, we demonstrate that substrate stiffness anisotropy coordinates contractility-driven collective cellular dynamics resulting in C2C12 myotube alignment over millimeter-scale distances. When cultured on mechanically anisotropic liquid crystalline polymer networks (LCNs) lacking topographic features that could confer contact guidance, C2C12 myoblasts collectively polarize in the stiffest direction of the substrate. Cellular coordination is amplified through reciprocal cell-ECM dynamics that emerge during fusion, driving global myotube-ECM ordering. Conversely, myotube alignment was restricted to small local domains with no directional preference on mechanically isotropic LCNs of same chemical formulation. These findings reveal a role for stiffness anisotropy in coordinating emergent collective cellular dynamics, with implications for understanding skeletal muscle tissue development and regeneration.

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.

Photoinduced Dithiolane Crosslinking for Multiresponsive Dynamic Hydrogels.

While many hydrogels are elastic networks crosslinked by covalent bonds, viscoelastic hydrogels with adaptable crosslinks are increasingly being developed to better recapitulate time and position-dependent processes found in many tissues. In this work, 1,2-dithiolanes are presented as dynamic covalent photocrosslinkers of hydrogels, resulting in disulfide bonds throughout the hydrogel that respond to multiple stimuli. Using lipoic acid as a model dithiolane, disulfide crosslinks are formed under physiological conditions, enabling cell encapsulation via an initiator-free light-induced dithiolane ring-opening photopolymerization. The resulting hydrogels allow for multiple photoinduced dynamic responses including stress relaxation, stiffening, softening, and network functionalization using a single chemistry, which can be supplemented by permanent reaction with alkenes to further control network properties and connectivity using irreversible thioether crosslinks. Moreover, complementary photochemical approaches are used to achieve rapid and complete sample degradation via radical scission and post-gelation network stiffening when irradiated in the presence of reactive gel precursor. The results herein demonstrate the versatility of this material chemistry to study and direct 2D and 3D cell-material interactions. This work highlights dithiolane-based hydrogel photocrosslinking as a robust method for generating adaptable hydrogels with a range of biologically relevant mechanical and chemical properties that are varied on demand.

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.

Myeloid cell subsets that express latency-associated peptide promote cancer growth by modulating T cells.

Myeloid suppressor cells promote tumor growth by a variety of mechanisms which are not fully characterized. We identified myeloid cells (MCs) expressing the latency-associated peptide (LAP) of TGF-ß on their surface and LAPHi MCs that stimulate Foxp3+ Tregs while inhibiting effector T cell proliferation and function. Blocking TGF-ß inhibits the tolerogenic ability of LAPHi MCs. Furthermore, adoptive transfer of LAPHi MCs promotes Treg accumulation and tumor growth in vivo. Conversely, anti-LAP antibody, which reduces LAPHi MCs, slows cancer progression. Single-cell RNA-Seq analysis on tumor-derived immune cells revealed LAPHi dominated cell subsets with distinct immunosuppressive signatures, including those with high levels of MHCII and PD-L1 genes. Analogous to mice, LAP is expressed on myeloid suppressor cells in humans, and these cells are increased in glioma patients. Thus, our results identify a previously unknown function by which LAPHi MCs promote tumor growth and offer therapeutic intervention to target these cells in cancer.