Open-Top Patterned Hydrogel-Laden 3D Glioma Cell Cultures for Creation of Dynamic Chemotactic Gradients to Direct Cell Migration.

The laminar flow profiles in microfluidic systems coupled to rapid diffusion at flow streamlines have been widely utilized to create well-controlled chemical gradients in cell cultures for spatially directing cell migration. However, within hydrogel-based closed microfluidic systems of limited depth (≤0.1 mm), the biomechanical cues for the cell culture are dominated by cell interactions with channel surfaces rather than with the hydrogel microenvironment. Also, leaching of poly(dimethylsiloxane) (PDMS) constituents in closed systems and the adsorption of small molecules to PDMS alter chemotactic profiles. To address these limitations, we present the patterning and integration of a PDMS-free open fluidic system, wherein the cell-laden hydrogel directly adjoins longitudinal channels that are designed to create chemotactic gradients across the 3D culture width, while maintaining uniformity across its ∼1 mm depth to enhance cell-biomaterial interactions. This hydrogel-based open fluidic system is assessed for its ability to direct migration of U87 glioma cells using a hybrid hydrogel that includes hyaluronic acid (HA) to mimic the brain tumor microenvironment and gelatin methacrylate (GelMA) to offer the adhesion motifs for promoting cell migration. Chemotactic gradients to induce cell migration across the hydrogel width are assessed using the chemokine CXCL12, and its inhibition by AMD3100 is validated. This open-top hydrogel-based fluidic system to deliver chemoattractant cues over square-centimeter-scale areas and millimeter-scale depths can potentially serve as a robust screening platform to assess emerging glioma models and chemotherapeutic agents to eradicate them.

Modular Multiwell Viscoelastic Hydrogel Platform for Two- and Three-Dimensional Cell Culture Applications.

Hydrogels have gained significant popularity as model platforms to study reciprocal interactions between cells and their microenvironment. While hydrogel tools to probe many characteristics of the extracellular space have been developed, fabrication approaches remain challenging and time-consuming, limiting multiplexing or widespread adoption. Thus, we have developed a modular fabrication approach to generate distinct hydrogel microenvironments within the same 96-well plate for increased throughput of fabrication as well as integration with existing high-throughput assay technologies. This approach enables in situ hydrogel mechanical characterization and is used to generate both elastic and viscoelastic hydrogels across a range of stiffnesses. Additionally, this fabrication method enabled a 3-fold reduction in polymer and up to an 8-fold reduction in fabrication time required per hydrogel replicate. The feasibility of this platform for two-dimensional (2D) cell culture applications was demonstrated by measuring both population-level and single-cell-level metrics via microplate reader and high-content imaging. Finally, a 96-well hydrogel array was utilized for three-dimensional (3D) cell culture, demonstrating the ability to support high cell viability. Together, this work demonstrates a versatile and easily adaptable fabrication approach that can support the ever-expanding tool kit of hydrogel technologies for cell culture applications.

Serial Passaging Affects Stromal Cell Mechanosensitivity on Hyaluronic Acid Hydrogels.

There is a tremendous interest in developing hydrogels as tunable in vitro cell culture platforms to study cell response to mechanical cues in a controlled manner. However, little is known about how common cell culture techniques, such as serial expansion on tissue culture plastic, affect subsequent cell behavior when cultured on hydrogels. In this work, a methacrylated hyaluronic acid hydrogel platform is leveraged to study stromal cell mechanotransduction. Hydrogels are first formed through thiol-Michael addition to model normal soft tissue (e.g., lung) stiffness (E ≈ 1 kPa). Secondary cross-linking via radical photopolymerization of unconsumed methacrylates allows matching of early- (E ≈ 6 kPa) and late-stage fibrotic tissue (E ≈ 50 kPa). Early passage (P1) human bone marrow mesenchymal stromal cells (hMSCs) display increased spreading, myocardin-related transcription factor-A (MRTF-A) nuclear localization, and focal adhesion size with increasing hydrogel stiffness. However, late passage (P5) hMSCs show reduced sensitivity to substrate mechanics with lower MRTF-A nuclear translocation and smaller focal adhesions on stiffer hydrogels compared to early passage hMSCs. Similar trends are observed in an immortalized human lung fibroblast line. Overall, this work highlights the implications of standard cell culture practices on investigating cell response to mechanical signals using in vitro hydrogel models.

Modular multiwell viscoelastic hydrogel platform for two- and three-dimensional cell culture applications.

Hydrogels have gained significant popularity as model platforms to study the reciprocal interactions between cells and their microenvironment. While hydrogel tools to probe many characteristics of the extracellular space have been developed, fabrication approaches remain challenging and time-consuming, limiting multiplexing or widespread adoption. Thus, we have developed a modular fabrication approach to generate distinct hydrogel microenvironments within 96-well plates for increased throughput of fabrication as well as integration with existing high-throughput assay technologies. This approach enables in situ hydrogel mechanical characterization and was used to generate both elastic and viscoelastic hydrogels across a range of stiffnesses. Additionally, this fabrication method enabled a 3-fold reduction in polymer and up to an 8-fold reduction in fabrication time required per hydrogel replicate. The feasibility of this platform for cell culture applications was demonstrated by measuring both population-level and single cell-level metrics via microplate reader and high-content imaging. Finally, the 96-well hydrogel array was utilized for 3D cell culture, demonstrating the ability to support high cell viability. Together, this work demonstrates a versatile and easily adoptable fabrication approach that can support the ever-expanding tool kit of hydrogel technologies for cell culture applications.

Supramolecular Fibrous Hydrogel Augmentation of Uterosacral Ligament Suspension for Treatment of Pelvic Organ Prolapse.

Uterosacral ligament suspension (USLS) is a common surgical treatment for pelvic organ prolapse (POP). However, the relatively high failure rate of up to 40% underscores a strong clinical need for complementary treatment strategies, such as biomaterial augmentation. Herein, the first hydrogel biomaterial augmentation of USLS in a recently established rat model is described using an injectable fibrous hydrogel composite. Supramolecularly-assembled hyaluronic acid (HA) hydrogel nanofibers encapsulated in a matrix metalloproteinase (MMP)-degradable HA hydrogel create an injectable scaffold showing excellent biocompatibility and hemocompatibility. The hydrogel can be successfully delivered and localized to the suture sites of the USLS procedure, where it gradually degrades over six weeks. In situ mechanical testing 24 weeks post-operative in the multiparous USLS rat model shows the ultimate load (load at failure) to be 1.70 ± 0.36 N for the intact uterosacral ligament (USL), 0.89 ± 0.28 N for the USLS repair, and 1.37 ± 0.31 N for the USLS + hydrogel (USLS+H) repair (n = 8). These results indicate that the hydrogel composite significantly improves load required for tissue failure compared to the standard USLS, even after the hydrogel degrades, and that this hydrogel-based approach can potentially reduce the high failure rate associated with USLS procedures.

Hydrogel mechanics regulate fibroblast DNA methylation and chromatin condensation.

Cellular mechanotransduction plays a central role in fibroblast activation during fibrotic disease progression, leading to increased tissue stiffness and reduced organ function. While the role of epigenetics in disease mechanotransduction has begun to be appreciated, little is known about how substrate mechanics, particularly the timing of mechanical inputs, regulate epigenetic changes such as DNA methylation and chromatin reorganization during fibroblast activation. In this work, we engineered a hyaluronic acid hydrogel platform with independently tunable stiffness and viscoelasticity to model normal (storage modulus, G' ∼ 0.5 kPa, loss modulus, G'' ∼ 0.05 kPa) to increasingly fibrotic (G' ∼ 2.5 and 8 kPa, G'' ∼ 0.05 kPa) lung mechanics. Human lung fibroblasts exhibited increased spreading and nuclear localization of myocardin-related transcription factor-A (MRTF-A) with increasing substrate stiffness within 1 day, with these trends holding steady for longer cultures. However, fibroblasts displayed time-dependent changes in global DNA methylation and chromatin organization. Fibroblasts initially displayed increased DNA methylation and chromatin decondensation on stiffer hydrogels, but both of these measures decreased with longer culture times. To investigate how culture time affected the responsiveness of fibroblast nuclear remodeling to mechanical signals, we engineered hydrogels amenable to in situ secondary crosslinking, enabling a transition from a compliant substrate mimicking normal tissue to a stiffer substrate resembling fibrotic tissue. When stiffening was initiated after only 1 day of culture, fibroblasts rapidly responded and displayed increased DNA methylation and chromatin decondensation, similar to fibroblasts on static stiffer hydrogels. Conversely, when fibroblasts experienced later stiffening at day 7, they showed no changes in DNA methylation and chromatin condensation, suggesting the induction of a persistent fibroblast phenotype. These results highlight the time-dependent nuclear changes associated with fibroblast activation in response to dynamic mechanical perturbations and may provide mechanisms to target for controlling fibroblast activation.

Serial passaging affects stromal cell mechanosensitivity on hyaluronic acid hydrogels.

There is tremendous interest in developing hydrogels as tunable in vitro cell culture platforms to study cell response to mechanical cues in a controlled manner. However, little is known about how common cell culture techniques, such as serial expansion on tissue culture plastic, affect subsequent cell behavior when cultured on hydrogels. In this work we leverage a methacrylated hyaluronic acid hydrogel platform to study stromal cell mechanotransduction. Hydrogels are first formed through thiol-Michael addition to model normal soft tissue (e.g., lung) stiffness ( E ~ 1 kPa). Secondary crosslinking via radical photopolymerization of unconsumed methacrylates allows matching of early- ( E ~ 6 kPa) and late-stage fibrotic tissue ( E ~ 50 kPa). Early passage (P1) primary human mesenchymal stromal cells (hMSCs) display increased spreading, myocardin-related transcription factor-A (MRTF-A) nuclear localization, and focal adhesion size with increasing hydrogel stiffness. However, late passage (P5) hMSCs show reduced sensitivity to substrate mechanics with lower MRTF-A nuclear translocation and smaller focal adhesions on stiffer hydrogels compared to early passage hMSCs. Similar trends are observed in an immortalized human lung fibroblast line. Overall, this work highlights the implications of standard cell culture practices on investigating cell response to mechanical signals using in vitro hydrogel models.

Development of a Uterosacral Ligament Suspension Rat Model.

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately 10%-20% of women undergo pelvic floor repair surgery to treat prolapse in the United States. PFD cases result in an overall $26.3 billion annual cost in the United States alone. This multifactorial condition has a negative impact on the quality of life and yet the treatment options have only dwindled in the recent past. One common surgical option is uterosacral ligament suspension (USLS), which is typically performed by affixing the vaginal vault to the uterosacral ligament in the pelvis. This repair has a lower incidence of complications compared to those with mesh augmentation, but is notable for a relatively high failure rate of up to 40%. Considering the lack of standard animal models to study pelvic floor dysfunction, there is an urgent clinical need for innovation in this field with a focus on developing cost-effective and accessible animal models. In this manuscript, we describe a rat model of USLS involving a complete hysterectomy followed by fixation of the remaining vaginal vault to the uterosacral ligament. The goal of this model is to mimic the procedure performed on women to be able to use the model to then investigate reparative strategies that improve the mechanical integrity of the ligament attachment. Importantly, we also describe the development of an in situ tensile testing procedure to characterize interface integrity at chosen time points following surgical intervention. Overall, this model will be a useful tool for future studies that investigate treatment options for POP repair via USLS.

Controlling scaffold conductivity and pore size to direct myogenic cell alignment and differentiation.

Skeletal muscle's combination of three-dimensional (3D) anisotropy and electrical excitability is critical for enabling normal movement. We previously developed a 3D aligned collagen scaffold incorporating conductive polypyrrole (PPy) particles to recapitulate these key muscle properties and showed that the scaffold facilitated enhanced myotube maturation compared with nonconductive controls. To further optimize this scaffold design, this work assessed the influence of conductive polymer incorporation and scaffold pore architecture on myogenic cell behavior. Conductive PPy and poly(3,4-ethylenedioxythiophene) (PEDOT) particles were synthesized and mixed into a suspension of type I collagen and chondroitin sulfate prior to directional freeze-drying to produce anisotropic scaffolds. Energy dispersive spectroscopy revealed homogenous distribution of conductive PEDOT particles throughout the scaffolds that resulted in a threefold increase in electrical conductivity while supporting similar myoblast metabolic activity compared to nonconductive scaffolds. Control of freezing temperature enabled fabrication of PEDOT-doped scaffolds with a range of pore diameters from 98 to 238 µm. Myoblasts conformed to the anisotropic contact guidance cues independent of pore size to display longitudinal cytoskeletal alignment. The increased specific surface area of the smaller pore scaffolds helped rescue the initial decrease in myoblast metabolic activity observed in larger pore conductive scaffolds while also promoting modestly increased expression levels of the myogenic marker myosin heavy chain (MHC) and gene expression of myoblast determination protein (MyoD). However, cell infiltration to the center of the scaffolds was marginally reduced compared with larger pore variants. Together these data underscore the potential of aligned and PEDOT-doped collagen scaffolds for promoting myogenic cell organization and differentiation.

Photoreactive Hydrogel Stiffness Influences Volumetric Muscle Loss Repair.

Volumetric muscle loss (VML) injuries are characterized by permanent loss of muscle mass, structure, and function. Hydrogel biomaterials provide an attractive platform for skeletal muscle tissue engineering due to the ability to easily modulate their biophysical and biochemical properties to match a range of tissue characteristics. In this work, we successfully developed a mechanically tunable hyaluronic acid (HA) hydrogel system to investigate the influence of hydrogel stiffness on VML repair. HA was functionalized with photoreactive norbornene groups to create hydrogel networks that rapidly crosslink through thiol-ene click chemistry with tailored mechanics. Mechanical properties were controlled by modulating the amount of matrix metalloproteinase-degradable peptide crosslinker to produce hydrogels with increasing elastic moduli of 1.1 ± 0.002, 3.0 ± 0.002, and 10.6 ± 0.006 kPa, mimicking a relevant range of developing and mature muscle stiffnesses. Functional muscle recovery was assessed following implantation of the HA hydrogels by in situ photopolymerization into rat latissimus dorsi (LD) VML defects at 12 and 24 weeks postinjury. After 12 weeks, muscles treated with medium stiffness (3.0 kPa) hydrogels produced maximum isometric forces most similar to contralateral healthy LD muscles. This trend persisted at 24 weeks postinjury, suggestive of sustained functional recovery. Histological analysis revealed a significantly larger zone of regeneration with more de novo muscle fibers following implantation of medium stiffness hydrogels in VML-injured muscles compared to other experimental groups. Lower (low and medium) stiffness hydrogels also appeared to attenuate the chronic inflammatory response characteristic of VML injuries, displaying similar levels of macrophage infiltration and polarization to healthy muscle. Together these findings illustrate the importance of hydrogel mechanical properties in supporting functional repair of VML injuries. Impact statement This report defines the role hydrogel mechanical properties play in the repair of volumetric muscle loss (VML) injuries. We show that an intermediate hydrogel stiffness (3 kPa) more compliant than adult muscle tissue facilitated improved and sustained regenerative outcomes up to 24 weeks postinjury in a rat latissimus dorsi model of VML. Muscles treated with 3 kPa hydrogels showed enhanced myogenesis and attenuation of the chronic inflammatory response characteristic of VML injuries. These results should help guide the future design of hydrogels for skeletal muscle tissue engineering and regeneration.

The Combined Influence of Viscoelastic and Adhesive Cues on Fibroblast Spreading and Focal Adhesion Organization.

INTRODUCTION: Tissue fibrosis is characterized by progressive extracellular matrix (ECM) stiffening and loss of viscoelasticity that ultimately impairs organ functionality. Cells bind to the ECM through integrins, where αv integrin engagement in particular has been correlated with fibroblast activation into contractile myofibroblasts that drive fibrosis progression. There is a significant unmet need for in vitro hydrogel systems that deconstruct the complexity of native tissues to better understand the individual and combined effects of stiffness, viscoelasticity, and integrin engagement on fibroblast behavior. METHODS: We developed hyaluronic acid hydrogels with independently tunable cell-instructive properties (stiffness, viscoelasticity, ligand presentation) to address this challenge. Hydrogels with mechanics matching normal or fibrotic lung tissue were synthesized using a combination of covalent crosslinks and supramolecular interactions to tune viscoelasticity. Cell adhesion was mediated through incorporation of either RGD peptide or engineered fibronectin fragments promoting preferential integrin engagement via αvß3 or α5ß1. RESULTS: On fibrosis-mimicking stiff elastic hydrogels, preferential αvß3 engagement promoted increased spreading, actin stress fiber organization, and focal adhesion maturation as indicated by paxillin organization in human lung fibroblasts. In contrast, preferential α5ß1 binding suppressed these metrics. Viscoelasticity, mimicking the mechanics of healthy tissue, largely curtailed fibroblast spreading and focal adhesion organization independent of adhesive ligand type, highlighting its role in reducing fibroblast-activating behaviors. CONCLUSIONS: Together, these results provide new insights into how mechanical and adhesive cues collectively guide disease-relevant cell behaviors. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00672-1.

Aligned and electrically conductive 3D collagen scaffolds for skeletal muscle tissue engineering.

Skeletal muscle is characterized by its three-dimensional (3D) anisotropic architecture composed of highly aligned and electrically-excitable muscle fibers that enable normal movement. Biomaterial-based tissue engineering approaches to repair skeletal muscle are limited due to difficulties combining 3D structural alignment (to guide cell/matrix organization) and electrical conductivity (to enable electrically-excitable myotube assembly and maturation). In this work we successfully produced aligned and electrically conductive 3D collagen scaffolds using a freeze-drying approach. Conductive polypyrrole (PPy) nanoparticles were synthesized and directly mixed into a suspension of type I collagen and chondroitin sulfate followed by directional lyophilization. Scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), and confocal microscopy showed that directional solidification resulted in scaffolds with longitudinally aligned pores with homogeneously-distributed PPy content. Chronopotentiometry verified that PPy incorporation resulted in a five-fold increase in conductivity compared to non-PPy-containing collagen scaffolds without detrimentally affecting myoblast metabolic activity. Furthermore, the aligned scaffold microstructure provided contact guidance cues that directed myoblast growth and organization. Incorporation of PPy also promoted enhanced myotube formation and maturation as measured by myosin heavy chain (MHC) expression and number of nuclei per myotube. Together these data suggest that aligned and electrically conductive 3D collagen scaffolds could be useful for skeletal muscle tissue engineering.

Guest-Host Supramolecular Assembly of Injectable Hydrogel Nanofibers for Cell Encapsulation.

The fibrous architecture of the extracellular matrix (ECM) is recognized as an integral regulator of cell function. However, there is an unmet need to develop mechanically robust biomaterials mimicking nanofibrous tissue topography that are also injectable to enable minimally invasive delivery. In this study, we have developed a fibrous hydrogel composed of supramolecularly assembled hyaluronic acid (HA) nanofibers that exhibits mechanical integrity, shear-thinning behavior, rapid self-healing, and cytocompatibility. HA was modified with methacrylates to permit fiber photo-cross-linking following electrospinning and either "guest" adamantane or "host" ß-cyclodextrin groups to guide supramolecular fibrous hydrogel assembly. Analysis of fibrous hydrogel rheological properties showed that the mixed guest-host fibrous hydrogel was more mechanically robust (6.6 ± 2.0 kPa, storage modulus (G')) than unmixed guest hydrogel fibers (1.0 ± 0.1 kPa) or host hydrogel fibers (1.1 ± 0.1 kPa) separately. The reversible nature of the guest-host supramolecular interactions also allowed for shear-thinning and self-healing behavior as demonstrated by cyclic deformation testing. Human mesenchymal stromal cells (hMSCs) encapsulated in fibrous hydrogels demonstrated satisfactory viability following injection and after 7 days of culture (>85%). Encapsulated hMSCs were more spread and elongated when cultured in viscoelastic guest-host hydrogels compared to nonfibrous elastic controls, with hMSCs also showing significantly decreased circularity in fibrous guest-host hydrogels compared to nonfibrous guest-host hydrogels. Together, these data highlight the potential of this injectable fibrous hydrogel platform for cell and tissue engineering applications requiring minimally invasive delivery.

Fabrication approaches for high-throughput and biomimetic disease modeling.

There is often a tradeoff between in vitro disease modeling platforms that capture pathophysiologic complexity and those that are amenable to high-throughput fabrication and analysis. However, this divide is closing through the application of a handful of fabrication approaches-parallel fabrication, automation, and flow-driven assembly-to design sophisticated cellular and biomaterial systems. The purpose of this review is to highlight methods for the fabrication of high-throughput biomaterial-based platforms and showcase examples that demonstrate their utility over a range of throughput and complexity. We conclude with a discussion of future considerations for the continued development of higher-throughput in vitro platforms that capture the appropriate level of biological complexity for the desired application. STATEMENT OF SIGNIFICANCE: There is a pressing need for new biomedical tools to study and understand disease. These platforms should mimic the complex properties of the body while also permitting investigation of many combinations of cells, extracellular cues, and/or therapeutics in high-throughput. This review summarizes emerging strategies to fabricate biomimetic disease models that bridge the gap between complex tissue-mimicking microenvironments and high-throughput screens for personalized medicine.

Click-functionalized hydrogel design for mechanobiology investigations.

The advancement of click-functionalized hydrogels in recent years has coincided with rapid growth in the fields of mechanobiology, tissue engineering, and regenerative medicine. Click chemistries represent a group of reactions that possess high reactivity and specificity, are cytocompatible, and generally proceed under physiologic conditions. Most notably, the high level of tunability afforded by these reactions enables the design of user-controlled and tissue-mimicking hydrogels in which the influence of important physical and biochemical cues on normal and aberrant cellular behaviors can be independently assessed. Several critical tissue properties, including stiffness, viscoelasticity, and biomolecule presentation, are known to regulate cell mechanobiology in the context of development, wound repair, and disease. However, many questions still remain about how the individual and combined effects of these instructive properties regulate the cellular and molecular mechanisms governing physiologic and pathologic processes. In this review, we discuss several click chemistries that have been adopted to design dynamic and instructive hydrogels for mechanobiology investigations. We also chart a path forward for how click hydrogels can help reveal important insights about complex tissue microenvironments.

3D Hyaluronic Acid Hydrogels for Modeling Oligodendrocyte Progenitor Cell Behavior as a Function of Matrix Stiffness.

The lack of regenerative solutions for demyelination within the central nervous system motivates the development of strategies to expand and drive the bioactivity of the cells, including oligodendrocyte progenitor cells (OPCs), that ultimately give rise to myelination. In this work, we introduce a 3D hyaluronic acid (HA) hydrogel system to study the effects of microenvironmental mechanical properties on the behavior of OPCs. We tuned the stiffness of the hydrogels to match the brain tissue (storage modulus 200-2000 Pa) and studied the effects of stiffness on metabolic activity, proliferation, and cell morphology of OPCs over a 7 day period. Although hydrogel mesh size decreased with increasing stiffness, all hydrogel groups facilitated OPC proliferation and mitochondrial metabolic activity to similar degrees. However, OPCs in the two lower stiffness hydrogel groups (170 ± 42 and 794 ± 203 Pa) supported greater adenosine triphosphate levels per cell than the highest stiffness hydrogels (2179 ± 127 Pa). Lower stiffness hydrogels also supported higher levels of cell viability and larger cell spheroid formation compared to the highest stiffness hydrogels. Together, these data suggest that 3D HA hydrogels are a useful platform for studying OPC behavior and that OPC growth/metabolic health may be favored in lower stiffness microenvironments mimicking brain tissue mechanics.

Spatiotemporal Control of Viscoelasticity in Phototunable Hyaluronic Acid Hydrogels.

Viscoelasticity has emerged as a critical regulator of cell behavior. However, there is an unmet need to develop biomaterials where viscoelasticity can be spatiotemporally controlled to mimic the dynamic and heterogeneous nature of tissue microenvironments. Toward this objective, we developed a modular hyaluronic acid hydrogel combining light-mediated covalent and supramolecular cross-linking to afford spatiotemporal control of network viscoelastic properties. Covalently cross-linked elastic hydrogels or viscoelastic hydrogels combining covalent and supramolecular interactions were fabricated to match healthy and fibrotic liver mechanics. LX-2 human hepatic stellate cells cultured on viscoelastic hydrogels displayed reductions in spreading, actin stress fiber organization, and myocardin-related transcription factor A (MRTF-A) nuclear localization compared to cells on elastic hydrogels. We further demonstrated the dynamic capabilities of our hydrogel system through photo-mediated secondary incorporation of either covalent or supramolecular cross-links to modulate viscoelastic properties. We used photopatterning to create hydrogels with well-controlled patterned regions of stiff elastic mechanics representing fibrotic tissue nodules surrounded by regions of soft viscoelastic hydrogel mimicking healthy tissue. Cells responded to the local mechanics of the patterned substrates with increased spreading in fibrosis-mimicking regions. Together, this work represents an important step forward toward the creation of hydrogel models with spatiotemporal control of both stiffness and viscoelastic cell-instructive cues.

Engineering biomaterial microenvironments to promote myelination in the central nervous system.

Promoting remyelination and/or minimizing demyelination are key therapeutic strategies under investigation for diseases and injuries like multiple sclerosis (MS), spinal cord injury, stroke, and virus-induced encephalopathy. Myelination is essential for efficacious neuronal signaling. This myelination process is originated by oligodendrocyte progenitor cells (OPCs) in the central nervous system (CNS). Resident OPCs are capable of both proliferation and differentiation, and also migration to demyelinated injury sites. OPCs can then engage with these unmyelinated or demyelinated axons and differentiate into myelin-forming oligodendrocytes (OLs). However this process is frequently incomplete and often does not occur at all. Biomaterial strategies can now be used to guide OPC and OL development with the goal of regenerating healthy myelin sheaths in formerly damaged CNS tissue. Growth and neurotrophic factors delivered from such materials can promote proliferation of OPCs or differentiation into OLs. While cell transplantation techniques have been used to replace damaged cells in wound sites, they have also resulted in poor transplant cell viability, uncontrollable differentiation, and poor integration into the host. Biomaterial scaffolds made from extracellular matrix (ECM) mimics that are naturally or synthetically derived can improve transplanted cell survival, support both transplanted and endogenous cell populations, and direct their fate. In particular, stiffness and degradability of these scaffolds are two parameters that can influence the fate of OPCs and OLs. The future outlook for biomaterials research includes 3D in vitro models of myelination / remyelination / demyelination to better mimic and study these processes. These models should provide simple relationships of myelination to microenvironmental biophysical and biochemical properties to inform improved therapeutic approaches.

Temperature-Dependent Complex Coacervation of Engineered Elastin-like Polypeptide and Hyaluronic Acid Polyelectrolytes.

Coacervates have enormous potential due to their diverse functional properties supporting a wide number of applications in personal care products, pharmaceuticals, and food processing. Normally, separation of coacervate phases is induced by changes in pH, ionic strength, and/or polyelectrolyte concentration. This study investigates the microphase separation and coacervate complex formation of two natural polyelectrolytes, elastin-like polypeptides (ELPs) and hyaluronic acid (HA), as simple models for biological coacervates. These complex coacervates are formed over a wide range of stoichiometric molar charge ratios without the presence of salt or changes in pH and are primarily induced by changes in temperature. Unlike pure ELP solutions, the ELP/HA coacervates result in well-formed spherical particles after the temperature-induced phase transition. We also note that the formation of these complex coacervates is reversible with low hysteresis. We have demonstrated via fluorescent imaging and dynamic light scattering that high positive/negative charge ratios at elevated temperatures produced 400-600 nm particles with relatively low polydispersity indices (PDIs) of ∼0.1. Furthermore, dynamic light scattering, fluorescence microscopy, and optical microscopy revealed that the ratio of the two polyions strongly influenced the size and structure of these ELP/HA complex coacervates. Finally, we showed that the ELP/HA coacervates were able to sequester the hydrophobic fluorescent molecule pyrene, highlighting their potential for use as delivery vehicles for hydrophobic payloads.

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.