Effects of Cell-Adhesive Ligand Presentation on Pentapeptide Supramolecular Assembly and Gelation: Simulations and Experiments.

The extracellular matrix (ECM) is a complex, hierarchical material containing structural and bioactive components. This complexity makes decoupling the effects of biomechanical properties and cell-matrix interactions difficult, especially when studying cellular processes in a 3D environment. Matrix mechanics and cell adhesion are both known regulators of specific cellular processes such as stem cell proliferation and differentiation. However, more information is required about how such variables impact various neural lineages that could, upon transplantation, therapeutically improve neural function after a central nervous system injury or disease. Rapidly Assembling Pentapeptides for Injectable Delivery (RAPID) hydrogels are one biomaterial approach to meet these goals, consisting of a family of peptide sequences that assemble into physical hydrogels in physiological media. In this study, we studied our previously reported supramolecularly-assembling RAPID hydrogels functionalized with the ECM-derived cell-adhesive peptide ligands RGD, IKVAV, and YIGSR. Using molecular dynamics simulations and experimental rheology, we demonstrated that these integrin-binding ligands at physiological concentrations (3-12 mm) did not impact the assembly of the KYFIL peptide system. In simulations, molecular measures of assembly such as hydrogen bonding and pi-pi interactions appeared unaffected by cell-adhesion sequence or concentration. Visualizations of clustering and analysis of solvent-accessible surface area indicated that the integrin-binding domains remained exposed. KYFIL or AYFIL hydrogels containing 3 mm of integrin-binding domains resulted in mechanical properties consistent with their non-functionalized equivalents. This strategy of doping RAPID gels with cell-adhesion sequences allows for the precise tuning of peptide ligand concentration, independent of the rheological properties. The controllability of the RAPID hydrogel system provides an opportunity to investigate the effect of integrin-binding interactions on encapsulated neural cells to discern how hydrogel microenvironment impacts growth, maturation, or differentiation.

Peptide Stereocomplexation Orchestrates Supramolecular Assembly of Hydrogel Biomaterials.

Stereocomplexation, or specific interactions among complementary stereoregular macromolecules, is burgeoning as an increasingly impactful design tool, exerting exquisite control of material structure and properties. Since stereocomplexation of polymers produces remarkable transformations in mechanics, morphology, and degradation, we sought to leverage stereocomplexation to tune these properties in peptide-based biomaterials. We found that blending the pentapeptides l- and d-KYFIL triggers dual mechanical and morphological transformations from stiff fibrous hydrogels into less stiff networks of plates, starkly contrasting prior reports that blending l- and d-peptides produces stiffer fibrous hydrogels than the individual constituents. The morphological transformation of KYFIL in phosphate-buffered saline from fibers that entangle into hydrogels to plates that cannot entangle explains the accompanying mechanical transformation. Moreover, the blends shield l-KYFIL from proteolytic degradation, producing materials with comparable proteolytic stability to d-KYFIL but with distinct 2D plate morphologies that in biomaterials may promote unique therapeutic release profiles and cell behavior. To confirm that these morphological, mechanical, and stability changes arise from differences in molecular packing as in polymer stereocomplexation, we acquired X-ray diffraction patterns, which showed l- and d-KYFIL to be amorphous and their blends to be crystalline. Stereocomplexation is particularly apparent in pure water, where l- and d-KYFIL are soluble random coils, and their blends form ß-sheets and gel within minutes. Our results highlight the role of molecular details, such as peptide sequence, in determining the material properties resulting from stereocomplexation. Looking forward, the ability of stereocomplexation to orchestrate supramolecular assembly and tune application-critical properties champions stereochemistry as a compelling design consideration.

Influence of Supraphysiologic Biomaterial Stiffness on Ventricular Mechanics and Myocardial Infarct Reinforcement.

Injectable intramyocardial biomaterials have promise to limit adverse ventricular remodeling through mechanical and biologic mechanisms. While some success has been observed by injecting materials to regenerate new tissue, optimal biomaterial stiffness to thicken and stiffen infarcted myocardium to limit adverse remodeling has not been determined. In this work, we present an in-vivo study of the impact of biomaterial stiffness over a wide range of stiffness moduli on ventricular mechanics. We utilized injectable methacrylated polyethylene glycol (PEG) hydrogels fabricated at 3 different mechanical moduli: 5 kPa (low), 25 kPa (medium/myocardium), and 250 kPa (high/supraphysiologic). We demonstrate that the supraphysiological high stiffness favorably alters post-infarct ventricular mechanics and prevents negative tissue remodeling. Lower stiffness materials do not alter mechanics and thus to be effective, must instead target biological reparative mechanisms. These results may influence rationale design criteria for biomaterials developed for infarct reinforcement therapy. STATEMENT OF SIGNIFICANCE: Acellular biomaterials for cardiac application can provide benefit via mechanical and biological mechanisms post myocardial infarction. We study the role of biomaterial mechanical characteristics on ventricular mechanics in myocardial infarcts. Previous studies have not measured the influence of injected biomaterials on ventricular mechanics, and consequently rational design criteria is unknown. By utilizing an in-vivo assessment of ventricular mechanics, we demonstrate that low stiffness biomaterial do not alter pathologic ventricular mechanics. Thus, to be effective, low stiffness biomaterials must target biological reparative mechanisms. Physiologic and supra-physiologic biomaterials favorably alter post-infarct mechanics and prevents adverse ventricular remodeling.

Biomaterials via peptide assembly: Design, characterization, and application in tissue engineering.

A core challenge in biomaterials, with both fundamental significance and technological relevance, concerns the rational design of bioactive microenvironments. Designed properly, peptides can undergo supramolecular assembly into dynamic, physical hydrogels that mimic the mechanical, topological, and biochemical features of native tissue microenvironments. The relatively facile, inexpensive, and automatable preparation of peptides, coupled with low batch-to-batch variability, motivates the expanded use of assembling peptide hydrogels for biomedical applications. Integral to realizing dynamic peptide assemblies as functional biomaterials for tissue engineering is an understanding of the molecular and macroscopic features that govern assembly, morphology, and biological interactions. In this review, we first discuss the design of assembling peptides, including primary structure (sequence), secondary structure (e.g., α-helix and ß-sheets), and molecular interactions that facilitate assembly into multiscale materials with desired properties. Next, we describe characterization tools for elucidating molecular structure and interactions, morphology, bulk properties, and biological functionality. Understanding of these characterization methods enables researchers to access a variety of approaches in this ever-expanding field. Finally, we discuss the biological properties and applications of peptide-based biomaterials for engineering several important tissues. By connecting molecular features and mechanisms of assembling peptides to the material and biological properties, we aim to guide the design and characterization of peptide-based biomaterials for tissue engineering and regenerative medicine. STATEMENT OF SIGNIFICANCE: Engineering peptide-based biomaterials that mimic the topological and mechanical properties of natural extracellular matrices provide excellent opportunities to direct cell behavior for regenerative medicine and tissue engineering. Here we review the molecular-scale features of assembling peptides that result in biomaterials that exhibit a variety of relevant extracellular matrix-mimetic properties and promote beneficial cell-biomaterial interactions. Aiming to inspire and guide researchers approaching this challenge from both the peptide biomaterial design and tissue engineering perspectives, we also present characterization tools for understanding the connection between peptide structure and properties and highlight the use of peptide-based biomaterials in neural, orthopedic, cardiac, muscular, and immune engineering applications.

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.

Guiding Oligodendrocyte Precursor Cell Maturation With Urokinase Plasminogen Activator-Degradable Elastin-like Protein Hydrogels.

Demyelinating injuries and diseases, like multiple sclerosis, affect millions of people worldwide. Oligodendrocyte precursor cells (OPCs) have the potential to repair demyelinated tissues because they can both self-renew and differentiate into oligodendrocytes (OLs), the myelin producing cells of the central nervous system (CNS). Cell-matrix interactions impact OPC differentiation into OLs, but the process is not fully understood. Biomaterial hydrogel systems help to elucidate cell-matrix interactions because they can mimic specific properties of native CNS tissues in an in vitro setting. We investigated whether OPC maturation into OLs is influenced by interacting with a urokinase plasminogen activator (uPA) degradable extracellular matrix (ECM). uPA is a proteolytic enzyme that is transiently upregulated in the developing rat brain, with peak uPA expression correlating with an increase in myelin production in vivo. OPC-like cells isolated through the Mosaic Analysis with Double Marker technique (MADM OPCs) produced low-molecular-weight uPA in culture. MADM OPCs were encapsulated into two otherwise similar elastin-like protein (ELP) hydrogel systems: one that was uPA degradable and one that was nondegradable. Encapsulated MADM OPCs had similar viability, proliferation, and metabolic activity in uPA degradable and nondegradable ELP hydrogels. Expression of OPC maturation-associated genes, however, indicated that uPA degradable ELP hydrogels promoted MADM OPC maturation although not sufficiently for these cells to differentiate into OLs.

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.

Impact of Elastin-like Protein Temperature Transition on PEG-ELP Hybrid Hydrogel Properties.

Biomaterial hydrogels are made by cross-linking either natural materials, which exhibit inherent bioactivity but suffer from batch-to-batch variations, or synthetic materials, which have a well-defined chemical structure but usually require chemical modification to exhibit bioactivity. Recombinant engineered proteins bridge the divide between natural and synthetic materials because proteins incorporate bioactive domains within the biopolymer backbone and have a well-defined amino acid structure and sequence. Recombinant engineered elastin-like proteins (ELPs) are modeled from the native tropoelastin sequence. ELPs are composed of repeating VPGxG penta-peptide sequences, where x is any guest residue except proline. ELPs undergo a lower critical solution temperature (LCST) transition above which they aggregate into a coacervate phase in aqueous solution. Here we show that the LCST transition impacts hydrogel microarchitecture which may serve as a useful design feature in engineering ELP-based hydrogels. We investigate how the ELP LCST transition contributes to the properties of hybrid poly(ethylene glycol) (PEG) and ELP (PEG-ELP) hydrogels. PEG-ELP hydrogels gelled below the LCST have a homogeneous distribution of ELP, while gelling above the LCST results in the formation of spherical ELP-rich regions within the bulk hydrogel. The ELP-rich microarchitecture is maintained when an amine-reactive cross-linker is incorporated during the gelation process. The formation of ELP-rich regions reduces PEG-ELP hydrogel bulk stiffness and increases optical density. Our characterizations of hydrogels created by using the LCST transition provide design criteria for incorporating microscale features. This may be a useful technique in understanding the role of localized bioactivity at the microscale level within hydrogel systems.

Matrix Remodeling Enhances the Differentiation Capacity of Neural Progenitor Cells in 3D Hydrogels.

Neural progenitor cells (NPCs) are a promising cell source to repair damaged nervous tissue. However, expansion of therapeutically relevant numbers of NPCs and their efficient differentiation into desired mature cell types remains a challenge. Material-based strategies, including culture within 3D hydrogels, have the potential to overcome these current limitations. An ideal material would enable both NPC expansion and subsequent differentiation within a single platform. It has recently been demonstrated that cell-mediated remodeling of 3D hydrogels is necessary to maintain the stem cell phenotype of NPCs during expansion, but the role of matrix remodeling on NPC differentiation and maturation remains unknown. By culturing NPCs within engineered protein hydrogels susceptible to degradation by NPC-secreted proteases, it is identified that a critical amount of remodeling is necessary to enable NPC differentiation, even in highly degradable gels. Chemical induction of differentiation after sufficient remodeling time results in differentiation into astrocytes and neurotransmitter-responsive neurons. Matrix remodeling modulates expression of the transcriptional co-activator Yes-associated protein, which drives expression of NPC stemness factors and maintains NPC differentiation capacity, in a cadherin-dependent manner. Thus, cell-remodelable hydrogels are an attractive platform to enable expansion of NPCs followed by differentiation of the cells into mature phenotypes for therapeutic use.

Stimuli-Responsive, Pentapeptide, Nanofiber Hydrogel for Tissue Engineering.

Short peptides are uniquely versatile building blocks for self-assembly. Supramolecular peptide assemblies can be used to construct functional hydrogel biomaterials-an attractive approach for neural tissue engineering. Here, we report a new class of short, five-residue peptides that form hydrogels with nanofiber structures. Using rheology and spectroscopy, we describe how sequence variations, pH, and peptide concentration alter the mechanical properties of our pentapeptide hydrogels. We find that this class of seven unmodified peptides forms robust hydrogels from 0.2-20 kPa at low weight percent (less than 3 wt %) in cell culture media and undergoes shear-thinning and rapid self-healing. The peptides self-assemble into long fibrils with sequence-dependent fibrillar morphologies. These fibrils exhibit a unique twisted ribbon shape, as visualized by transmission electron microscopy (TEM) and Cryo-EM imaging, with diameters in the low tens of nanometers and periodicities similar to amyloid fibrils. Experimental gelation behavior corroborates our molecular dynamics simulations, which demonstrate peptide assembly behavior, an increase in ß-sheet content, and patterns of variation in solvent accessibility. Our rapidly assembling pentapeptides for injectable delivery (RAPID) hydrogels are syringe-injectable and support cytocompatible encapsulation of oligodendrocyte progenitor cells (OPCs), as well as their proliferation and three-dimensional process extension. Furthermore, RAPID gels protect OPCs from mechanical membrane disruption and acute loss of viability when ejected from a syringe needle, highlighting the protective capability of the hydrogel as potential cell carriers for transplantation therapies. The tunable mechanical and structural properties of these supramolecular assemblies are shown to be permissive to cell expansion and remodeling, making this hydrogel system suitable as an injectable material for cell delivery and tissue engineering applications.

Rapidly Assembling Pentapeptides for Injectable Delivery (RAPID) Hydrogels as Cytoprotective Cell Carriers.

Low cell survival after syringe injection hampers the success of preclinical and clinical cell transplantation trials. During syringe injection, cells experience mechanical forces that lead to cell-membrane disruption and decreased viability. To improve cell survival, we designed rapidly assembling pentapeptides for injectable delivery (RAPID) hydrogels that shear-thin, protect cells from extensional flow, form fibers, and provide mechanical properties similar to native tissue. We found that 1.5 wt % RAPID hydrogels mitigate the damaging effects of extensional flow, resulting in significantly greater cell viability (of common laboratory cell lines, primary cells, and human cells) than cells injected in PBS.

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.

Encapsulated oligodendrocyte precursor cell fate is dependent on PDGF-AA release kinetics in a 3D microparticle-hydrogel drug delivery system.

Biomaterial drug delivery systems (DDS) can be used to regulate growth factor release and combat the limited intrinsic regeneration capabilities of central nervous system (CNS) tissue following injury and disease. Of particular interest are systems that aid in oligodendrocyte regeneration, as oligodendrocytes generate myelin which surrounds neuronal axons and helps transmit signals throughout the CNS. Oligodendrocyte precursor cells (OPCs) are found in small numbers in the adult CNS, but are unable to effectively differentiate following CNS injury. Delivery of signaling molecules can initiate a favorable OPC response, such as proliferation or differentiation. Here, we investigate the delivery of one such molecule, platelet derived growth factor-AA (PDGF-AA), from poly(lactic-co-glycolic) acid microparticles to OPCs in a 3D polyethylene glycol-based hydrogel. The goal of this DDS was to better understand the relationship between PDGF-AA release kinetics and OPC fate. The system approximates native brain tissue stiffness, while incorporating PDGF-AA under seven different delivery scenarios. Within this DDS, supply of PDGF-AA followed by PDGF-AA withdrawal caused OPCs to upregulate gene expression of myelin basic protein (MBP) by factors of 1.6-9.2, whereas continuous supply of PDGF-AA caused OPCs to remain proliferative. At the protein expression level, we observed an upregulation in O1, a marker for mature oligodendrocytes. Together, these results show that burst release followed by withdrawal of PDGF-AA from a hydrogel DDS stimulates survival, proliferation, and differentiation of OPCs in vitro. Our results could inform the development of improved neural regeneration strategies that incorporate delivery of PDGF-AA to the injured CNS. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2402-2411, 2018.

From de novo peptides to native proteins: advancements in biomaterial scaffolds for acute ischemic stroke repair.

An active area of research in the field of regenerative medicine involves the development of bioactive matrices that can promote cellular interactions and elicit desirable regenerative behavior in vivo. This is particularly important in the context of ischemic stroke where a focal lesion forms forestalling the regrowth of brain tissue. Protein-based molecules have been used as building blocks to create supramolecular structures that emulate the properties of the native healthy extracellular matrix (ECM) within the central nervous system (CNS). In this review, we briefly describe the relevant biological aspect of stroke and the techniques found in molecular biology and biochemical synthesis methodologies used in the design and synthesis of novel biomaterials. Within these biomaterials, researchers are able to incorporate a number of different domains that trigger assembly or promote cell growth and survival and direct transplanted or endogenous stem cell behavior within the 3D scaffolds. Such domains may also yield stimuli-responsive biomaterial scaffolds where the structure of the hydrogel undergoes a change in response to the local environment. These highly modular proteinaceous materials allow incorporation of diverse biofunctional motifs and structural elements comparable to those found in native ECM. We explore CNS relevant biomaterials that promote cell survival and host tissue integration and discuss their applications to stem cell therapy in the treatment of stroke.

Fabricating PLGA microparticles with high loads of the small molecule antioxidant N-acetylcysteine that rescue oligodendrocyte progenitor cells from oxidative stress.

Reactive oxygen species (ROS), encompassing all oxygen radical or non-radical oxidizing agents, play key roles in disease progression. Controlled delivery of antioxidants is therapeutically relevant in such oxidant-stressed environments. Encapsulating small hydrophilic molecules into hydrophobic polymer microparticles via traditional emulsion methods has long been a challenge due to rapid mass transport of small molecules out of particle pores. We have developed a simple alteration to the existing water-in-oil-in-water (W/O/W) drug encapsulation method that dramatically improves loading efficiency: doping external water phases with drug to mitigate drug diffusion out of the particle during fabrication. PLGA microparticles with diameters ranging from 0.6 to 0.9 micrometers were fabricated, encapsulating high loads of 0.6-0.9 µm diameter PLGA microparticles were fabricated, encapsulating high loads of the antioxidant N-acetylcysteine (NAC), and released active, ROS-scavenging NAC for up to 5 weeks. Encapsulation efficiencies, normalized to the theoretical load of traditional encapsulation without doping, ranged from 96% to 400%, indicating that NAC-loaded external water phases not only prevented drug loss due to diffusion, but also doped the particles with additional drug. Antioxidant-doped particles positively affected the metabolism of oligodendrocyte progenitor cells (OPCs) under H2 O2 -mediated oxidative stress when administered both before (protection) or after (rescue) injury. Antioxidant doped particles improved outcomes of OPCs experiencing multiple doses of H2 O2 by increasing the intracellular glutathione content and preserving cellular viability relative to the injury control. Furthermore, antioxidant-doped particles preserve cell number, number of process extensions, cytoskeletal morphology, and nuclear size of H2 O2 -stressed OPCs relative to the injury control. These NAC-doped particles have the potential to provide temporally-controlled antioxidant therapy in neurodegenerative disorders such as multiple sclerosis (MS) that are characterized by continuous oxidative stress.

Maintenance of neural progenitor cell stemness in 3D hydrogels requires matrix remodelling.

Neural progenitor cell (NPC) culture within three-dimensional (3D) hydrogels is an attractive strategy for expanding a therapeutically relevant number of stem cells. However, relatively little is known about how 3D material properties such as stiffness and degradability affect the maintenance of NPC stemness in the absence of differentiation factors. Over a physiologically relevant range of stiffness from ∼0.5 to 50 kPa, stemness maintenance did not correlate with initial hydrogel stiffness. In contrast, hydrogel degradation was both correlated with, and necessary for, maintenance of NPC stemness. This requirement for degradation was independent of cytoskeletal tension generation and presentation of engineered adhesive ligands, instead relying on matrix remodelling to facilitate cadherin-mediated cell-cell contact and promote ß-catenin signalling. In two additional hydrogel systems, permitting NPC-mediated matrix remodelling proved to be a generalizable strategy for stemness maintenance in 3D. Our findings have identified matrix remodelling, in the absence of cytoskeletal tension generation, as a previously unknown strategy to maintain stemness in 3D.

Oligodendrocyte Precursor Cell Viability, Proliferation, and Morphology is Dependent on Mesh Size and Storage Modulus in 3D Poly(ethylene glycol)-Based Hydrogels.

Oligodendrocytes in the central nervous system (CNS) are responsible for generating myelin, an electrically insulating layer around neuronal axons. When myelin is damaged, neurons are incapable of sustaining normal communications, which can manifest in patients as pain and loss of mobility and vision. A plethora of research has used biomaterials to promote neuronal regeneration, but despite the wide implications of a disrupted myelin sheath, very little is known about how biomaterial environments impact proliferation of oligodendrocyte precursor cells (OPCs) or their differentiation into myelinating oligodendrocytes. This work investigates how the storage modulus and mesh size of a polyethylene glycol (PEG)-based hydrogel, varied via two different mechanisms, directly affect the proliferation of two OPC lines encapsulated and cultured in 3D. Viability and proliferation of both OPC lines was dependent on hydrogel swelling and stiffness, where the concentration of ATP increased more in the more compliant gels. OPCs multiplied in the 3D hydrogels, creating significantly larger spheroids in the less cross-linked conditions. Stiffer, more highly cross-linked materials lead to greater expression of PDGFRα, an OPC receptor, indicating that fewer cells were committed to the oligodendrocyte lineage or had dedifferentiated in compliant materials. Laminin incorporation in the 3D matrix was found to have little effect on viability or proliferation. These findings provide valuable information on how mesh size and stiffness affect OPCs where more compliant materials favor proliferation of OPCs with less commitment to a mature oligodendrocyte lineage. Such information will be useful in the development of translational biomaterials to stimulate oligodendrocyte maturation for neural regeneration.

Engineering Biomaterials to Influence Oligodendroglial Growth, Maturation, and Myelin Production.

Millions of people suffer from damage or disease to the nervous system that results in a loss of myelin, such as through a spinal cord injury or multiple sclerosis. Diminished myelin levels lead to further cell death in which unmyelinated neurons die. In the central nervous system, a loss of myelin is especially detrimental because of its poor ability to regenerate. Cell therapies such as stem or precursor cell injection have been investigated as stem cells are able to grow and differentiate into the damaged cells; however, stem cell injection alone has been unsuccessful in many areas of neural regeneration. Therefore, researchers have begun exploring combined therapies with biomaterials that promote cell growth and differentiation while localizing cells in the injured area. The regrowth of myelinating oligodendrocytes from neural stem cells through a biomaterials approach may prove to be a beneficial strategy following the onset of demyelination. This article reviews recent advancements in biomaterial strategies for the differentiation of neural stem cells into oligodendrocytes, and presents new data indicating appropriate properties for oligodendrocyte precursor cell growth. In some cases, an increase in oligodendrocyte differentiation alongside neurons is further highlighted for functional improvements where the biomaterial was then tested for increased myelination both in vitro and in vivo.

Toward a Designable Extracellular Matrix: Molecular Dynamics Simulations of an Engineered Laminin-Mimetic, Elastin-Like Fusion Protein.

Native extracellular matrices (ECMs) exhibit networks of molecular interactions between specific matrix proteins and other tissue components. Guided by these naturally self-assembling supramolecular systems, we have designed a matrix-derived protein chimera that contains a laminin globular-like (LG) domain fused to an elastin-like polypeptide (ELP). This bipartite design offers a flexible protein engineering platform: (i) laminin is a key multifunctional component of the ECM in human brains and other neural tissues, making it an ideal bioactive component of our fusion, and (ii) ELPs, known to be well-tolerated in vivo, provide a self-assembly scaffold with tunable physicochemical (viscoelastic, thermoresponsive) properties. Experimental characterization of novel proteins is resource-intensive, and examining many conceivable designs would be a formidable challenge in the laboratory. Computational approaches offer a way forward: molecular dynamics (MD) simulations can be used to analyze the structural/physical behavior of candidate LG-ELP fusion proteins, particularly in terms of conformational properties salient to our design goals, such as assembly propensity in a temperature range spanning the inverse temperature transition. As a first step in examining the physical characteristics of a model LG-ELP fusion protein, including its temperature-dependent structural behavior, we simulated the protein over a range of physiologically relevant temperatures (290-320 K). We find that the ELP region, built upon the archetypal (VPGXG)5 scaffold, is quite flexible and has a propensity for ß-rich secondary structures near physiological (310-315 K) temperatures. Our trajectories indicate that the temperature-dependent burial of hydrophobic patches in the ELP region, coupled to the local water structure dynamics and mediated by intramolecular contacts between aliphatic side chains, correlates with the temperature-dependent structural transitions in known ELP polymers. Because of the link between compaction of ELP segments into ß-rich structures and differential solvation properties of this region, we posit that future variation of ELP sequence and composition can be used to systematically alter the phase transition profiles and, thus, the general functionality of our LG-ELP fusion protein system.

Mimicking biological phenomena in hydrogel-based biomaterials to promote dynamic cellular responses.

Hydrogel-based biomaterials, often classified as synthetic or natural, have long been pursued for cell culture, tissue engineering, regenerative medicine, and drug delivery. This classification system is now being blurred as hybrid partially synthetic systems using elements of native and designer molecules gain traction. Partially synthetic polymer gels can offer protection of encapsulated cells or drugs and provide instructional biochemical and/or biophysical cues to cells. To enable cellular interaction however, they must be endowed with bioactive elements. The extracellular matrix (ECM) provides a "toolbox" of bioactive moieties that can be incorporated into user-defined synthetic polymer scaffolds to promote cell responses such as migration or cell-derived material responses such as matrix multimerization. Incorporating bioactive elements like cell-adhesive peptides, protease-cleavable sites, and ECM-mimetic mechanical properties such as stiffness and porosity into robust well-characterized hydrogels has been pursued for decades. Through careful selection of linkage chemistries and structured design of material subunits, hydrogels have been created that facilitate a great deal of native cellular functions while retaining the customizable nature of engineered materials. A new thrust has emerged to engineer materials with the innate, dynamic bioresponsive activity of native ECM materials. This review characterizes the cell responsive units of native materials and the literature that has recently incorporated these elements into hydrogel tissue engineering and drug delivery materials to promote cell-controlled dynamic responses, a defining characteristic of native functional tissue.