Impact of Confinement within a Hydrogel Mesh on Protein Thermodynamic Stability and Aggregation Kinetics.

Though protein stability and aggregation have been well characterized in dilute solutions, the influence of a confining environment that exists (e.g., in intercellular and tissue spaces and therapeutic formulations) on the protein structure is largely unknown. Herein, the effects of confinement on stability and aggregation were explored for proteins of different sizes, stability, and hydrophobicity when encapsulated in hydrophilic poly(ethylene glycol) hydrogels. Denaturation curves show linear correlations between confinement size (mesh size) and thermodynamic stability, i.e., unfolding free energy and surface area accessible for solvation (represented by m-value). Two clusters of protein types are identifiable from these correlations; the clusters are defined by differences in protein stability, surface area, and aggregation propensity. Proteins with higher stability, larger surface area, and lower aggregation propensity (e.g., lysozyme and myoglobin) are less affected by the confinement imposed by mesh size than proteins with lower stability, smaller surface area, and higher aggregation propensity (e.g., growth hormone and aldehyde dehydrogenase). According to aggregation kinetics measured by thioflavin T fluorescence, confinement in smaller mesh sizes resulted in slower aggregation rates than that in larger mesh sizes. Compared to that in buffer solution, the confinement of a hydrophobic protein (e.g., human insulin) in the hydrogels accelerates protein aggregation. Conversely, the confinement of a hydrophilic protein (e.g., human amylin) in the hydrogels decelerates or prevents aggregation, with the rates of aggregation inversely proportional to mesh size. These findings provide new insights into protein conformational stability in confined microenvironments relevant to various cellular, tissue, and therapeutics scenarios.

Microglial Depletion with CSF1R Inhibitor During Chronic Phase of Experimental Traumatic Brain Injury Reduces Neurodegeneration and Neurological Deficits.

Chronic neuroinflammation with sustained microglial activation occurs following severe traumatic brain injury (TBI) and is believed to contribute to subsequent neurodegeneration and neurological deficits. Microglia, the primary innate immune cells in brain, are dependent on colony stimulating factor 1 receptor (CSF1R) signaling for their survival. In this preclinical study, we examined the effects of delayed depletion of chronically activated microglia on functional recovery and neurodegeneration up to 3 months postinjury. A CSF1R inhibitor, Plexxikon (PLX) 5622, was administered to adult male C57BL/6J mice at 1 month after controlled cortical impact to remove chronically activated microglia, and the inhibitor was withdrawn 1-week later to allow for microglial repopulation. Following TBI, the repopulated microglia displayed a ramified morphology similar to that of Sham uninjured mice, whereas microglia in vehicle-treated TBI mice showed the typical chronic posttraumatic hypertrophic morphology. PLX5622 treatment limited TBI-associated neuropathological changes at 3 months postinjury; these included a smaller cortical lesion, reduced hippocampal neuron cell death, and decreased NOX2- and NLRP3 inflammasome-associated neuroinflammation. Furthermore, delayed depletion of chronically activated microglia after TBI led to widespread changes in the cortical transcriptome and altered gene pathways involved in neuroinflammation, oxidative stress, and neuroplasticity. Using a variety of complementary neurobehavioral tests, PLX5622-treated TBI mice also had improved long-term motor and cognitive function recovery through 3 months postinjury. Together, these studies demonstrate that chronic phase removal of neurotoxic microglia after TBI using CSF1R inhibitors markedly reduce chronic neuroinflammation and associated neurodegeneration, as well as related motor and cognitive deficits.SIGNIFICANCE STATEMENT Traumatic brain injury (TBI) is a debilitating neurological disorder that can seriously impact the patient's quality of life. Microglial-mediated neuroinflammation is induced after severe TBI and contributes to neurological deficits and on-going neurodegenerative processes. Here, we investigated the effect of breaking the neurotoxic neuroinflammatory loop at 1-month after controlled cortical impact in mice by pharmacological removal of chronically activated microglia using a colony stimulating factor 1 receptor (CSF1R) inhibitor, Plexxikon 5622. Overall, we show that short-term elimination of microglia during the chronic phase of TBI followed by repopulation results in long-term improvements in neurological function, suppression of neuroinflammatory and oxidative stress pathways, and a reduction in persistent neurodegenerative processes. These studies are clinically relevant and support new concepts that the therapeutic window for TBI may be far longer than traditionally believed if chronic and evolving microglial-mediated neuroinflammation can be inhibited or regulated in a precise manner.

Stability of proteins encapsulated in Michael-type addition polyethylene glycol hydrogels.

Degradable polyethylene glycol (PEG) hydrogels are excellent vehicles for sustained drug release due to their biocompatibility, tunable physical properties, and customizable degradation. However, protein therapeutics are unstable under physiological conditions and releasing degraded or inactive therapeutics can induce immunogenic effects. While controlling protein release from PEG hydrogels has been extensively investigated, few studies have detailed protein stability long-term or under stress conditions. Here, lysozyme and alcohol dehydrogenase (ADH) stability were explored upon encapsulation in PEG hydrogels formed through Michael-type addition. The stability and structure of the two model proteins were monitored by measuring the free energy of unfolding and fluoresce quenching when confined in a hydrogel and compared to PEG solution and buffer. Hydrogels destabilized lysozyme structure at low denaturant concentrations but prevented complete unfolding at high concentrations. ADH was stabilized as the confining mesh size approached the protein radius of gyration. Both proteins retained enzymatic activity within the hydrogels under stress conditions, including denaturant, high temperature, and agitation. Conjugation between lysozyme and PEG-acrylate was identified at long reaction times but no conjugation was observed in the time required for complete gelation. Studies of protein stability in PEG hydrogels, as the one detailed here, can lead to designer technologies for the improved formulation, storage, and delivery of protein therapeutics.

Real-time local oxygen measurements for high resolution cellular imaging.

Single-cell metabolic investigations are hampered by the absence of flexible tools to measure local partial pressure of O2 (pO2) at high spatial-temporal resolution. To this end, we developed an optical sensor capable of measuring local pericellular pO2 for subcellular resolution measurements with confocal imaging while simultaneously carrying out electrophysiological and/or chemo-mechanical single cell experiments. Here we present the OxySplot optrode, a ratiometric fluorescent O2-micro-sensor created by adsorbing O2-sensitive and O2-insensitive fluorophores onto micro-particles of silica. To protect the OxySplot optrode from the components and reactants of liquid environment without compromising access to O2, the micro-particles are coated with an optically clear silicone polymer (PDMS, polydimethylsiloxane). The PDMS coated OxySplot micro-particles are used alone or in a thin (~50 µm) PDMS layer of arbitrary shape referred to as the OxyMat. Additional top coatings on the OxyMat (e.g., fibronectin, laminin, polylysine, special photoactivatable surfaces etc.) facilitate adherence of cells. The OxySplots report the cellular pO2 and micro-gradients of pO2 without disrupting the flow of extracellular solutions or interfering with patch-clamp pipettes, mechanical attachments, and micro-superfusion. Since OxySplots and a cell can be imaged and spatially resolved, calibrated changes of pO2 and intracellular events can be imaged simultaneously. In addition, the response-time (t0.5 = 0.7 s, 0-160 mmHg) of OxySplots is ~100 times faster than amperometric Clark-type polarization microelectrodes. Two usage example of OxySplots with cardiomyocytes show (1) OxySplots measuring pericellular pO2 while tetramethylrhodamine methyl-ester (TMRM) was used to measure mitochondrial membrane potential (ΔΨm); and (2) OxySplots measuring pO2 during ischemia and reperfusion while rhod-2 was used to measure cytosolic [Ca2+]i levels simultaneously. The OxySplot/OxyMat optrode system provides an affordable and highly adaptable optical sensor system for monitoring pO2 with a diverse array of imaging systems, including high-speed, high-resolution confocal microscopes while physiological features are measured simultaneously.

Enhancement of human neural stem cell self-renewal in 3D hypoxic culture.

The pathology of neurological disorders is associated with the loss of neuronal and glial cells that results in functional impairments. Human neural stem cells (hNSCs), due to their self-renewing and multipotent characteristics, possess enormous tissue-specific regenerative potential. However, the efficacy of clinical applications is restricted due to the lack of standardized in vitro cell production methods with the capability of generating hNSC populations with well-defined cellular compositions. At any point, a population of hNSCs may include undifferentiated stem cells, intermediate and terminally differentiated progenies, and dead cells. Due to the plasticity of hNSCs, environmental cues play crucial roles in determining the cellular composition of hNSC cultures over time. Here, we investigated the independent and synergistic effect of three important environmental factors (i.e., culture dimensionality, oxygen concentration, and growth factors) on the survival, renewal potential, and differentiation of hNSCs. Our experimental design included two dimensional (2D) versus three dimensional (3D) cultures and normoxic (21% O2 ) versus hypoxic (3% O2 ) conditions in the presence and absence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Additionally, we discuss the feasibility of mathematical models that predict hNSC growth and differentiation under these culture conditions by adopting a negative feedback regulatory term. Our results indicate that the synergistic effect of culture dimensionality and hypoxic oxygen concentration in the presence of growth factors enhances the proliferation of viable, undifferentiated hNSCs. Moreover, the same synergistic effect in the absence of growth factors promotes the differentiation of hNSCs. Biotechnol. Bioeng. 2017;114: 1096-1106. © 2016 Wiley Periodicals, Inc.

Fluorescent silica particles for monitoring oxygen levels in three-dimensional heterogeneous cellular structures.

Bacterial biofilms are a major obstacle challenging the development of more effective therapies to treat implant infections. Oxygen availability to bacterial cells has been implicated in biofilm formation and planktonic cell detachment; however, there are insufficient tools available to measure oxygen concentrations within complex three-dimensional structures with ∼ 1 µm resolution. Such measurements may complement measures of biofilm structure and cell activity to provide a more comprehensive understanding of biofilm biology. Thus, we developed oxygen-sensing microparticles specifically designed to characterize oxygen transport through the volume of bacterial biofilms. The Stöber method was used to synthesize monodisperse silica microparticles of approximately the same size as a bacterium (∼ 1 µm). Two fluorophores, oxygen-sensitive Ru(Ph(2) phen(3))Cl(2), and the reference fluorophore Nile blue chloride were immobilized on the surface of the particles. We demonstrate application of the microparticles toward measuring the oxygen concentration profiles within a live Staphylococcus aureus biofilm.

Characterization of protein release from hydrolytically degradable poly(ethylene glycol) hydrogels.

We present a novel fully hydrophilic, hydrolytically degradable poly(ethylene glycol) (PEG) hydrogel suitable for soft tissue engineering and delivery of protein drugs. The gels were designed to overcome drawbacks associated with current PEG hydrogels (i.e., reaction mechanisms or degradation products that compromise protein stability): the highly selective and mild cross-linking reaction allowed for encapsulating proteins prior to gelation without altering their secondary structure as shown by circular dichroism experiments. Further, hydrogel degradation and structure, represented by mesh size, were correlated to protein release. It was determined that polymer density had the most profound effect on protein diffusivity, followed by the polymer molecular weight, and finally by the specific chemical structure of the cross-linker. By examining the diffusion of several model proteins, we confirmed that the protein diffusivity was dependent on protein size as smaller proteins (e.g., lysozyme) diffused faster than larger proteins (e.g., Ig). Furthermore, we demonstrated that the protein physical state was preserved upon encapsulation and subsequent release from the PEG hydrogels and contained negligible aggregation or protein-polymer adducts. These initial studies indicate that the developed PEG hydrogels are suitable for release of stable proteins in drug delivery and tissue engineering applications.

Fate of transition metals in PO4-based in vitro assays: equilibrium modeling and macroscopic studies.

Transition metals are thought to be among the most toxic components in atmospheric particulate matter (PM) due to their role in catalyzing reactive oxygen species (ROS) formation. We show that precipitation of the transition metals Fe(ii), Fe(iii), and Mn(ii) are thermodynamically favored in phosphate-based assays used to measure the oxidative potential (OP) - a surrogate for toxicity - of PM. Fe and Mn precipitation is likely to occur at extremely low metal concentrations (<0.5 µM), levels that are imperceptible to the naked eye. The concentration of each metal (other than Cu) in aqueous PM filter extracts often exceeds the solubility limit in OP assays, indicating favorable thermodynamic conditions for precipitation. Macroscopic experimental results at higher metal concentrations (>100 µM) with visible precipitates provide quasi-validation of the thermodynamic modeling. Oxidation of Fe(ii) to Fe(iii) is likely to be rapid in all in vitro OP assays, transforming Fe to a much less soluble form. Fe precipitates are likely to increase the rate of precipitation of other metals and possibly induce co-precipitation. These results have direct relevance for all PO4-based assays; the implications for studies of PM toxicity are discussed.

Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties.

The objective of this work was to create 3D hydrogel matrices with defined mechanical properties as well as tunable degradability for use in applications involving protein delivery and cell encapsulation. Therefore, we report the synthesis and characterization of a novel hydrolytically degradable poly(ethylene glycol) (PEG) hydrogel composed of PEG vinyl sulfone (PEG-VS) cross-linked with PEG-diester-dithiol. Unlike previously reported degradable PEG-based hydrogels, these materials are homogeneous in structure, fully hydrophilic, and have highly specific cross-linking chemistry. We characterized hydrogel degradation and associated trends in mechanical properties, that is, storage modulus (G'), swelling ratio (Q(M)), and mesh size (xi). Degradation time and the monitored mechanical properties of the hydrogel correlated with cross-linker molecular weight, cross-linker functionality, and total polymer density; these properties changed predictably as degradation proceeded (G' decreased, whereas Q(M) and xi increased) until the gels reached complete degradation. Balb/3T3 fibroblast adhesion and proliferation within the 3D hydrogel matrices were also verified. In sum, these unique properties indicate that the reported degradable PEG hydrogels are well poised for specific applications in protein and cell delivery to repair soft tissue.

Solute diffusion and interactions in cross-linked poly(ethylene glycol) hydrogels studied by Fluorescence Correlation Spectroscopy.

Controlled diffusion and release of soluble molecules is one of the key challenges in developing three-dimensional (3D) scaffolds for tissue engineering and drug delivery applications in part because current methods to measure dynamic transport properties are difficult to perform directly, are strongly affected by the experimental setup, and therefore can be a subject to various artifacts. In this work we present a method for direct measurement of translational diffusion of solutes, namely Fluorescence Correlation Spectroscopy (FCS), by characterizing the diffusion of model proteins through a 3D cross-linked poly(ethylene glycol) (PEG) hydrogel scaffold. We examined both the dynamics of hydrogel structure (e.g., cross-linking and swelling) as well as protein size and their effect on protein diffusivity. For example, we demonstrated that protein diffusivity was closely related to protein size as smaller proteins (e.g., lysozyme) diffused faster than larger proteins (e.g., γ-globulin or Ig). We validated the FCS protein diffusivity results by comparison to standard bulk diffusion assays. Additionally, due to the nature of FCS measurements, we were able to probe for hydrogel-protein interactions during cross-linking that may contribute to the obstructed protein diffusion in the 3D scaffold. We determined that such interactions in this system were not covalent (i.e., were independent of the cross-linking chemistry) but may be due to weaker hydrogen bonding or ionic interactions. Also, these interactions were protein specific and contributed up to 25% of the total decrease in protein diffusivity in the hydrogel as compared to diffusivity in water. Though interactions between various proteins and PEG have been reported, this is the first study that has explored these effects in detail in cross-linked PEG hydrogels using FCS; our findings question the assumption that PEG hydrogels are completely inert to protein interactions when applied as drug delivery matrices and tissue engineering scaffolds.

Protein folding and assembly in confined environments: Implications for protein aggregation in hydrogels and tissues.

In the biological milieu of a cell, soluble crowding molecules and rigid confined environments strongly influence whether the protein is properly folded, intrinsically disordered proteins assemble into distinct phases, or a denatured or aggregated protein species is favored. Such crowding and confinement factors act to exclude solvent volume from the protein molecules, resulting in an increased local protein concentration and decreased protein entropy. A protein's structure is inherently tied to its function. Examples of processes where crowding and confinement may strongly influence protein function include transmembrane protein dimerization, enzymatic activity, assembly of supramolecular structures (e.g., microtubules), nuclear condensates containing transcriptional machinery, protein aggregation in the contexts of disease and protein therapeutics. Historically, most protein structures have been determined from pure, dilute protein solutions or pure crystals. However, these are not the environments in which these proteins function. Thus, there has been an increased emphasis on analyzing protein structure and dynamics in more "in vivo-like" environments. Complex in vitro models using hydrogel scaffolds to study proteins may better mimic features of the in vivo environment. Therefore, analytical techniques need to be optimized for real-time analysis of proteins within hydrogel scaffolds.

Collagen hydrogel confinement of Amyloid-ß (Aß) accelerates aggregation and reduces cytotoxic effects.

Alzheimer's disease (AD) is the most common form of dementia and is associated with the accumulation of amyloid-ß (Aß), a peptide whose aggregation has been associated with neurotoxicity. Drugs targeting Aß have shown great promise in 2D in vitro models and mouse models, yet preclinical and clinical trials for AD have been highly disappointing. We propose that current in vitro culture systems for discovering and developing AD drugs have significant limitations; specifically, that Aß aggregation is vastly different in these 2D cultures carried out on flat plastic or glass substrates vs. in a 3D environment, such as brain tissue, where Aß confinement alters aggregation kinetics and thermodynamics. In this work, we identified attenuation of Aß cytotoxicity in 3D hydrogel culture compared to 2D cell culture. We investigated Aß structure and aggregation in solution vs. hydrogel using Transmission Electron Microscopy (TEM), Fluorescence Correlation Spectroscopy (FCS), and Thioflavin T (ThT) assays. Our results reveal that the equilibrium is shifted to stable extended ß-sheet (ThT positive) aggregates in hydrogels and away from the relatively unstable/unstructured presumed toxic oligomeric Aß species in solution. Volume exclusion imparted by hydrogel confinement stabilizes unfolded, presumably toxic species, promoting stable extended ß-sheet fibrils. STATEMENT OF SIGNIFICANCE: Alzheimer's disease (AD) is a devastating disease and has been studied for over 100 years. Yet, no cure exists and only 5 prescription drugs are FDA-approved to temporarily treat the AD symptoms of declining brain functions related to thinking and memory. Why don't we have more effective treatments to cure AD or relieve AD symptoms? We propose that current culture methods based upon cells cultured on flat, stiff substrates have significant limitations for discovering and developing AD drugs. This study provides strong evidence that AD drugs should be tested in 3D culture systems as a step along the development pathway towards new, more effective drugs to treat AD.

Impact of Four Common Hydrogels on Amyloid-ß (Aß) Aggregation and Cytotoxicity: Implications for 3D Models of Alzheimer's Disease.

The physiochemical properties of hydrogels utilized in 3D culture can be used to modulate cell phenotype and morphology with a striking resemblance to cellular processes that occur in vivo. Indeed, research areas including regenerative medicine, tissue engineering, in vitro cancer models, and stem cell differentiation have readily utilized 3D biomaterials to investigate cell biological questions. However, cells are only one component of this biomimetic milieu. In many models of disease such as Alzheimer's disease (AD) that could benefit from the in vivo-like cell morphology associated with 3D culture, other aspects of the disease such as protein aggregation have yet to be methodically considered in this 3D context. A hallmark of AD is the accumulation of the peptide amyloid-ß (Aß), whose aggregation is associated with neurotoxicity. We have previously demonstrated the attenuation of Aß cytotoxicity when cells were cultured within type I collagen hydrogels versus on 2D substrates. In this work, we investigated the extent to which this phenomenon is conserved when Aß is confined within hydrogels of varying physiochemical properties, notably mesh size and bioactivity. We investigated the Aß structure and aggregation kinetics in solution and hydrogels composed of type I collagen, agarose, hyaluronic acid, and polyethylene glycol using fluorescence correlation spectroscopy and thioflavin T assays. Our results reveal that all hydrogels tested were associated with enhanced Aß aggregation and Aß cytotoxicity attenuation. We suggest that confinement itself imparts a profound effect, possibly by stabilizing Aß structures and shifting the aggregate equilibrium toward larger species. If this phenomenon of altered protein aggregation in 3D hydrogels can be generalized to other contexts including the in vivo environment, it may be necessary to reevaluate aspects of protein aggregation disease models used for drug discovery.

Direct, Real-Time Detection of Adenosine Triphosphate Release from Astrocytes in Three-Dimensional Culture Using an Integrated Electrochemical Aptamer-Based Sensor.

In this manuscript, we describe the development and application of electrochemical aptamer-based (E-AB) sensors directly interfaced with astrocytes in three-dimensional (3D) cell culture to monitor stimulated release of adenosine triphosphate (ATP). The aptamer-based sensor couples specific detection of ATP, selective performance directly in cell culture media, and seconds time resolution using squarewave voltammetry for quantitative ATP-release measurements. More specifically, we demonstrate the ability to quantitatively monitor ATP release into the extracellular environment after stimulation by the addition of calcium (Ca2+), ionomycin, and glutamate. The sensor response is confirmed to be specific to ATP and requires the presence of astrocytes in culture. For example, PC12 cells do not elicit a sensor response after stimulation with the same stimulants. In addition, we confirmed cell viability in the collagen matrix for all conditions tested. Our hydrogel-sensor interface offers the potential to study the release of small molecule messengers in 3D environments. Given the generality of electrochemical aptamer-based sensors and the demonstrated successful interfacing of sensors with tissue scaffold material, in the long term, we anticipate our sensors will be able to translate from in vitro to in vivo small molecule recordings.

Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity.

Rationally designed matrices for nerve tissue engineering and encapsulated cell therapies critically rely on a comprehensive understanding of neural response to biochemical as well as biophysical cues. Whereas biochemical cues are established mediators of neuronal behavior (e.g., outgrowth), physical cues such as substrate stiffness have only recently been recognized to influence cell behavior. In this work, we examine the response of PC12 neurites to substrate stiffness. We quantified and controlled fibronectin density on the substrates and measured multiple neurite behaviors (e.g., growth, branching, neurites per cell, per cent cells expressing neurites) in a large sample population. We found that PC12 neurons display a threshold response to substrate stiffness. On the softest substrates tested (shear modulus approximately 10 Pa), neurites were relatively few, short in length and unbranched. On stiffer substrates (shear modulus approximately 10(2)-10(4) Pa), neurites were longer and more branched and a greater percentage of cells expressed neurites; significant differences in these measures were not found on substrates with a shear modulus >10(2) Pa. Based on these data and comparisons with published neurobiology and neuroengineering reports of neurite mechanotransduction, we hypothesize that results from studies of neuronal response to compliant substrates are cell-type dependent and sensitive to ligand density, sample size and the range of stiffness investigated.

The effect of hypoxia and laminin-rich substrates on the proliferative behavior of human neural stem cells.

Human neural stem cells (hNSCs) possess an enormous potential to be utilized in novel cell-replacement therapies for neurodegenerative diseases and injuries. The hNSCs are a renewable source of cells with the capacity to generate the major cell types of the central nervous system (CNS). However, the translational potential of cell-based therapy is constrained due to the limited availability of scalable methods to rapidly expand numbers of stem cells in vitro. Here, we investigated the possible synergistic effect of oxygen concentration and substrate composition on hNSC growth. The hNSCs were cultured on six different substrates (i.e., collagen I, collagen IV, poly-l-ornithine, fibronectin, laminin, and Matrigel) under normoxic (21% oxygen concentration) and hypoxic (3% oxygen concentration) conditions and then total cell numbers were determined after 2 and 4 d. The percentages of cells undergoing proliferation (EdU+) and apoptosis (TUNEL+) varied with culture conditions, with a synergistic interaction between Matrigel substrate and hypoxia that resulted in the greatest number of hNSCs after 4 d compared to other conditions. These findings inform new methods to scale up NSC production by identifying potential substrate biomaterial design criteria as well as culture conditions that favor the generation of larger numbers of undifferentiated cells.

Three-Dimensional Environment Sustains Morphological Heterogeneity and Promotes Phenotypic Progression During Astrocyte Development.

Astrocytes are critical for coordinating normal brain function by regulating brain metabolic homeostasis, synaptogenesis and neurotransmission, and blood-brain barrier permeability and maintenance. Dysregulation of normal astrocyte ontogeny contributes to neurodevelopmental and neurodegenerative disorders, epilepsies, and adverse responses to injury. To achieve these multiple essential roles, astrocyte phenotypes are regionally, morphologically, and functionally heterogeneous. Therefore, the best regenerative medicine strategies may require selective production of distinct astrocyte subpopulations at defined maturation levels. However, little is known about the mechanisms that direct astrocyte diversity or whether heterogeneity is represented in biomaterials. In vitro studies report lack of normal morphologies and overrepresentation of the glial scar type of reactive astrocyte morphology and expression of markers, questioning how well the in vitro astrocytes represent glia in vivo and whether in vitro tissue engineering methods are suitable for regenerative medicine applications. Our previous work with neurons suggests that the three-dimensional (3D) environment, when compared with standard two-dimensional (2D) substrate, yields cellular and molecular behaviors that more closely approximately normal ontogeny. To specifically study the effects of dimensionality, we used purified glial fibrillary acidic protein (GFAP)-expressing primary cerebral cortical astrocyte cultures from single pups and characterized the cellular maturation profiles in 2D and 3D milieu. We identified four morphological groups in vitro: round, bipolar, stellate, and putative perivascular. In the 3D hydrogel culture environment, postnatal astrocytes transitioned from a population of nearly all round cells and very few bipolar cells toward a population with significant fractions of round, stellate, and putative perivascular cells within a few days, following the in vivo ontogeny. In 2D, however, the population shift from round and bipolar to stellate and perivascular was rarely observed. The transition to distinct cellular morphologies in 3D corresponded to the in vivo expression of phenotypic markers, supporting the generation of mature heterogeneous glial populations in vitro. This study presents quantitative data supporting that 3D culture is critical for sustaining the heterogeneity of astrocytes in vitro and for generating a representation of the in vivo portfolio of heterogeneous populations of astrocytes required for therapeutic interventions in neurodevelopmental disorders, epilepsy, and brain injury.

Characterization of protein release from photocrosslinkable hyaluronic acid-polyethylene glycol hydrogel tissue engineering scaffolds.

The goal of this work was to utilize the naturally derived bioactive polymer hyaluronic acid (HA) to create a combination tissue engineering scaffold and protein delivery device. HA is a non-immunogenic, non-adhesive glycosaminoglycan that plays significant roles in several cellular processes, including angiogenesis and the regulation of inflammation. In previous work, we created photopolymerizable glycidyl methacrylate-hyaluronic acid (GMHA) hydrogels that had controlled degradation rates, were cytocompatible, and were able to be modified with peptide moieties. In the present studies, we characterized the release of a model protein, bovine serum albumin (BSA), from GMHA and GMHA-polyethylene glycol (PEG) hydrogels. Although BSA could be released rapidly (> 60% within 6 h) from 1% GMHA hydrogels, we found that increasing either the GMHA or the PEG concentrations could lengthen the duration of protein delivery. Preliminary size exclusion chromatography studies indicated that the released BSA was almost entirely in its native monomeric form. Lastly, protein release was extended to several weeks by suspending BSA-poly(lactic-co-glycolic acid) microspheres within the hydrogel bulk. These initial studies indicate that the naturally derived biopolymer HA can be employed to design novel photopolymerizable composites that are suitable for delivering stable proteins from scaffolding in tissue engineering applications.

Crosslinked alpha-elastin biomaterials: towards a processable elastin mimetic scaffold.

Elastin is a critical biochemical and biomechanical component of vascular tissue. However, elastin is also highly insoluble and therefore difficult to process into new biomaterials. We present a simple approach for synthesizing elastin-based materials from two commercially available and water-soluble components: alpha-elastin and a diepoxy crosslinker. Reaction pH was shown to modulate the degree of crosslinking, as demonstrated by materials characterized with a range of swelling ratios (approximately 10-25), enzymatic degradation rates (approximately 8-50% per h in 0.1 u/ml elastase), and elastic moduli (approximately 4-120 kPa). Crosslinking with a combination alkaline and neutral pH process results in materials with the highest degree of crosslinks, as indicated by a swelling ratio of 10, slow degradation rate, and high elastic moduli (approximately 120 kPa). Furthermore, the crosslinked alpha-elastin materials support vascular smooth muscle cell (VSMC) adhesion and a decreased proliferation rate compared to polystyrene controls. The functional outcomes of the crosslinking reaction, including the dependence of structure-function properties on reaction pH, are discussed. Our approach towards 'processable' elastin-based materials is versatile and could be integrated into existing tissue engineering methodologies to enhance biomaterial performance by providing a natural elastomeric and biofunctional component.

Development of photocrosslinkable hyaluronic acid-polyethylene glycol-peptide composite hydrogels for soft tissue engineering.

Hyaluronic acid (HA; also called hyaluronan) is a naturally derived, nonimmunogenic, nonadhesive glycosaminoglycan that has important roles in several wound-healing processes. In previous work, we created photocrosslinkable glycidyl methacrylate-HA (GMHA) hydrogel biomaterials that were cytocompatible, biologically active, and had a decreased rate of hyaluronidase degradation compared with native HA. The goal of the studies presented herein was to explore peptide conjugation techniques to further adjust the material and biological properties of the GMHA hydrogels. We conjugated GMHA with acrylated forms of polyethylene glycol (PEG) and PEG-peptides to yield GMHA-PEG-peptide composite hydrogels. By varying the reactant concentrations, we created stable hydrogels with high peptide conjugation efficiencies (up to 80%), controllable peptide concentrations (in the range of 1-6 micromol peptide per milliliter of hydrogel), and defined physicochemical properties (e.g., swelling ratio, enzymatic degradation rate). These composite hydrogels may prove to be a promising scaffolding biomaterial for a variety of soft tissue engineering applications.