Improved 3D Hydrogel Cultures of Primary Glial Cells for <em>In Vitro</em> Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

University of Alberta

Poultry feather quills have been extruded in a twin screw extruder with sodium sulfite treatment as a reducing agent. The effect of four different plasticizers (ethylene glycol, propylene glycol, glycerol, and diethyl tartrate) on the thermoplastic properties was then investigated. Conformational changes and plasticizer-protein interactions in the extruded resins were assessed by Fourier transform infrared spectroscopy (FTIR), while viscoelastic behavior of the quill keratin plasticized with different plasticizers was investigated by dynamic mechanical analysis (DMA). Differential scanning calorimetry (DSC) was used to determine the effect of different plasticizers on protein denaturation. Thermal degradation patterns of the extrudates were studied by thermogravimetric analysis (TGA). The effect of plasticizers on the mechanical properties of resins was also assessed by tensile strength measurements. Results indicated that ethylene glycol was able to interact more effectively with quill keratin at the molecular level, exhibiting only one sharp glass transition, better mechanical properties, and higher transparency compared to other plasticized resins. The two phases found in glycerol plasticized material were attributed to glycerol-rich and protein-rich zones. Propylene glycol and diethyl tartrate exhibited lower H-bonding interactions and showed wide transition regions in DMA profiles during heating, suggesting weak and heterogeneous interactions between quill keratin and these plasticizers.

Bioplastics from feather quill.

We report the development of a surrogate spinal cord for evaluating the mechanical suitability of electrode arrays for intraspinal implants. The mechanical and interfacial properties of candidate materials (including silicone elastomers and gelatin hydrogels) for the surrogate cord were tested. The elastic modulus was characterized using dynamic mechanical analysis, and compared with values of actual human spinal cords from the literature. Forces required to indent the surrogate cords to specified depths were measured to obtain values under static conditions. Importantly, to quantify surface properties in addition to mechanical properties normally considered, interfacial frictional forces were measured by pulling a needle out of each cord at a controlled rate. The measured forces were then compared to those obtained from rat spinal cords. Formaldehyde-crosslinked gelatin, 12 wt% in water, was identified as the most suitable material for the construction of surrogate spinal cords. To demonstrate the utility of surrogate spinal cords in evaluating the behavior of various electrode arrays, cords were implanted with two types of intraspinal electrode arrays (one made of individual microwires and another of microwires anchored with a solid base), and cord deformation under elongation was evaluated. The results demonstrate that the surrogate model simulates the mechanical and interfacial properties of the spinal cord, and enables in vitro screening of intraspinal implants.

Development of surrogate spinal cords for the evaluation of electrode arrays used in intraspinal implants.

This work describes the development of a robust and repeatable in vitro 3D culture model of glial scarring, which may be used to evaluate the foreign body response to electrodes and other implants in the central nervous system. The model is based on methacrylated hyaluronic acid, a hydrogel that may be photopolymerized to form an insoluble network. Hydrogel scaffolds were formed at four different macromer concentrations (0.50, 0.75, 1.00, and 1.50% (w/v)). As expected, the elastic modulus of the scaffolds increased with increasing macromer weight fraction. Adult rat brain tested under identical conditions had an elastic modulus range that spanned the elastic modulus of both the 0.50 and 0.75% (w/v) hydrogel samples. Gels formed with higher macromer weight fraction had decreased equilibrium swelling ratio and visibly thicker pore walls relative to gels formed with lower macromer weight fractions. Mixed glial cells (microglia and astrocytes) were then encapsulated in the HA scaffolds. Viability of the mixed cultures was most stable at a cell density of 1 × 10(7) cells/mL. Cell viability at the highest macromer weight fraction tested (1.50% (w/v)) was significantly lower than other tested gels (0.50, 0.75 and 1.00% (w/v)). The inflammatory response of microglia and astrocytes to a microelectrode inserted into the scaffold was assessed over a period of 2 weeks and closely represented that reported in vivo. Microglia responded first to the electrode (increased cell density at the electrode, and activated morphology) followed by astrocytes (appeared to line the electrode in a manner similar to glial scarring). All together, these results demonstrate the potential of the 3D in vitro model system to assess glial scarring in a robust and repeatable manner.

Hyaluronic acid-based 3D culture model for in vitro testing of electrode biocompatibility.

Spinal cord injuries (SCI) can disrupt communications between the brain and the body, resulting in loss of control over otherwise intact neuromuscular systems. Functional electrical stimulation (FES) of the central and peripheral nervous system can use these intact neuromuscular systems to provide therapeutic exercise options to allow functional restoration and to manage medical complications following SCI. The use of FES for the restoration of muscular and organ functions may significantly decrease the morbidity and mortality following SCI. Many FES devices are commercially available and should be considered as part of the lifelong rehabilitation care plan for all eligible persons with SCI.

Functional electrical stimulation and spinal cord injury.

The goal of this study is to improve the integration of implanted microdevices with tissue in the central nervous system (CNS). The long-term utility of neuroprosthetic devices implanted in the CNS is affected by the formation of a scar by resident glial cells (astrocytes and microglia), limiting the viability and functional stability of the devices. Reduction in the proliferation of glial cells is expected to enhance the biocompatibility of devices. We demonstrate the modification of polyimide-insulated microelectrodes with a bioactive peptide KHIFSDDSSE. Microelectrode wires were functionalized with (3-aminopropyl) triethoxy silane (APTES); the peptide was then covalently bonded to the APTES. The soluble peptide was tested in 2D mixed cultures of astrocytes and microglia, and reduced the proliferation of both cell types. The interactions of glial cells with the peptide-modified wires was then examined in 3D cell-laden hydrogels by immunofluorescence microscopy. As expected for uncoated wires, the microglia were first attracted to the wire (7days) followed by astrocyte recruitment and hypertrophy (14days). For the peptide-treated wires, astrocytes coated the wires directly (24h), and formed a thin, stable coating without evidence of hypertrophy, and the attraction of microglia to the wire was significantly reduced. The results suggest a mechanism to improve tissue integration by promoting uniform coating of astrocytes on a foreign body while lessening the reactive response of microglia. We conclude that the bioactive peptide KHIFSDDSSE may be effective in improving the biocompatibility of neural interfaces by both reducing acute glial reactivity and generating stable integration with tissue.

Peptide modification of polyimide-insulated microwires: Towards improved biocompatibility through reduced glial scarring.

An enzyme activated time-temperature indicator (TTI) which produces a direct colour change concomitant to variations in integrated time and temperature conditions is described. This direct colour change is realised by degrading a dye-loaded polyhydroxybutyrate (PHB) film by a depolymerase enzyme. The degradation of the PHB film by the enzyme causes the release of the dye in solution, which in turn undergoes an optical transition from clear to coloured with elapsing time. Macroscopic and microscopic optical observations confirms the uniform distribution of the dye in the PHB film. The dye release kinetics, mediated by the enzymatic reaction, are tested at different temperatures ranging from 4 to 37 °C, and are used to determine the suitability of a dye-loaded PHB as a time-temperature indicator for fresh food products based on kinetic parameters previously reported. The kinetic analysis shows that the activation energy of the dye release process is 74 kJ mol(-1) , and that, at 37 °C, the dye would be totally released within 6 h. However, when incubated at 4 °C, the TTI requires in the range of 168 h (7 days) to release all the dye. These kinetics values highlight the potential of the TTI for monitoring fresh food products that have optimum shelf life around 4 °C.

Time-Temperature Indicator Based on Enzymatic Degradation of Dye-Loaded Polyhydroxybutyrate.

The effect of dimensional constraint, imparted by a variation in film thickness, on the enzymatic degradation of polyhydroxybutyrate (PHB) is reported. The characterization of the crystalline structure and the surface topography of solvent-cast PHB thin films revealed strong correlations between film thickness and both crystallinity and crystal anisotropy, with the polymer film becoming more amorphous with decreasing thickness. The enzymatic degradation of the PHB films was characterized using a high precision diffraction metrology, which enabled the visualization of small variations in the degradation behavior. The results show that the degradation rate increases with decreasing thickness due to the corresponding decrease in crystallinity. However, in a nanoscopic ultra-thin PHB specimen, produced by μ-transfer molding, enzymatic degradation was impeded. The enzymatic degradation rate of the PHB films therefore was found to exhibit a discontinuous trend with respect to film thickness: initially increasing as film thickness was reduced, and then decreasing dramatically once the thickness was reduced to tens of nanometers. In this regime, enzymatic degradation was hindered by the absence of crystalline regions in the films. These results show that a nano-dimensional constraint on PHB films can result in specimens with a tunable response to extracellular enzymes.

Anastasia L. Elias

Department of Chemical and Materials Engineering

University of Alberta

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Anastasia L. Elias

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemical and Materials Engineering

University of Alberta

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Department of Chemical and Materials Engineering