Polyhydroxybutyrate (PHB) biodegradation using bacterial strains with demonstrated and predicted PHB depolymerase activity.

The biodegradation of polyhydroxybutyrate (PHB) has been broadly investigated, but studies typically focus on a single strain or enzyme and little attention has been paid to comparing the interaction of different PHB depolymerase (PhaZ)-producing strains with this biopolymer. In this work, we selected nine bacterial strains-five with demonstrated and four with predicted PhaZ activity-to compare their effectiveness at degrading PHB film provided as sole carbon source. Each of the strains with demonstrated activity were able to use the PHB film (maximum mass losses ranging from 12% after 2 days for Paucimonas lemoignei to 90% after 4 days for Cupriavidus sp.), and to a lower extent Marinobacter algicola DG893 (with a predicted PhaZ) achieved PHB film mass loss of 11% after 2 weeks of exposure. Among the strains with proven PhaZ activity, Ralstonia sp. showed the highest specific activity since less biomass was required to degrade the polymer in comparison to the other strains. In the case of Ralstonia sp., PHB continued to be degraded at pH values as low as pH 3.3-3.7. In addition, analysis of the extracellular fractions of the strains with demonstrated activity showed that Comamonas testosteroni, Cupriavidus sp., and Ralstonia sp. readily degraded both PHB film and PHB particles in agar suspensions. This study highlights that whole cell cultures and enzymatic (extracellular) fractions display different levels of activity, an important factor in the development of PHB-based applications and in understanding the fate of PHB and other PHAs released in the environment. Furthermore, predictions of PhaZ functionality from genome sequencing analyses remain to be validated by experimental results; PHB-degrading ability could not be proven for three of four investigated species predicted by the polyhydroxyalkanoates (PHA) depolymerase engineering database.

Enzymatic degradation of dimensionally constrained polyhydroxybutyrate films.

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.

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

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.

Accelerating the Biodegradation of Poly(lactic acid) through the Inclusion of Plant Fibers: A Review of Recent Advances.

As the global demand for plastics continues to grow, plastic waste is accumulating at an alarming rate with negative effects on the natural environment. The industrially compostable biopolymer poly(lactic acid) (PLA) is therefore being adopted for use in many applications, but the degradation of this material is slow under many end-of-life conditions. This Perspective explores the feasibility of accelerating the degradation of PLA through the formation of PLA-plant fiber composites. Topics include: (a) key properties of PLA, plant-based fibers, and biocomposites; (b) mechanisms of both hydrolytic degradation and biodegradation of PLA-fiber composites; (c) end-of-life degradation of PLA and PLA-plant fiber composites in aerobic and anaerobic conditions, relevant to compost, soil and seawater (aerobic), and landfills (anaerobic); and (d) sustainability and environmental impact of PLA and PLA-plant fiber composites, as evaluated using life cycle assessment. Additional degradation modes, including thermal and photodegradation, which are relevant during processing and use, have been omitted for clarity, as have other types of PLA biocomposites. Multiple studies have shown that the addition of some types of plant fibers to PLA (to form PLA biocomposites) accelerates both water transport in the material and hydrolysis, presenting a possible avenue for improving the end-of-life degradation of these materials. To facilitate the continued development of materials with enhanced biodegradability, we identify a need to implement testing protocols that can distinguish between different degradation mechanisms.

Hemicellulose Films from Curaua Fibers (Ananas erectifolius): Extraction and Thermal and Mechanical Characterization.

With growing environmental concerns over synthetic polymers, natural polymeric materials, such as hemicellulose, are considered a good sustainable alternative. Curaua fibers could be an excellent source of biopolymer as they have a relatively high hemicellulose content (15 wt%) and only a small amount of lignin (7 wt%). In this work, hemicellulose was extracted by an alkaline medium using KOH and the influence of the alkali concentration, temperature, and time was studied. A hemicellulose film was produced by water casting and its mechanical, thermal, and morphological properties were characterized. The results show that the best method, which resulted in the highest hemicellulose yield and lowest contamination from lignin, was using 10% (w/v) KOH concentration, 25 °C, and time of 3 h. The hemicellulose film exhibited better thermal stability and elongation at break than other polymeric films. It also exhibited lower rigidity and higher flexibility than other biodegradable polymers, including polylactic acid (PLA) and polyhydroxybutyrate (PHB).

Bioplastics from feather quill.

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.

High surface area nitrogen-functionalized Ni nanozymes for efficient peroxidase-like catalytic activity.

Nitrogen-functionalization is an effective means of improving the catalytic performances of nanozymes. In the present work, plasma-assisted nitrogen modification of nanocolumnar Ni GLAD films was performed using an ammonia plasma, resulting in an improvement in the peroxidase-like catalytic performance of the porous, nanostructured Ni films. The plasma-treated nanozymes were characterized by TEM, SEM, XRD, and XPS, revealing a nitrogen-rich surface composition. Increased surface wettability was observed after ammonia plasma treatment, and the resulting nitrogen-functionalized Ni GLAD films presented dramatically enhanced peroxidase-like catalytic activity. The optimal time for plasma treatment was determined to be 120 s; when used to catalyze the oxidation of the colorimetric substrate TMB in the presence of H2O2, Ni films subjected to 120 s of plasma treatment yielded a much higher maximum reaction velocity (3.7⊆10-8 M/s vs. 2.3⊆10-8 M/s) and lower Michaelis-Menten coefficient (0.17 mM vs. 0.23 mM) than pristine Ni films with the same morphology. Additionally, we demonstrate the application of the nanozyme in a gravity-driven, continuous catalytic reaction device. Such a controllable plasma treatment strategy may open a new door toward surface-functionalized nanozymes with improved catalytic performance and potential applications in flow-driven point-of-care devices.

Valorization of Hemp Hurds as Bio-Sourced Additives in PLA-Based Biocomposites.

Sourced from agricultural waste, hemp hurds are a low-cost renewable material with high stiffness; however, despite their potential to be used as low-cost filler in natural fiber reinforced polymer biocomposites, they are often discarded. In this study, the potential to add value to hemp hurds by incorporating them into poly(lactic acid) (PLA) biopolymer to form bio-based materials for packaging applications is investigated. However, as with many plant fibers, the inherent hydrophilicity of hemp hurds leads to inferior filler-matrix interfacial interactions, compromising the mechanical properties of the resulting biocomposites. In this study, two chemical treatments, alkaline (NaOH) and alkaline/peroxide (NaOH/H2O2) were employed to treat hemp hurds to improve their miscibility with poly(lactic acid) (PLA) for the formation of biocomposites. The effects of reinforcement content (5, 10, and 15 wt. %), chemical treatments (purely alkaline vs. alkaline/peroxide) and treatment cycles (1 and 3 cycles) on the mechanical and thermal properties of the biocomposites were investigated. The biocomposites of treated hemp hurd powder exhibited enhanced thermal stability in the temperature range commonly used to process PLA (130-180 °C). The biocomposites containing 15 wt. % hemp hurd powder prepared using a single-cycle alkaline/peroxide treatment (PLA/15APHH1) exhibited a Young's modulus of 2674 MPa, which is 70% higher than that of neat PLA and 9.3% higher than that of biocomposites comprised of PLA containing the same wt. % of untreated hemp hurd powder (PLA/15UHH). Furthermore, the tensile strength of the PLA/15APHH1 biocomposite was found to be 62.6 MPa, which was 6.5% lower than that of neat PLA and 23% higher than that of the PLA/15UHH sample. The results suggest that the fabricated PLA/hemp hurd powder biocomposites have great potential to be utilized in green and sustainable packaging applications.

Colorimetric indicators for volatile amines based on succinic anhydride (SAh)-grafted poly (lactic acid) (PLA).

Smart materials that can undergo changes in color upon the detection of amines have the potential to provide information on the freshness of fish and meat. To engineer a material that undergoes a change in color upon exposure to biogenic amines, succinic anhydride functional groups - which are deprotonated in the presence of amines - are grafted onto a biopolymer poly(lactic acid) backbone. This material is then blended with a pH sensitive dye, yielding a material that shows a highly specific response to amines. In this system, the reaction between SAh and amines protonates the dye and leads to an irreversible change in color in the indicators. The resulting change in color was recorded and monitored with standardized photos and UV-Vis spectroscopy. Initially, indicators of different degrees of SAh (from 5 wt% to 45 wt%) grafted onto PLA were exposed to the vapours from a 400 ppm amine solution. The samples with higher degrees of grafting underwent the most visible changes in color. A more detailed study of the effect of temperature and amine concentration was performed on indicators with 30 wt% SAh. The limits of detection, half-time and kinetics of the response are also presented. Higher temperatures and concentrations were found to increase the degree of the color change while decreasing the half-time of the response of the indicators. This work shows potential opportunities for the development of simple real-time amine indicators.

Flexible and Stretchable Temperature Sensors Fabricated Using Solution-Processable Conductive Polymer Composites.

Accurate monitoring of physiological temperatures is important for the diagnosis and tracking of various medical conditions. This work presents the design, fabrication, and characterization of temperature sensors using conductive polymer composites (CPCs) patterned on both flexible and stretchable substrates through both drop coating and direct ink writing (DIW). These composites were formed using a high melting point biopolymer polyhydroxybutyrate (PHB) as the matrix and the graphenic nanomaterial reduced graphene oxide (rGO) as the nanofiller (from 3 to 12 wt%), resulting in a material that exhibits a temperature-dependent resistivity. At room temperature the composites exhibited electrical percolation behavior. Around the percolation threshold, both the carrier concentration and mobility were found to increase sharply. Sensors were fabricated by drop-coating PHB-rGO composites onto ink-jet printed silver electrodes. The temperature coefficient of resistance was determined to be 0.018 /°C for pressed rGO powders and 0.008 /°C for the 3 wt% samples (the highest responsivity of all composites). Composites were found to have good selectivity to temperature with respect to pressure and moisture. Thermal mapping was demonstrated using 6 × 7 arrays of sensing elements. Stretchable devices with a meandering pattern were fabricated using DIW, demonstrating the potential for these materials in healthcare monitoring devices.

Peroxidase-Like Behavior of Ni Thin Films Deposited by Glancing Angle Deposition for Enzyme-Free Uric Acid Sensing.

We present a nanozyme-based biosensor fabricated from nanostructured Ni films deposited onto a silicon wafer by glancing angle deposition (GLAD) for enzyme-free colorimetric monitoring of uric acid (UA), a biomarker for gout, high blood pressure, heart disease, and kidney disease. The helically structured Ni GLAD nanozymes exhibit excellent peroxidase-like activity to accelerate the oxidation reaction of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to a blue product, oxidized TMB (oxTMB), mediated by H2O2. In the presence of UA, oxTMB is reduced, decreasing the optical absorbance by an amount determined by the concentration of UA in the solution. The nanozyme not only mimics peroxidase but also possesses the notable qualities of reusability, simple operation, and reliability, making it environment-friendly and suitable for on-demand analysis. We optimized essential working parameters (pH, TMB concentration, and H2O2 concentration) to maximize the initial color change of the TMB solution. The catalytic activity of this nanozyme was compared with conventional nanofilms using the Michaelis-Menten theory. Based on this, enzyme-free biosensors were developed for colorimetric detection of UA, providing a wide detection range and a limit of detection (3.3 µM) suitable for measurements of UA concentration in sweat. Furthermore, interference from glucose and urea was studied so as to explore the potential of the biosensor for use in the clinical diagnosis of UA biomarkers.

Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases.

Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His-tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta-gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate-based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation.

Amine Responsive Poly(lactic acid) (PLA) and Succinic Anhydride (SAh) Graft-Polymer: Synthesis and Characterization.

Amines are known to react with succinic anhydride (SAh), which in reactions near room temperature, undergoes a ring opening amidation reaction to form succinamic acid (succinic acid-amine). In this work, we propose to form an amine-responsive polymer by grafting SAh to a poly(lactic acid) (PLA) backbone, such that the PLA can provide chemical and mechanical stability for the functional SAh during the amidation reaction. Grafting is performed in a toluene solution at mass content from 10 wt% to 75 wt% maleic anhydride (MAh) (with respect to PLA and initiator), and films are then cast. The molecular weight and thermal properties of the various grafted polymers are measured by gel permeation chromatography and differential scanning calorimetry, and the chemical modification of these materials is examined using infrared spectroscopy. The efficiency of the grafting reaction is estimated with thermogravimetric analysis. The degree of grafting is determined to range from 5% to 42%; this high degree of grafting is desirable to engineer an amine-responsive material. The response of the graft-polymers to amines is characterized using X-ray photoelectron spectroscopy, infrared spectroscopy, and differential scanning calorimetry. Changes in the chemical and thermal properties of the graft-polymers are observed after exposure to the vapors from a 400 ppm methylamine solution. In contrast to these changes, control samples of neat PLA do not undergo comparable changes in properties upon exposure to methylamine vapor. In addition, the PLA-g-SAh do not undergo changes in structure when exposed to vapors from deionized water without amines. This work presents potential opportunities for the development of real-time amine sensors.

Influence of Epoxidized Canola Oil (eCO) and Cellulose Nanocrystals (CNCs) on the Mechanical and Thermal Properties of Polyhydroxybutyrate (PHB)-Poly(lactic acid) (PLA) Blends.

Two major obstacles to utilizing polyhydroxybutyrate (PHB)-a biodegradable and biocompatible polymer-in commercial applications are its low tensile yield strength (<10 MPa) and elongation at break (~5%). In this work, we investigated the modification of the mechanical properties of PHB through the use of a variety of bio-derived additives. Poly(lactic acid) (PLA) and sugarcane-sourced cellulose nanocrystals (CNCs) were proposed as mechanical reinforcing elements, and epoxidized canola oil (eCO) was utilized as a green plasticizer. Zinc acetate was added to PHB and PLA blends in order to improve blending. Composites were mixed in a micro-extruder, and the resulting filaments were molded into 2-mm sheets utilizing a hot-press prior to characterization. The inclusion of the various additives was found to influence the crystallization process of PHB without affecting thermal stability. In general, the addition of PLA and, to a lesser degree, CNCs, resulted in an increase in the Young's modulus of the material, while the addition of eCO improved the strain at break. Overall, samples containing eCO and PLA (at concentrations of 10 wt %, and 25 wt %, respectively) demonstrated the best mechanical properties in terms of Young's modulus, tensile strength and strain at break.

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

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.

Improved response time of flexible microelectromechanical sensors employing eco-friendly nanomaterials.

Flexible force/pressure sensors are of interest for academia and industry and have applications in wearable technologies. Most of such sensors on the market or reported in journal publications are based on the operation mechanism of probing capacitance or resistance changes of the materials under pressure. Recently, we reported the microelectromechanical (MEM) sensors based on a different mechanism: mechanical switches. Multiples of such MEM sensors can be integrated to achieve the same function of regular force/pressure sensors while having the advantages of ease of fabrication and long-term stability in operation. Herein, we report the dramatically improved response time (more than one order of magnitude) of these MEM sensors by employing eco-friendly nanomaterials-cellulose nanocrystals. For instance, the incorporation of polydimethysiloxane filled with cellulose nanocrystals shortened the response time of MEM sensors from sub-seconds to several milliseconds, leading to the detection of both diastolic and systolic pressures in the radial arterial blood pressure measurement. Comprehensive mechanical and electrical characterization of the materials and the devices reveal that greatly enhanced storage modulus and loss modulus play key roles in this improved response time. The demonstrated fast-response flexible sensors enabled continuous monitoring of heart rate and complex cardiovascular signals using pressure sensors for future wearable sensing platforms.

A pH-Indicating Colorimetric Tough Hydrogel Patch towards Applications in a Substrate for Smart Wound Dressings.

The physiological milieu of healthy skin is slightly acidic, with a pH value between 4 and 6, whereas for skin with chronic or infected wounds, the pH value is above 7.3. As testing pH value is an effective way to monitor the status of wounds, a novel smart hydrogel wound patch incorporating modified pH indicator dyes was developed in this study. Phenol red (PR), the dye molecule, was successfully modified with methacrylate (MA) to allow a copolymerization with the alginate/polyacrylamide (PAAm) hydrogel matrix. This covalent attachment prevented the dye from leaching out of the matrix. The prepared pH-responsive hydrogel patch exhibited a porous internal structure, excellent mechanical property, and high swelling ratio, as well as an appropriate water vapour transmission rate. Mechanical responses of alginate/P(AAm-MAPR) hydrogel patches under different calcium and water contents were also investigated to consider the case of exudate accumulation into hydrogels. Results showed that increased calcium amount and reduced water content significantly improved the Young's modulus and elongation at break of the hydrogels. These characteristics indicated the suitability of hydrogels as wound dressing materials. When pH increased, the color of the hydrogel patches underwent a transition from yellow (pH 5, 6 and 7) to orange (7.4 and 8), and finally to red (pH 9). This range of color change matches the clinically-meaningful pH range of chronic or infected wounds. Therefore, our developed hydrogels could be applied as promising wound dressing materials to monitor the wound healing process by a simple colorimetric display, thus providing a desirable substrate for printed electronics for smart wound dressing.

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

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. STATEMENT OF SIGNIFICANCE: The peptide KHIFSDDSSE has previously been shown in vitro to both reduce the proliferation of astrocytes, and to increase the adhesion of astrocyte to glass substrates. Here, we demonstrate a method to apply uniform coatings of peptides to microwires, which could readily be generalized to other peptides and surfaces. We then show that when peptide-modified wires are inserted into 3D cell-laden hydrogels, the normal cellular reaction (microglial activation followed by astrocyte recruitment and hypertrophy) does not occur, rather astrocytes are attracted directly to the surface of the wire, forming a relatively thin and uniform coating. This suggests a method to improve tissue integration of implanted devices to reduce glial scarring and ultimately reduce failure of neural interfaces.

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

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Immobilization of Active Bacteriophages on Polyhydroxyalkanoate Surfaces.

A rapid, efficient technique for the attachment of bacteriophages (phages) onto polyhydroxyalkanoate (PHA) surfaces has been developed and compared to three reported methods for phage immobilization. Polymer surfaces were modified to facilitate phage attachment using (1) plasma treatment alone, (2) plasma treatment followed by activation by 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), (3) plasma-initiated acrylic acid grafting, or (4) plasma-initiated acrylic acid grafting with activation by EDC and sulfo-NHS. The impact of each method on the surface chemistry of PHA was investigated using contact angle analysis and X-ray photoelectron spectroscopy. Each of the four treatments was shown to result in both increased hydrophilicity and in the modification of the surface functional groups. Modified surfaces were immersed in suspensions of phage T4 for immobilization. The highest level of phage binding was observed for the surfaces modified by plasma treatment alone. The change in chemical bond states observed for surfaces that underwent plasma treatment is suspected to be the cause of the increased binding of active phages. Plasma-treated surfaces were further analyzed through phage-staining and fluorescence microscopy to assess the surface density of immobilized phages and their capacity to capture hosts. The infective capability of attached phages was confirmed by exposing the phage-immobilized surfaces to the host bacteria Escherichia coli in both plaque and infection dynamic assays. Plasma-treated surfaces with immobilized phages displayed higher infectivity than surfaces treated with other methods; in fact, the equivalent initial multiplicity of infection was 2 orders of magnitude greater than with other methods. Control samples - prepared by immersing polymer surfaces in phage suspensions (without prior plasma treatment) - did not show any bacterial growth inhibition, suggesting they did not bind phages from the suspension.