Miniaturized multi-enzyme biosensors integrated with pH sensors on flexible polymer carriers for in vivo applications

An electrochemical glucose sensor has been integrated, together with a pH sensor, on a flexible polyimide substrate for in vivo applications. The glucose sensor is based on the measurement of H₂O₂ produced by the membrane-entrapped enzyme glucose oxidase (GOD). To minimize electrochemical interference, an electrode configuration was designed to perform differential measurements. The solid-state pH sensor employs a PVC-based neutral carrier membrane. The enzymes GOD and catalase were immobilized into two layers of photolithographically patterned hydrogels. The intended use of this device is the short-term monitoring of glucose and pH in intensive care units and operating theatres, especially for neurosurgical applications. The developed immobilization technique can also be used to create integrated multi-sensor chips for clinical analysers. The glucose and pH sensor exhibited excellent performance during tests in buffer solutions, serum and whole blood.     

Live reporting for hypoxia: Hypoxia sensor–modified mesenchymal stem cells as in vitro reporters

Natural oxygen gradients occur in tissues of biological organisms and also in the context of three-dimensional (3D) in vitro cultivation. Oxygen diffusion limitation and metabolic oxygen consumption by embedded cells produce areas of hypoxia in the tissue/matrix. However, reliable systems to detect oxygen gradients and cellular response to hypoxia in 3D cell culture systems are still missing. In this study, we developed a system for visualization of oxygen gradients in 3D using human adipose tissue–derived mesenchymal stem cells (hAD-MSCs) modified to stably express a fluorescent genetically engineered hypoxia sensor HRE-dUnaG. Modified cells retained their stem cell characteristics in terms of proliferation and differentiation capacity. The hypoxia-reporter cells were evaluated by fluorescence microscopy and flow cytometry under variable oxygen levels (2.5%, 5%, and 7.5% O₂). We demonstrated that reporter hAD-MSCs output is sensitive to different oxygen levels and displays fast decay kinetics after reoxygenation. Additionally, the reporter cells were encapsulated in bulk hydrogels with a variable cell number, to investigate the sensor response in model 3D cell culture applications. The use of hypoxia-reporting cells based on MSCs represents a valuable tool for approaching the genuine in vivo cellular microenvironment and will allow a better understanding of the regenerative potential of AD-MSCs.     

Hydrogel baits with low‐dose thiamethoxam for sustainable Argentine ant management in commercial orchards

Argentine ants, Linepithema humile (Mayr) (Hymenoptera: Formicidae), are a significant pest in various agricultural systems around the world, and are often associated with outbreaks of phloem‐feeding hemipteran insects. Previous research has evaluated a number of active ingredients and management approaches for controlling Argentine ant populations in agricultural systems, but various regulatory and economic issues have limited the development of effective management tools. Current chemical controls rely on residual sprays or toxic baits, each one posing unique disadvantages that limit their usefulness and efficacy. This study evaluated the potential of water‐storing crystals to effectively deliver liquid baits to Argentine ants. The efficacy of bait crystals containing 0.007% thiamethoxam was first evaluated in laboratory colonies. In addition, field studies were performed in a commercial plum orchard to determine the efficacy of the bait crystals. Protein marking was used within the orchard to examine the distribution of the bait in Argentine ant populations when delivered via water‐storing crystals. Results of laboratory tests showed that water‐storing crystals containing 0.007% thiamethoxam are highly attractive and effective against Argentine ants and require ca. 3–5 days to kill all castes and life stages. Results of the protein‐marking study demonstrated that the percentage of ants carrying protein‐labeled sugar water decreases sharply with increasing distance from the bait station. Bait movement was limited to within 17 m of the bait dispenser. Furthermore, bait efficacy tests in the field showed that Argentine ants can be effectively controlled using liquid thiamethoxam baits deployed via water‐storing crystals. The bait was highly effective and ant densities throughout the baited plots declined by 94 ± 2% within 14 days. The results of this study demonstrate that (1) thiamethoxam is highly effective for Argentine ant control in fruit orchards when used in low concentrations (0.007%), and (2) water‐storing crystals are an effective tool for delivering liquid baits to Argentine ants in agricultural settings.     

A graphene/zinc oxide nanocomposite film protects dental implant surfaces against cariogenic Streptococcus mutans

Oral biofilms play a crucial role in the development of dental caries and other periodontal diseases. Streptococcus mutans is one of the primary etiological agents in dental caries. Implant systems are regularly employed to replace missing teeth. Oral biofilms accumulate on these implants and are the chief cause of dental implant failure. In the present study, the potential of graphene/zinc oxide nanocomposite (GZNC) against the cariogenic properties of Streptococcus mutans was explored and the anti-biofilm behaviour of artificial acrylic teeth surfaces coated with GZNC was examined. Acrylic teeth are a good choice for implants as they are low cost, have low density and can resist fracture. Microscopic studies and anti-biofilm assays showed a significant reduction in biofilm in the presence GZNC. GZNC was also found to be nontoxic against HEK-293 (human embryonic kidney cell line). The results indicate the potential of GZNC as an effective coating agent for dental implants by efficiently inhibiting S. mutans biofilms.     

Mechanical properties of modern calcite- (Mergerlia truncata) and phosphate-shelled brachiopods (Discradisca stella and Lingula anatina) determined by nanoindentation

We measured distribution patterns of hardness and elastic modulus by nanoindentation on shells of the rhynchonelliform brachiopod Mergerlia truncata and the linguliform brachiopods Discradisca stella and Lingula anatina. The rhynchonelliformea produce calcitic shells while the linguliformea produce chitinophosphatic shells. Dorsal and ventral valves, commissure and hinge of the calcitic shell of M. truncata show different nanohardness values (from 2.3 to 4.6 GPa) and E-modulus (from 52 to 76 GPa). The hardness of the biocalcite is always increased compared to inorganic calcite. We attribute the effects to different amounts of inter- and intracrystalline organic matrix. Profiles parallel to the radius of curvature of the valves cutting through the different layers of shell material surprisingly show quite uniform values of nanohardness and modulus of elasticity. Nanoindentation tests on the chitinophosphatic brachiopods D. stella and L. anatina reflect the hierarchical structure composed of laminae with varying degree of mineralization. As a result of the two-phase composite of biopolymer nanofibrils reinforced with Ca-phosphate nanoparticles, nanohardness, and E-modulus correlate almost linearly from (H = 0.25 GPa, E = 2.5 GPa) to (H = 2.5 GPa, E = 50 GPa). The mineral provides stiffness and hardness, the biopolymer provides flexibility; and the composite provides fracture toughness. Gradients in the degree of mineralization reduce potential stress concentrations at the interface between stiff mineralized and soft non-mineralized laminae. For the epibenthic chitinophosphatic D. stella the lamination is also present but less pronounced than for the infaunal L. anatina, and the overall distribution of material strength in the cross-sectional profile shows a maximum in the center and a decrease towards the inner and outer shell margins (modulus of elasticity from 30 to 12 GPa, hardness from 1.7 to 0.5 GPa). Accordingly, the two epibenthic forms, calcitic M. truncata and chitinophosphatic D. stella display fairly bulky (homogeneous) nanomechanical properties of their shell materials, while the burrowing infaunal L. anatina is distinctively laminated. The strongly mineralized laminae, which provide the strength to the shell, are also brittle, but keeping them as thin as possible, allows some bending flexibility. This flexibility is not required for the epibenthic life style.     

Cross-linking density alters early metabolic activities in chondrocytes encapsulated in poly(ethylene glycol) hydrogels and cultured in the rotating wall vessel

In designing a tissue engineering strategy for cartilage repair, selection of both the bioreactor, and scaffold is important to the development of a mechanically functional tissue. The hydrodynamic environment associated with many bioreactors enhances nutrient transport, but also introduces fluid shear stress, which may influence cellular response. This study examined the combined effects of hydrogel cross-linking and the hydrodynamic environment on early chondrocyte response. Specifically, chondrocytes were encapsulated in poly(ethylene glycol) (PEG) hydrogels having two different cross-linked structures, corresponding to a low and high cross-linking density. Both cross-linked gels yielded high water contents (92% and 79%, respectively) and mesh sizes of 150 and 60 A respectively. Cell-laden PEG hydrogels were cultured in rotating wall vessels (RWV) or under static cultures for up to 5 days. Rotating cultures yielded low fluid shear stresses (< or = 0.11 Pa) at the hydrogel periphery indicating a laminar hydrodynamic environment. Chondrocyte response was measured through total DNA content, total nitric oxide (NO) production, and matrix deposition for glycosaminoglycans (GAG). In static cultures, gel cross-linking had no effect on DNA content, NO production, or GAG production; although GAG production increased with culture time for both cross-linked gels. In rotating cultures, DNA content increased, NO production decreased, and overall GAG production decreased when compared to static controls for the low cross-linked gels. For the high cross-linked gels, the hydrodynamic environment had no effect on DNA content, but exhibited similar results to the low cross-linked gel for NO production, and matrix production. Our findings demonstrated that at early culture times, when there is limited matrix production, the hydrodynamic environment dramatically influences cell response in a manner dependent on the gel cross-linking, which may impact long-term tissue development.     

Probing vocal fold fibroblast response to hyaluronan in 3D contexts

A number of treatments are being investigated for vocal fold (VF) scar, including designer implants. The aim of the present study was to validate a 3D model system for probing the effects of various bioactive moieties on VF fibroblast (VFF) behavior toward rational implant design. We selected poly(ethylene glycol) diacrylate (PEGDA) hydrogels as our base‐scaffold due to their broadly tunable material properties. However, since cells encapsulated in PEGDA hydrogels are generally forced to take on rounded/stellate morphologies, validation of PEGDA gels as a 3D VFF model system required that the present work directly parallel previous studies involving more permissive scaffolds. We therefore chose to focus on hyaluronan (HA), a polysaccharide that has been a particular focus of the VF community. Toward this end, porcine VFFs were encapsulated in PEGDA hydrogels containing consistent levels of high M _w HA (${\rm HA}_{{\rm H}{M}_{\rm W} } $ ), intermediate M_w HA (${\rm HA}_{{\rm I}{M}_{\rm W} } $ ), or the control polysaccharide, alginate, and cultured for 7 and 21 days. ${\rm HA}_{{\rm H}{M}_{\rm W} } $ promoted sustained increases in active ERK1/2 relative to ${\rm HA}_{{\rm I}{M}_{\rm W} } $ . Furthermore, VFFs in ${\rm HA}_{{\rm I}{M}_{\rm W} } $ gels displayed a more myofibroblast‐like phenotype, higher elastin production, and greater protein kinase C (PkC) levels at day 21 than VFFs in ${\rm HA}_{{\rm H}{M}_{\rm W} } $ and alginate gels. The present results are in agreement with a previous 3D study of VFF responses to ${\rm HA}_{{\rm I}{M}_{\rm W} } $ relative to alginate in collagen‐based scaffolds permissive of cell elongation, indicating that PEGDA hydrogels may serve as an effective 3D model system for probing at least certain aspects of VFF behavior. Biotechnol. Bioeng. 2009; 104: 821–831 © 2009 Wiley Periodicals, Inc.     

Chondroitin sulfate and dynamic loading alter chondrogenesis of human MSCs in PEG hydrogels

While biochemical and biomechanical cues are known to play important roles in directing stem cell differentiation, there remains little known regarding how these inextricably linked biological cues impact the differentiation fate of human marrow stromal cells (hMSCs). This study investigates the chondrogenic differentiation potential of hMSCs when encapsulated in a three dimensional (3D) hydrogel and exposed to a biochemical cue, chondroitin sulfate (ChS), a biomechanical cue, dynamic loading, and their combination. hMSCs were encapsulated in bioinert poly(ethylene glycol) (PEG) hydrogels only, PEG hydrogels modified with covalently incorporated methacrylated ChS and cultured under free swelling conditions or subjected to delayed intermittent dynamic loading for 2 weeks. The 3D hydrogel environment led to the expression of chondrogenic genes (SOX9) and proteins (aggrecan and collagen II), but also upregulated hypertrophic genes (RUNX2 and Col X mRNA) and proteins (collagen X), while the application of loading generally led to a downregulation in chondrogenic proteins (collagen II). The presence of ChS led to elevated levels of aggrecan, but also collagen I, protein expression and when combined with dynamic loading downregulated, but did not suppress, hypertrophic genes (Col X and RUNX2) and collagen I protein expression. Taken together, this study demonstrates that while the 3D environment induces early terminal differentiation during chondrogenesis of hMSCs, the incorporation of ChS into PEG hydrogels may slow the terminal differentiation process down the hypertrophic lineage particularly when dynamic loading is applied. Biotechnol. Bioeng. 2012; 109: 2671–2682. © 2012 Wiley Periodicals, Inc.     

Development of a CrN/Cu nanocomposite coating on titanium-modified stainless steel for antibacterial activity against Pseudomonas aeruginosa

A relatively simple method was developed to fabricate CrN/Cu nanocomposite coatings using pulsed DC magnetron sputtering for application in antibacterial activity. These nanocomposite coatings were applied on titanium (Ti)-modified stainless steel substrata (D-9 alloy) and the antibacterial activity of these coating with respect to the Gram-negative bacterium Pseudomonas aeruginosa was investigated qualitatively and quantitatively. Scanning electron microscopy, epifluorescence microscope analyses, and total viable counts confirmed that inclusion of copper in the CrN/Cu nanocomposite coatings provided antibacterial activity against P. aeruginosa. The quantitative examination of the bacterial activity of P. aeruginosa was estimated by the survival ratio as calculated from the number of viable cells which formed colonies on nutrient agar plates.