Two for the Price of One: Heterobivalent Ligand Design Targeting Two Binding Sites on Voltage-Gated Sodium Channels Slows Ligand Dissociation and Enhances Potency.

Voltage-gated sodium (NaV) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of NaV channel ligands. We conjugated µ-conotoxin KIIIA, which occludes the pore of the NaV channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide-alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40-120 Å. The ligand with an 80 Å linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4-24-fold higher potency compared to those of the monovalent peptides for the human NaV1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of NaV channels.

In vivo assessment of high-molecular-weight polyethylene core suture tape for intra-articular ligament reconstruction: an animal study.

AIMS: Options for the treatment of intra-articular ligament injuries are limited, and insufficient ligament reconstruction can cause painful joint instability, loss of function, and progressive development of degenerative arthritis. This study aimed to assess the capability of a biologically enhanced matrix material for ligament reconstruction to withstand tensile forces within the joint and enhance ligament regeneration needed to regain joint function. MATERIALS AND METHODS: A total of 18 New Zealand rabbits underwent bilateral anterior cruciate ligament reconstruction by autograft, FiberTape, or FiberTape-augmented autograft. Primary outcomes were biomechanical assessment (n = 17), microCT (µCT) assessment (n = 12), histological evaluation (n = 12), and quantitative polymerase chain reaction (qPCR) analysis (n = 6). RESULTS: At eight weeks, FiberTape alone or FiberTape-augmented autograft demonstrated increased biomechanical stability compared with autograft regarding ultimate load to failure (p = 0.035), elongation (p = 0.006), and energy absorption (p = 0.022). FiberTape-grafted samples also demonstrated increased bone mineral density in the bone tunnel (p = 0.039). Histological evaluation showed integration of all grafts in the bone tunnels by new bone formation, and limited signs of inflammation overall. A lack of prolonged inflammation in all samples was confirmed by quantification of inflammation biomarkers. However, no regeneration of ligament-like tissue was observed along the suture tape materials. Except for one autograft failure, no adverse events were detected. CONCLUSION: Our results indicate that FiberTape increases the biomechanical performance of intra-articular ligament reconstructions in a verified rabbit model at eight weeks. Within this period, FiberTape did not adversely affect bone tunnel healing or invoke a prolonged elevation in inflammation. Cite this article: Bone Joint J 2019;101-B:1238-1247.

Impedimetric array in polymer microfluidic cartridge for low cost point-of-care diagnostics.

Deep Vein Thrombosis and pulmonary embolism (DVT/PE) is one of the most common causes of unexpected death for hospital in-patients. D-dimer is used as a biomarker within blood for the diagnosis of DVT/PE. We report a low-cost microfluidic device with a conveniently biofunctionalised interdigitated electrode (IDE) array and a portable impedimetric reader as a point-of-care (POC) device for the detection of D-dimer to aid diagnosis of DVT/PE. The IDE array elements, fabricated on a polyethylenenaphtalate (PEN) substrate, are biofunctionalised in situ after assembly of the microfluidic device by electropolymerisation of a copolymer of polypyrrole to which is immobilised a histidine tag anti-D-Dimer antibody. The most consistent copolymer films were produced using chronopotentiometry with an applied current of 5µA for a period of 50 s using a two-electrode system. The quality of the biofunctionalisation was monitored using optical microscopy, chronopotentiometry curves and impedimetric analysis. Measurement of clinical plasma sample with a D-dimer at concentration of 437 ng/mL with 15 biofunctionalised IDE array electrodes gave a ratiometric percentage of sample reading against the blank with an average value of 124 ±â€¯15 at 95% confidence. We have demonstrated the concept of a low cost disposable microfluidic device with a receptor functionalised on the IDE array for impedimetric detection towards POC diagnostics. Changing the receptor on the IDE array would allow this approach to be used for the direct detection of a wide range of analytes in a low cost manner.

Enzymatic glucose/oxygen biofuel cells: Use of oxygen-rich cathodes for operation under severe oxygen-deficit conditions.

A glucose/oxygen biofuel cell (BFC) that can operate continuously under oxygen-free conditions is described. The oxygen-deficit limitations of metabolite/oxygen enzymatic BFCs have been addressed by using an oxygen-rich cathode binder material, polychlorotrifluoroethylene (PCTFE), which provides an internal oxygen supply for the BFC reduction reaction. This oxygen-rich cathode component mitigates the potential power loss in oxygen-free medium or during external oxygen fluctuations through internal supply of oxygen, while the bioanode employs glucose oxidase-mediated reactions. The internal oxygen supply leads to a prolonged energy-harvesting in oxygen-free solutions, e.g., maintaining over 90% and 70% of its initial power during 10- and 24-h operations, respectively, in the absence of oxygen. The new strategy holds considerable promise for energy-harvesting and self-powered biosensing applications in oxygen-deficient conditions.

Development of biomimetic in vitro fatigue assessment for UHMWPE implant materials.

An important research goal in the field of biomaterials lies in the progressive amendment of in vivo tests with suitable in vitro experiments. Such approaches are gaining more significance nowadays because of an increasing demand on life sciences and the ethical issues bound to the sacrifice of animals for the sake of scientific research. Another advantage of transferring the experiments to the in vitro field is the possibility of accurately control the boundary conditions and experimental parameters in order to reduce the need of validation tests involving animals. With the aim to reduce the amount of needed in vivo studies for this cause, a short-time in vitro test procedure using instrumented load increase tests with superimposed environmental loading has been developed at TUD to assess the mechanical long-term durability of ultra-high molecular weight polyethylene (UHMWPE) under fatigue loading in a biological environment.

Radiopaque UHMWPE sublaminar cables for spinal deformity correction: Preclinical mechanical and radiopacifier leaching assessment.

Polymeric sublaminar cables have a number of advantages over metal cables in the field of spinal deformity surgery, with decreased risk of neurological injury and potential for higher correction forces as the two most predominant. However, currently available polymer cables are radiolucent, precluding postoperative radiological assessment of instrumentation stability and integrity. This study provides a preclinical assessment of a woven UHMWPE cable made with radiopaque UHMWPE fibers. Our primary goal was to determine if the addition of a radiopacifier negatively affects the mechanical properties of UHMWPE woven cables. Tensile mechanical properties were determined and compared to suitable controls. Radiopacity was evaluated and radiopacifier leaching was assessed in vitro and in vivo. Finally, in vivo bismuth organ content was quantified after a 24-week implantation period in sheep. Results show that the mechanical properties of woven UHMWPE cables were not deleteriously affected by the addition of homogenously dispersed bismuth oxide particles within each fiber. Limited amounts of bismuth oxide were released in vitro, well below the toxicological threshold. Tissue concentrations lower than generally accepted therapeutic dosages for use against gastrointestinal disorders, well below toxic levels, were discovered in vivo. These results substantiate controlled clinical introduction of these radiopaque UHMWPE cables. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 771-779, 2018.

Cardiovascular toxicity assessment of poly (ethylene imine)- based cationic polymers on zebrafish model.

Poly(ethylene imine)s (PEIs) have gained enormous attention in designing novel drug delivery systems for cancer treatment. High molecular weight of PEIs such as PEI 25 kD are promising for their drug carrying capacity. However, increased molecular weight is associated with toxicity. Currently, the toxicity evaluation of PEIs is mainly focused on the culture cell models, with very few studies investigating the risk assessment in vivo. Herein, the systemic evaluation of branched PEI 25 kD and PEI-CyD (PC) composed of low molecular PEI (Mw 600) and ß-cyclodextrin (ß-CyD) is performed in zebrafish model and endothelial cells. Our finding indicate that exposure of PC and PEI 25 kD can induce high mortality rate, shorten hatching time, promote malformations and cell apoptosis of zebrafish embryos in a dose-dependent manner. Most significantly, the cationic polymer PC and PEI 25 kD can decrease heart rate of zebrafish embryos and down-regulate the expression of heart development-related genes, which demonstrate their cardiovasculature toxicity. In this case, we further investigate the effect of PC on endothelial cells. Indeed, PC can induce endothelial cells dysfunction, including oxidative stress and apoptosis which are involved in cardiovascular diseases. These fundamental studies provide valuable insights into the biocompatible evaluation of PEI based drug carriers.

Micro X-Ray Computed Tomography Mass Loss Assessment of Different UHMWPE: A Hip Joint Simulator Study on Standard vs. Cross-Linked Polyethylene.

More than 60.000 hip arthroplasty are performed every year in Italy. Although Ultra-High-Molecular-Weight-Polyethylene remains the most used material as acetabular cup, wear of this material induces over time in vivo a foreign-body response and consequently osteolysis, pain, and the need of implant revision. Furthermore, oxidative wear of the polyethylene provoke several and severe failures. To solve these problems, highly cross-linked polyethylene and Vitamin-E-stabilized polyethylene were introduced in the last years. In in vitro experiments, various efforts have been made to compare the wear behavior of standard PE and vitamin-E infused liners. In this study we compared the in vitro wear behavior of two different configurations of cross-linked polyethylene (with and without the add of Vitamin E) vs. the standard polyethylene acetabular cups. The aim of the present study was to validate a micro X-ray computed tomography technique to assess the wear of different commercially available, polyethylene's acetabular cups after wear simulation; in particular, the gravimetric method was used to provide reference wear values. The agreement between the two methods is documented in this paper.

Quantification of structural changes of UHMWPE components in total joint replacements.

BACKGROUND: At present time the number of implantations of joint replacements as well as their revisions increases. Higher demands are required on the quality and longevity of implants. The aim of this work was to determine the degree of oxidative degradation and the amount of free/residual radicals in selected ultra-high molecular weight polyethylene (UHMWPE) components of the joint replacements and demonstrate that the measured values are closely connected with quality and lifetime of the polymer components. METHODS: We tested both new (4 samples) and explanted (4 samples) UHMWPE polymers for total joint replacements. The samples were characterized by infrared spectroscopy (IR), electron spin resonance (ESR) and microhardness (MH) test. The IR measurements yielded the values of oxidation index and trans-vinylene index. The ESR measurements gave the free radicals concentration. RESULTS: In the group of new polyethylene components, we found oxidation index values ranging from 0.00-0.03 to 0.24. The trans-vinylene index values ranged from 0.044 to 0.080. The value of free radical concentration was zero in virgin and also in sample of Beznoska Company and non-zero in the other samples. In the group of explanted components, the measured values were associated with their history, micromechanical properties and performance in vivo. CONCLUSIONS: We demonstrated that measuring of oxidative damage may help the orthopaedic surgeon in estimating the quality of UHMWPE replacement component and thus radically to avoid early joint replacement failure due to worse polyethylene quality.

A novel method for the assessment of targeted PEI-based nanoparticle binding based on a static surface plasmon resonance system.

The delivery of nucleic acids is a major hurdle in gene therapy or therapeutic gene knockdown, and the development of intelligent and safe nanoparticles as carrier systems is thus under intense investigation. The introduction of ligands for their targeted delivery is of major interest. Here, we describe a novel approach for the analysis of the binding properties of antibody-functionalized nanoparticles, using surface plasmon resonance (SPR) in a static cuvette system. By chemical coupling of the Epidermal Growth Factor Receptor (EGFR)-specific antibody cetuximab to poly(ethylene imine) (PEI) via a PEG-spacer and subsequent DNA or siRNA complexation, we generated targeted nanoplexes with low surface charge. Antibody-mediated uptake into EGFR overexpressing cells was observed. SPR measurements with use of a novel, protein A-based sandwich system for the immobilization of the target receptor in its correct steric orientation allowed the analysis of the specific PEI-PEG-cetuximab binding to EGFR and the determination of binding affinities. Importantly, our cuvette-based SPR assay system was also suitable for the monitoring of ligand-mediated nanoparticle binding, without convection or shear stress. We conclude that our SPR sandwich system allows the precise analysis of the binding of ligand-functionalized nanoparticles in real-time, and we thus establish SPR for the in vitro evaluation of ligand modifications for generating targeted nanoparticles.

Layer-by-layer self assembly of a water-soluble phthalocyanine on gold. Application to the electrochemical determination of hydrogen peroxide.

A self-assembled molecular film of a water-soluble cobalt tetrasulfophthalocyanine was deposited on a gold substrate premodified with poly(diallyldimethylammonium chloride). The process of layer-by-layer assembly on the gold substrate was characterized using UV-Vis, Raman spectroscopy, ellipsometry, contact angle measurements, atomic force microscopy and electrochemical methods. Results demonstrate the formation of a completely covered phthalocyanine film on the gold surface. UV-Vis spectra indicated the formation of monolayer film of the phthalocyanine on the surface. The functionalized surface is uniformly covered and becomes hydrophilic after modification. The modified gold surface exhibits a reversible redox behavior and acts as an electronic conductor for the electrochemical reduction of hydrogen peroxide in pH 7.0 phosphate buffer. A linear increase in the catalytic current is observed for the reduction of hydrogen peroxide in the concentration range from 1 to 20 µM, with a detection limit of 0.4 µM. Hydrogen peroxide spiked pond water sample showed a recovery of 94% indicating the method is selective and can be applied for various applications. The present method is a simple, cost effective and sensitive electrochemical method for the detection and quantification of hydrogen peroxide.

Assessment of bulk and surface properties of medical grade UHMWPE based nanocomposites using Nanoindentation and microtensile testing.

A thrust on the enhancement of the mechanical properties of ultra high molecular weight polyethylene (UHMWPE) to enhance its longevity has taken a new direction with the advent of nanomaterials and carbon nanotubes. In the present work, UHMWPE was reinforced by chemically treated multi walled carbon nanotubes (MWCNTs) at different concentrations such as 0.5, 1.0, 1.5, 2.0, 2.5 and 5 wt%. The mechanical properties of nanocomposites were studied using a Nanoindentation technique and micro-tensile testing. It is observed that the toughness, ultimate stress, fracture strain, and yield stress of medical grade UHMWPE were enhanced by 176, 93, 70, and 44%, respectively at an optimum concentration of 2 wt% MWCNTs reinforcement. The mechanism for the enhancement of mechanical properties was confirmed by the micro-Raman and calorimetric technique. The reduction of the mechanical properties of nanocomposites beyond optimum concentration of MWCNTs was confirmed by the rheological studies. The generation of microvoids on the nanocomposites was verified by the scanning electron microscopy technique. Nanoindentation characteristics revealed that the surface hardness of UHMWPE was increased by 75% by the reinforcement of 2 wt% of MWCNTs. The Young's modulus obtained at the surface of nanocomposites was observed to be 9.8% higher than that of surface layer removed sample for 2 wt% nanocomposite. It is concluded that the presence of MWCNTs enhanced the mechanical properties and surface properties of medical grade UHMWPE.

Rapid photogeneration of silver nanoparticles in ethanolic solution: a kinetic study.

Ag nanoparticles have been synthesized via UV irradiation of ethanolic solution of AgNO3 in presence of pluronic F127 surfactant. This study is aimed at developing a rapid, simple and green method to prepare Ag nanoparticles and understanding its generation kinetics. The formation dependency of silver nanoparticles on the concentration of reactants, UV exposure time and temperature has been investigated by using UV-vis spectroscopy. The 2D map technique has been used for the first time to estimate the switching time between the nucleation and growth of Ag nanoparticles. Appropriate kinetic models were used for modelling of both stages.

Probabilistic assessment of fatigue initiation data on highly crosslinked ultrahigh molecular weight polyethylenes.

Ultrahigh molecular weight polyethylenes (UHMWPE) showing wear resistance, oxidative stability and good mechanical performance go on being a relevant research area in biomaterials for total joint replacements, where fatigue happens to be a recurrent damage mode that needs to be investigated. While crack propagation lifetime has been extensively studied, fatigue initiation data are scarcely offered in the literature, often due to the higher costs implied in the experimental programs. Moreover, their analysis is not always suitable to obtain reliable guidance. Different deterministic and probabilistic methods, generally resting on empirical bases have been previously used to analyze the fatigue initiation data. In this work, the probabilistic Weibull regression model of Castillo et al., based on both physical and statistical conditions, such as weakest link principle and the necessary compatibility between life-time and stress range distributions, is applied for the first time in the assessment of fatigue results of polymers, particularly to highly crosslinked UHMWPEs (HXLPEs). Accordingly, different published experimental data corresponding to HXLPE stabilized by thermal treatments and with α-tocopherol (vitamin E) are re-analyzed. Additional data are incorporated to assess the influence of notched HXLPE on fatigue performance. New conclusions are drawn from this revision.

Final report of the Amended Safety Assessment of PVM/MA copolymer and its related salts and esters as used in cosmetics.

Polyvinyl methyl ether/maleic acid (PVM/MA) copolymer, and its related salts and esters, are used in cosmetics, mainly as binders, film formers, and hair fixatives. Animal and human data relevant to the use of these ingredients in cosmetic products were reviewed by the CIR Expert Panel. The Panel concluded that these ingredients are safe for use in cosmetic products.

On the assessment of oxidative and microstructural changes after in vivo degradation of historical UHMWPE knee components by means of vibrational spectroscopies and nanoindentation.

This study reports on the suitability of different experimental techniques to evaluate chemical, microstructural, and mechanical changes associated with in vivo oxidation encountered in historical polyethylene components. To accomplish this aim, eight traceable tibial inserts were analyzed after revision surgery. The knee bearings were gamma sterilized in air and implanted for an average of 11.5 years after a shelf life of no longer than 1 year. Characterization of oxidation and transvinylene indexes, crystallinity, amorphous, and intermediate phase fractions, along with hardness and surface modulus, were performed in transverse sections of each bearing using Fourier transform infrared spectroscopy, Raman spectroscopy, and nanoindentation, respectively. Generally, subsurface maxima in the crystallinity, oxidation index, and hardness were observed at a depth of about 1 mm in all of the bearings. The superior surfaces and anterior-posterior faces of the inserts exhibited significantly higher oxidation and greater crystallinity than the inferior side. These observations suggest that the metallic tray may limit the access of molecular oxygen to the backside of the tibial inserts. We conclude that chemical, physical, and mechanical properties data confirm the occurrence of in vivo degradation in the long-term implanted knee components following gamma irradiation in air. Furthermore, infrared spectroscopy alone appeared to provide excellent insight into the oxidation and crystallization state of the in vivo oxidized polyethylene.

Tribological assessment of a flexible carbon-fibre-reinforced poly(ether-ether-ketone) acetabular cup articulating against an alumina femoral head.

New material combinations have been introduced as the bearing surfaces of hip prostheses in an attempt to prolong their life by overcoming the problems of failure due to wear-particle-induced osteolysis. This will hopefully reduce the need for revision surgery. The study detailed here used a hip simulator to assess the volumetric wear rates of large-diameter carbon-fibre-reinforced pitch-based poly(ether-ether-ketone) (CFR-PEEK) acetabular cups articulating against alumina femoral heads. The joints were tested for 25 x 10(6) cycles. Friction tests were also performed on these joints to determine the lubrication regime under which they operate. The average volumetric wear rate of the CFR-PEEK acetabular component of 54 mm diameter was 1.16 mm(3)/10(6) cycles, compared with 38.6 mm(3)/10(6) cycles for an ultra-high-molecular-weight polyethylene acetabular component of 28 mm diameter worn against a ceramic head. This extremely low wear rate was sustained over 25 x 10(6) cycles (the equivalent of up to approximately 25 years in vivo). The frictional studies showed that the joints worked under the mixed-boundary lubrication regime. The low wear produced by these joints showed that this novel joint couple offers low wear rates and therefore may be an alternative material choice for the reduction of osteolysis.

Improvement in the assessment of wear of total knee replacements using coordinate-measuring machine techniques.

Total joint replacement is one of the most common elective surgical procedures performed worldwide, with an estimate of 1.5x 10(6) operations performed annually. Currently joint replacements are expected to function for 10-15 years; however, with an increase in life expectancy, and a greater call for knee replacement due to increased activity levels, there is a requirement to improve their function to offer longer-term improved quality of life for patients. Wear analysis of total joint replacements has long been an important means in determining failure mechanisms and improving longevity of these devices. The effectiveness of the coordinate-measuring machine (CMM) technique for assessing volumetric material loss during simulated life testing of a replacement knee joint has been proved previously by the present authors. The purpose of the current work is to present an improvement to this method for situations where no pre-wear data are available. To validate the method, simulator tests were run and gravimetric measurements taken throughout the test, such that the components measured had a known wear value. The implications of the results are then discussed in terms of assessment of joint functionality and development of standardized CMM-based product standards. The method was then expanded to allow assessment of clinically retrieved bearings so as to ascertain a measure of true clinical wear.

A new method of in vitro wear assessment of the UHMWPE tibial insert in total knee replacement.

The wear of the ultra-high-molecular-weight polyethylene (UHMWPE) tibial insert was determined using a four-station knee simulator. The bearings were subjected to flexion/extension (between 0 and 58 degrees ), anterior-posterior translation (between 0 and -5.2 mm), internal-external rotation (between -1.9 degrees and +5.7 degrees ), and a maximum axial load of 2.6 KN, as per ISO 14243-1,2,3. The wear tests were run at a frequency of 1.1 Hz for 5 million cycles, and the wear of the inserts (n = 3) was determined using the gravimetric method. The novelty of the study was a special setup developed to simulate, as realistically as possible, in vivo conditions; this involved fixing the femoral component to the distal end of a synthetic femur model by a qualified orthopedic surgeon using an approved method. After 5 million cycles, the mean weight losses were 11.16, 19.74, and 12.61 mg for specimens #1, #2, and #3, respectively. Visual and nondestructive inspections for each of the test specimens showed similar wear tracks and these were very similar to those seen on inserts retrieved after 2 years in vivo. These results show the efficacy of the new in vitro UHMWPE wear assessment method. Furthermore, a comparison between the present in vitro results and those reported in a relevant previous study provide some insight into the influence of the method used to fix the femoral component to the simulator on the wear magnitude and patterns of the tibial insert.

Subjective assessment by patients of the efficiency of two denture adhesive pastes.

PURPOSE: This study evaluated the subjective responses of 30 denture wearers with regard to the effectiveness of two denture adhesive pastes. MATERIALS AND METHODS: Thirty patients responded to questions related to retention, chewing ability, taste, duration of adhesives in the mouth, and removal; comparing the use of two denture adhesives based on polymethylvinylether-maleic anhydride compounds or carboxymethyl cellulose. RESULTS: The denture adhesive paste based on polymethylvinylether-maleic anhydride (PVM-MA) compound was rated higher (73% and 87%) than carboxymethyl cellulose (CC) adhesive paste (60% and 37%) on chewing ability and duration in the mouth (p= 0.0001 and p= 0.0001, respectively) by experienced denture wearers in both maxilla and mandible. There were no statistically significant differences between the two denture adhesive pastes on the retention of maxillary dentures, taste, and removal of the adhesive (p= 0.08, p= 0.67, and p= 0.41, respectively). CONCLUSIONS: All subjects responded that the retention of their dentures was either a little better or much better when using either of the adhesive pastes.