Long-term in vitro 3D hydrogel co-culture model of inflammatory bowel disease

The in vitro study of the pathogenesis of inflammatory bowel disease (IBD) requires a cell model which closely reflects the characteristics of the in vivo intestinal epithelium. This study aimed to investigate the application of L-pNIPAM hydrogel as a scaffold to develop a long-term 3D co-culture model of Caco-2 and HT29-MTX cells under conditions analogous to inflammation, to determine its potential use in studying IBD. Monocultures and co-cultures were layered on L-pNIPAM hydrogel scaffolds and maintained under dynamic culture conditions for up to 12 weeks. Treatments with IL-1β, TNFα, and hypoxia for 1 week were used to create an inflammatory environment. Following prolonged culture, the metabolic activity of Caco-2 monoculture and 90% Caco-2/10% HT29-MTX co-cultures on L-pNIPAM hydrogels were increased, and finger-like structures, similar in appearance to villi were observed. Following treatment with IL-1β, TNFα and hypoxia, ALP and ZO-1 were decreased, MUC2 increased, and MUC5AC remained unchanged. ADAMTS1 was increased in response to hypoxia. Caspase 3 expression was increased in response to TNFα and hypoxic conditions. In conclusion, L-pNIPAM hydrogel supported long-term co-culture within a 3D model. Furthermore, stimulation with factors seen during inflammation recapitulated features seen during IBD

An indirect Raman spectroscopy method for the quantitative measurement of respirable crystalline silica collected on filters inside respiratory equipment

This article describes the development of an analytical method to measure respirable crystalline silica (RCS) collected on filters by a miniature sampler placed behind respirators worn by workers to evaluate their ‘true’ exposure. Test samples were prepared by aerosolising a calibration powder (Quin B) and by pipetting aliquots from suspensions of bulk material (NIST 1878a and Quin B) onto filters. Samples of aerosolised RCS collected onto polyvinyl chloride PVC filters were ashed and their residue was suspended in isopropanol and filtered into a 10 mm diameter area onto silver filters. Samples were also collected by the Health and Safety Executive's (HSE) miniature sampler from within the facepiece of a respirator on a breathing manikin during a simulated work activity. Results obtained using Raman spectroscopy were compared with X-ray diffraction (XRD) measurements, which was used as a reference method and a linear relationship was obtained. Raman has similar estimates of uncertainty when compared with the XRD methods over the measurement range from 5 to 50 mg and obtained the lowest limit of detection (LOD) of 0.26 mg when compared with XRD and Fourier Transform Infrared FTIR methods. A significant intercept and slope coefficient greatly influenced the higher LOD for indirect XRD method. The level of precision and low LOD for Raman spectroscopy will potentially enable workplace measurements at lower concentrations below the Workplace Exposure Limit (WEL) than are achieved using current analytical instrumentation. Different inward leakage ratio (ILR) measurement approaches were compared using six aerosolised sandstone dust tests. For the three highest inward leakage ratios the Portacount® obtained higher values than the RCS mass or the miniWRAS ratios, the latter of which reporting both particle number and quartz mass concentration. However, these limited ILR data were insufficient to establish statistical correlations between the measurement methods

Vibrational spectroscopic studies of degradation and diffusion process in poly(ethylene terephthalate).

The interaction of polyethylene terephthalate (PET) with water at both ambient and elevated temperatures has been studied. The diffusion of water, at ambient temperatures, into PET films, of the order of 10 pm thick, has been followed using Fourier transform infrared attenuated total reflectance, FT-IR ATR, spectroscopy. Films of differing degrees of ciystallinity were prepared using two different methods. One method involved the annealing of the cast films at 85-90&deg; C for different lengths of time, to obtain a range of crystallinities. The other method involved the incorporation of different amounts of an /sophthalate group, to obtain a range of ciystallinities. The rate of water diffusion with time was then measured as a function of ciystallinity. The diffusion was shown to be classically Fickian in nature and the diffusion coefficients decreased with increased polymer crystallinity for both sets of films. The perturbation of the v(OH) band of the water in the polymer matrix was studied as both a function of time (i.e. concentration) and crystallinity. The water band was shown to be decoupled at low concentrations within the polymer matrix, indicating a breaking up of the water hydrogen bonding network. At higher concentrations, longer times, the v(OH) band gained more 'pure water' like character, but remained at higher frequency than pure water even at equilibrium water content, suggesting clustering of the water molecules, but an overall weakening of the hydrogen bonding network relative to the pure water spectrum.The study of the interactions of water at elevated temperatures including the degradative hydrolysis of PET at 90&deg; C was undertaken using reflection absorption infrared spectroscopy (RAIRS). Films of < 150 nm were immersed in pure water at 52, 62, 70, 80 and 90&deg; C and the effect on the polymeric structure was examined. At temperatures below 90&deg; C the effects noted, on the time scales studied, were annealing effects, resulting in an increase in crystallinity. Estimations of the apparent activation energy of the gauche to trans isomerisation, for different degrees of crystallinity, were calculated and were found to be lower than those reported in the literature in air. This difference was thought to be a result of the plasticisation effects of water.At 90&deg; C, during several days of immersion, the polymer was found to undergo hydrolysis. Complex changes in the RAIRS spectrum were related to changes in the polymeric structure, resulting from degradation. The autocatalytic nature of the degradation was highlighted, as was the loss of (small) mobile species from the polymer matrix. A mechanism involving the preferred site of hydrolysis being a terminal ester group was proposed. Comparisons with hot alkaline hydrolysis were made. This occurred much faster and with more random chain scission.The diffusion of two organic liquids, methanol and ethylene glycol, into PET was studied. The diffusion was shown to be non-Fickian in nature due to the swelling and crystallisation that accompanied the diffusion. For the amorphous PET films, diffusion was accompanied by swelling and crystallisation for both molecules and was fitted to a dual sorption model. There was spectroscopic evidence for both a 'bound' alcohol - PET moiety and a 'free' alcohol species within the polymer matrix. For methanol in PET, the proportion of the sorbed alcohol which was 'bound' was found to increase with crystallinity, but for ethylene glycol the reverse was true. For methanol diffusion, increasing the crystallinity was shown to have a drastic effect on both the rate of diffusion and degree of swelling. For ethylene glycol diffusion, the degree of crystallinity appeared to affect the rate of swelling and the initial rate of sorption of penetrant, but the rate of the subsequent diffusion seemed to be unaffected by morphology.The interface between two layers of 20 and 30 pm co-extruded PET laminates of PET and PET with an /.sophthalate comonomer, were examined using confocal Raman microscopy. The methods of confocal depth profiling through the polymer laminate and scanning, step-wise, along a cut edge were compared. The interface was examined using the carbonyl band width of PET as an indicator of crystallinity. The interface was shown to be 2 - 3 pm thick, independent of film thickness and contain a gradient of [equation] v(C=0) band width, indicating either interdiffusion or a trans-esterification reaction between the two polymer layers during co-extrusion

Application of a Fourier Transform Infrared (FTIR) Principal Component Regression (PCR) Chemometric Method for the Quantification of Respirable Crystalline Silica (Quartz), Kaolinite, and Coal in Coal Mine Dusts from Australia, UK, and South Africa

This article describes the approach used to assess the performance of a Fourier transform infrared (FTIR) and principal component regression (PCR) chemometric method when measuring respirable quartz, kaolinite, and coal in samples from a variety of mines from different countries; relative to target assigned values determined using X-ray diffraction (XRD). For comparison, FTIR results using the partial least squares regression (PLSR) method are also available. Bulk dusts from 10 Australian mines were scanned using XRD and grouped into three sets based on the levels of quartz, kaolinite, and feldspar within their crystalline mineral composition. Prediction samples were generated from 5 of these Australian mine dusts, Durrans coal dust, 2 mine dusts from the UK, and a single South African mine dust (71 samples in total) by collecting the aerosolized respirable dust onto 25-mm diameter polyvinylchloride filters using the Safety in Mines Personal Dust Sampler (SIMPEDS) operating at 2.2 l min−1. The predicted values from the FTIR chemometric methods were compared with assigned target values determined using a direct on-aerosol filter XRD analysis method described in Method for the Determination of Hazardous Substances (MDHS) 101. Limits of detection (LOD) and uncertainty values for each analyte were calculated from a linear regression between target and predicted values. The uncertainty was determined using the calibration uncertainty equation for an unweighted regression. FTIR results from PCR and PLSR are very similar. For the PCR method, the LOD for quartz, kaolinite, and coal were 5, 25, and 71 µg, respectively. For quartz, an LOD of 5 µg corresponds to an airborne quartz concentration of 10 µg m−3, assuming a 4-h sampling time and collection flow rate of 2.2 l min−1. The FTIR measurement met the expected performance criteria outlined in ISO 20581 when sampling quartz for more than 4 h using a flow rate of 2.2 l min−1 at a concentration of 0.1 mg m−3 (100 µg m−3), the current workplace exposure limit in Great Britain. This method met the same performance criteria when measuring exposures at the Australian Workplace Exposure Standard (WES) concentration of 0.05 mg m−3, although in this case a sampling period greater than 8 h was needed

Early changes in the extracellular matrix of the degenerating intervertebral disc, assessed by Fourier transform infrared imaging.

Mechanical overloading induces a degenerative cell response in the intervertebral disc. However, early changes in the extracellular matrix (ECM) are challenging to assess with conventional techniques. Fourier Transform Infrared (FTIR) imaging allows visualization and quantification of the ECM. We aim to identify markers for disc degeneration and apply these to investigate early degenerative changes due to overloading and katabolic cell activity. Three experiments were conducted; Exp 1.: In vivo, lumbar spines of seven goats were operated: one disc was injected with chondroitinase ABC (mild degeneration) and compared to the adjacent disc (control) after 24 weeks. Exp 2a: Ex vivo, caprine discs received physiological loading (n=10) or overloading (n=10) in a bioreactor. Exp 2b: Cell activity was diminished prior to testing by freeze-thaw cycles, 18 discs were then tested as in Exp 2a. In all experiments, FTIR images (spectral region: 1000-1300 cm ) of mid-sagittal slices were analyzed using multivariate curve resolution. In vivo, FTIR was more sensitive than biochemical and histological analysis in identifying reduced proteoglycan content (p=0.046) and increased collagen content in degenerated discs (p<0.01). Notably, FTIR analysis additionally showed disorganization of the ECM, indicated by increased collagen entropy (p=0.011). Ex vivo, the proteoglycan/collagen ratio decreased due to overloading (p=0.047) and collagen entropy increased (p=0.047). Cell activity affected collagen content only (p=0.044). FTIR imaging allows a more detailed investigation of early disc degeneration than traditional measures. Changes due to mild overloading could be assessed and quantified. Matrix remodeling is the first detectable step towards intervertebral disc degeneration. [Abstract copyright: Copyright © 2018. Published by Elsevier Ltd.

Injectable hydrogel induces regeneration of naturally degenerate human intervertebral discs in a loaded organ culture model.

Low back pain resulting from disc degeneration is a leading cause of disability worldwide. However, to date few therapies target the cause and fail to repair the intervertebral disc (IVD). This study investigates the ability of an injectable hydrogel (NPgel), to inhibit catabolic protein expression and promote matrix expression in human nucleus pulposus (NP) cells within a tissue explant culture model isolated from degenerate discs. Furthermore, the injection capacity of NPgel into naturally degenerate whole human discs, effects on mechanical function, and resistance to extrusion during loading were investigated. Finally, the induction of potential regenerative effects in a physiologically loaded human organ culture system was investigated following injection of NPgel with or without bone marrow progenitor cells. Injection of NPgel into naturally degenerate human IVDs increased disc height and Young's modulus, and was retained during extrusion testing. Injection into cadaveric discs followed by culture under physiological loading increased MRI signal intensity, restored natural biomechanical properties and showed evidence of increased anabolism and decreased catabolism with tissue integration observed. These results provide essential proof of concept data supporting the use of NPgel as an injectable therapy for disc regeneration. STATEMENT OF SIGNIFICANCE: Low back pain resulting from disc degeneration is a leading cause of disability worldwide. However, to date few therapies target the cause and fail to repair the intervertebral disc. This study investigated the potential regenerative properties of an injectable hydrogel system (NPgel) within human tissue samples. To mimic the human in vivo conditions and the unique IVD niche, a dynamically loaded intact human disc culture system was utilised. NPgel improved the biomechanical properties, increased MRI intensity and decreased degree of degeneration. Furthermore, NPgel induced matrix production and decreased catabolic factors by the native cells of the disc. This manuscript provides evidence for the potential use of NPgel as a regenerative biomaterial for intervertebral disc degeneration

Injectable biomaterial induces regeneration of the intervertebral disc in a caprine loaded disc culture model †

Back pain is the leading cause of disability with half of cases attributed to intervertebral disc (IVD) degeneration, yet currently no therapies target this cause. We previously reported an ex vivo caprine loaded disc culture system (LDCS) that accurately represents the cellular phenotype and biomechanical environment of human IVD degeneration. Here, the efficacy of an injectable hydrogel system (LAPONITE® crosslinked pNIPAM-co-DMAc, (NPgel)) to halt or reverse the catabolic processes of IVD degeneration was investigated within the LDCS. Following enzymatic induction of degeneration using 1 mg mL−1 collagenase and 2 U mL−1 chondroitinase ABC within the LDCS for 7 days, IVDs were injected with NPgel alone or with encapsulated human bone marrow progenitor cells (BMPCs). Un-injected caprine discs served as degenerate controls. IVDs were cultured for a further 21 days within the LDCS. Tissues were then processed for histology and immunohistochemistry. No extrusion of NPgel was observed during culture. A significant decrease in histological grade of degeneration was seen in both IVDs injected with NPgel alone and NPgel seeded with BMPCs, compared to un-injected controls. Fissures within degenerate tissue were filled by NPgel and there was evidence of native cell migration into injected NPgel. The expression of healthy NP matrix markers (collagen type II and aggrecan) was increased, whereas the expression of catabolic proteins (MMP3, ADAMTS4, IL-1β and IL-8) was decreased in NPgel (±BMPCs) injected discs, compared to degenerate controls. This demonstrates that NPgel promotes new matrix production at the same time as halting the degenerative cascade within a physiologically relevant testing platform. This highlights the potential of NPgel as a future therapy for IVD degeneration

Arginine–glycine–aspartic acid functional branched semi-interpenetrating hydrogels

For the first time a series of functional hydrogels based on semi-interpenetrating networks with both branched and crosslinked polymer components have been prepared and we show the successful use of these materials as substrates for cell culture. The materials consist of highly branched poly(N-isopropyl acrylamide)s with peptide functionalised end groups in a continuous phase of crosslinked poly(vinyl pyrrolidone). Functionalisation of the end groups of the branched polymer component with the GRGDS peptide produces a hydrogel that supports cell adhesion and proliferation. The materials provide a new synthetic functional biomaterial that has many of the features of extracellular matrix, and as such can be used to support tissue regeneration and cell culture. This class of high water content hydrogel material has important advantages over other functional hydrogels in its synthesis and does not require postprocessing modifications nor are functional-monomers, which change the polymerisation process, required. Thus, the systems are amenable to large scale and bespoke manufacturing using conventional moulding or additive manufacturing techniques. Processing using additive manufacturing is exemplified by producing tubes using microstereolithography

Epithelialization of hydrogels achieved by amine functionalization and co-culture with stromal cells

The aim of this study was to develop a hydrogel which would be suitable for corneal cell re-epithelialization when used as a corneal implant. To achieve this, a series of hydrogels were functionalized with primary amines by post-polymerization reactions between amine compounds and glycidyl ether groups attached to the hydrogels. We report a strong correlation between the structure of the amine and the viability of stromal cells and epithelial cells cultured on these hydrogels. Subsequent co-culture of epithelial and stromal cells on the amine modified hydrogels allowed successful expansion of epithelial cells on surfaces functionalized with alkyl α–ω diamines with carbon chain lengths of between 3 and 6. Analysis of variance showed that corneal epithelial cells had a strong preference for surfaces functionalized by the reaction of excess 1,3 diaminopropane with units of glycidyl methacrylate compared to the reaction products of other amines (ammonia; 1,2-diaminoethane; 1,4-diaminobutane or 1,6-diaminohexane). We suggest this approach of amine functionalization combined with stromal/epithelial co-culture offers a promising new approach to achieving a secure corneal epithelium. Keywords: Epithelial cell

Data on Medicare eligibility and cancer screening utilization

Health insurance is associated with increased utilization of cancer screening services. Data on breast, prostate and colorectal cancer screening were abstracted from the 2012 Behavioral Risk Factor and Surveillance System. This data in brief includes two sets of analyses: (i) the use of cancer screening in individuals within the low-income bracket and (ii) determinants for each of the three approaches to colorectal cancer screening (fecal occult blood test, colonoscopy and sigmoidoscopy+fecal occult blood test). Covariates included education attainment, residency, and access to health care provider. The data supplement our original research article on the effect of Medicare eligibility on cancer screening utilization “The impact of Medicare eligibility on cancer screening behaviors” [1]