In vitro electroretinogram for the study of the functionality of differentiated retinal pigment epithelium cells

The purpose of this study is to develop and test a method to reveal if the retinal pigment epithelium (RPE) cells differentiated from human embryonic stem cells (hESC) support the functions of photoreceptors. hESC-derived RPE (hESC–RPE) cells offer a potent cell source for cell replacement therapy that may be used to prevent certain eye diseases. Methods to assure the functionality of the RPE cells are well warranted. Electroretinograms (ERG) measure the electrophysiological response of the retina to light stimuli. A setup was developed that enables the measurement of ERG in vitro from mice retinas cultured together with hESC–RPE cells. The co-culture of RPE and retinas seems to be a viable tool to assess the functionality of RPE in vitro. However, owing to limited sample size results were somewhat mixed, and thus it was not possible to prove that hESC–RPE cells enhance the ERG response of a mouse retina in vitro. The long-term culturing of the retinas needs to be refined to acquire more conclusive evidence of the supporting role of the RPE and to explore the full potential of the co-culture and ERG methods in assessing RPE functionality.     

Adaptation of canine femoral head articular cartilage to long distance running exercise in young beagles.

OBJECTIVE--To study the effects of long term (one year), long distance (up to 40 km/day) running on the metabolism of articular cartilage the biosynthesis of proteoglycans was examined by in vitro labelling of anterior (weight bearing) and posterior (less weight bearing) areas of the femoral head from young beagles. METHODS--Total sulphate incorporation rates were determined and distribution of the incorporated sulphate was localised by quantitative autoradiography. Concentration and extractability of the proteoglycans were determined, and proteoglycan structures were investigated by gel filtration chromatography, agarose gel electrophoresis, and chemical determinations. RESULTS--In the less weight bearing area the amount of extractable proteoglycans was decreased (p < or = 0.02), simultaneously with an increased concentration of residual glycosaminoglycans in the tissue after 4 M GuCl extraction (p < or = 0.05). In control animals proteoglycan synthesis was most active in the deep zone of the cartilage, whereas exercise increased synthesis in the intermediate zone. There was a tendency to a lower keratan: chondroitin sulphate ratio in the running dogs. No macroscopical or microscopical signs of articular degeneration or injury were visible in any of the animals. CONCLUSION--The articular cartilage of the femoral head showed a great capacity to adapt to the increased mechanical loading. The reduced proteoglycan extractability in the less weight bearing area changed it similar to the weight bearing area, suggesting that the low extractability of proteoglycans reflects the long term loading history of articular cartilage. The congruency of the femoral head with acetabulum seems to protect the cartilage from the untoward alterations that occur in the femoral condyles subjected to a similar running programme.     

Analysis of lapine cartilage matrix after radiosynovectomy with holmium-166 ferric hydroxide macroaggregate

OBJECTIVE: To study the short and long term effects of radiosynovectomy on articular cartilage in growing and mature rabbits. METHODS: The articular cartilage of the distal femurs of rabbits was examined four days, two months, and one year after radiosynovectomy with holmium-166 ferric hydroxide macroaggregate ([(166)Ho]FHMA). Arthritic changes were evaluated from histological sections by conventional and polarised light microscopy, and glycosaminoglycan measurements using safranin O staining, digital densitometry, and uronic acid determination. Proteoglycan synthesis was studied by metabolic [(35)]sulphate labelling followed by autoradiography, and electrophoretic analysis of extracted proteoglycans. Northern analyses were performed to determine the mRNA levels of type II collagen, aggrecan, and Sox9 in cartilage samples. RESULTS: Radiosynovectomy had no major effect on the histological appearance of articular cartilage in mature rabbits, whereas more fibrillation was seen in [(166)Ho]FHMA radiosynovectomised knee joints of growing rabbits two months after treatment, but not after one year. Radiosynovectomy did not cause changes in the glycosaminoglycan content of cartilage or in the synthesis or chemical structure of proteoglycans. No radiosynovectomy related changes were seen in the mRNA levels of type II collagen, whereas a transient down regulation of aggrecan and Sox9 mRNA levels was seen in young rabbits two months after [(166)Ho]FHMA radiosynovectomy. CONCLUSIONS: [(166)Ho]FHMA radiosynovectomy caused no obvious chondrocyte damage or osteoarthritic changes in mature rabbits, but in growing rabbits some transient radiation induced effects were seen--for example, mild cartilage fibrillation and down regulation of cartilage-specific genes.     

Effects of chitosan and bioactive glass modifications of knitted and rolled polylactide‐based 96/4 L/D scaffolds on chondrogenic differentiation of adipose stem cells

The performance of biodegradable knitted and rolled 3‐dimensional (3D) polylactide‐based 96/4 scaffolds modified with bioactive glass (BaG) 13‐93, chitosan and both was compared with regard to the viability, proliferation and chondrogenic differentiation of rabbit adipose stem cells (ASCs). Scaffold porosities were determined by micro‐computed tomography (μCT). Water absorption and degradation of scaffolds were studied during 28‐day hydrolysis in Tris‐buffer. Viability, number and differentiation of ASCs in PLA96/4 scaffolds were examined in vitro. The dimensions of the scaffolds were maintained during hydrolysis and mass loss was detected only in the BaG13‐93 containing scaffolds. ASCs adhered and proliferated on each scaffold type. Cell aggregation and expression of chondral matrix components improved in all scaffold types in chondrogenic medium. Signs of hypertrophy were detected in the modified scaffolds but not in the plain PLA96/4 scaffold. Chondrogenic differentiation was most enhanced in the presence of chitosan. These findings indicate that the plain P scaffold provided a good 3D‐matrix for ASC proliferation whereas the addition of chitosan to the PLA96/4 scaffold induced chondrogenic differentiation independent of the medium. Accordingly, a PLA96/4 scaffold modified by chitosan could provide a functional and bioactive basis for tissue‐engineered chondral implants. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparison of Chondroitin Sulfate and Hyaluronic Acid Doped Conductive Polypyrrole Films for Adipose Stem Cells

Polypyrrole (PPy) is a conductive polymer that has aroused interest due to its biocompatibility with several cell types and high tailorability as an electroconductive scaffold coating. This study compares the effect of hyaluronic acid (HA) and chondroitin sulfate (CS) doped PPy films on human adipose stem cells (hASCs) under electrical stimulation. The PPy films were synthetized electrochemically. The surface morphology of PPy–HA and PPy–CS was characterized by an atomic force microscope. A pulsed biphasic electric current (BEC) was applied via PPy films non-stimulated samples acting as controls. Viability, attachment, proliferation and osteogenic differentiation of hASCs were evaluated by live/dead staining, DNA content, Alkaline phosphatase activity and mineralization assays. Human ASCs grew as a homogenous cell sheet on PPy–CS surfaces, whereas on PPy–HA cells clustered into small spherical structures. PPy–CS supported hASC proliferation significantly better than PPy–HA at the 7 day time point. Both substrates equally triggered early osteogenic differentiation of hASCs, although mineralization was significantly induced on PPy–CS compared to PPy–HA under BEC. These differences may be due to different surface morphologies originating from the CS and HA dopants. Our results suggest that PPy–CS in particular is a potential osteogenic scaffold coating for bone tissue engineering.     

Arterial pulse wave velocity in relation to carotid intima-media thickness, brachial flow-mediated dilation and carotid artery distensibility: the Cardiovascular Risk in Young Finns Study and the Health 2000 Survey

ObjectiveIncreased arterial pulse wave velocity (PWV) is a strong predictor of cardiovascular events and mortality. The data regarding the relationships between PWV and other indices of vascular damage is limited and partly controversial. We conducted the present study to examine PWV in relation to non-invasive measures of early atherosclerosis (brachial flow-mediated dilation [FMD], carotid intima-media thickness [IMT]) and local arterial stiffness (carotid artery distensibility [Cdist]).MethodsThe study population consisted of 1754 young adults (aged 30–45 years, 45.5% males) participating in the Cardiovascular Risk in Young Finns Study (YFS), and of 336 older adults (aged 46–76 years, 43.2% males) participating in the Health 2000 Survey. FMD was measured only in the YFS cohort. FMD, IMT and Cdist were assessed by ultrasound, and PWV was measured using the whole-body impedance cardiography device.ResultsIn young adults, FMD and IMT were not associated with PWV independently of cardiovascular risk factors. Moreover, FMD status was not found to modulate the association between cardiovascular risk factors and PWV. In older adults, PWV and IMT were directly and independently associated (β = 1.233, p = 0.019). In both cohorts, PWV was inversely related with Cdist, and this relation remained significant (p < 0.04) in models adjusted for cardiovascular risk factors.ConclusionsThe current findings suggest that PWV reflects a different aspect of vascular damage than FMD or IMT in young adults, whereas in older adults the information provided by PWV and IMT may be, to some extent, similar as regards subclinical vascular damage. The present observations also suggest that PWV and Cdist represent, at least in part, a similar adverse vascular wall process.     

Human articular cartilage proteoglycans are not undersulfated in osteoarthritis

Chondroitin sulfate is the major constituent of cartilage. Inadequate sulfate availability results in the production of undersulfated proteoglycans. In osteoarthritis, there is a net loss of articular cartilage proteoglycans. Theoretically, it is possible that during the progress of disease undersulfated glycosaminoglycans are synthesized producing proteoglycans with poorer biological properties. In this study, we tested whether in early human osteoarthritic articular cartilage (Mankin's score of 2 and 3) or more advanced disease (Mankin's score over 3), there are proteoglycans that contain a higher relative amount of nonsulfated chondroitin disaccharide isomer in their chondroitin sulfate chains by analyzing the molar ratios of chondroitin sulfate disaccharide isoforms with fluorophore-assisted carbohydrate electrophoresis. Our results indicated that the nonsulfated disaccharide of chondroitin sulfate formed in average only 1-2% of the total chondroitin sulfate. More important, the molar ratio of nonsulfated disaccharide did not appear to be increased in the osteoarthritic articular cartilage. We conclude that undersulfation of articular cartilage proteoglycans is not present in the human osteoarthritic joint.     

Diet,cow's milk protein antibodies and the risk of IDDM in Finnish children

Summary Associations of infant feeding patterns and milk consumption with cow's milk protein antibody titres were studied in 697 newly-diagnosed diabetic children, 415 sibling-control children and 86 birth-date-and sex-matched population-based control children in the nationwide Childhood Diabetes in Finland study. IgA and IgG antibody titres to the proteins of cow's milk formula, BLG and BSA, and IgM antibody titres to cow's milk formula proteins were measured by ELISA. Several inverse correlations were observed between the duration of breast-feeding or age at introduction of dairy products and antibody titres, and positive correlations were observed between milk consumption and antibody titres in all three populations studied. Multivariate analyses which included the infant feeding variables, milk consumption and current age simultaneously showed that the earlier the introduction of dairy products and the greater the consumption of milk was, the higher several antibody titres were. High IgA antibody titres to cow's milk formula were associated with a greater risk of IDDM both among diabeticpopulation-control and diabetic-sibling-control pairs when adjusted for other cow's milk antibody titres, dietary variables and in diabetic-sibling-control pairs also for ICA. The results suggest that young age at introduction of dairy products and high milk consumption during childhood increase the levels of cow's milk antibodies and that high IgA antibodies to cow's milk formula are independently associated with increased risk of IDDM.Abbreviations IDDM Insulin-dependent diabetes mellitus - BLG betalactoglobulin - BSA bovine serum albumin - ICA islet cell antibodies - IAA insulin autoantibodies - OR odds ratio - CI confidence interval     

Spatial aspects of oncogenic signalling determine the response to combination therapy in slice explants from Kras‐driven lung tumours

A key question in precision medicine is how functional heterogeneity in solid tumours informs therapeutic sensitivity. We demonstrate that spatial characteristics of oncogenic signalling and therapy response can be modelled in precision‐cut slices from Kras‐driven non‐small‐cell lung cancer with varying histopathologies. Unexpectedly, profiling of in situ tumours demonstrated that signalling stratifies mostly according to histopathology, showing enhanced AKT and SRC activity in adenosquamous carcinoma, and mitogen‐activated protein kinase (MAPK) activity in adenocarcinoma. In addition, high intertumour and intratumour variability was detected, particularly of MAPK and mammalian target of rapamycin (mTOR) complex 1 activity. Using short‐term treatment of slice explants, we showed that cytotoxic responses to combination MAPK and phosphoinositide 3‐kinase–mTOR inhibition correlate with the spatially defined activities of both pathways. Thus, whereas genetic drivers determine histopathology spectra, histopathology‐associated and spatially variable signalling activities determine drug sensitivity. Our study is in support of spatial aspects of signalling heterogeneity being considered in clinical diagnostic settings, particularly to guide the selection of drug combinations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.