Creep assessment in hyperelastic material by a 3D Neural Network Reconstructor using bulge testing

A new methodology based on a Neural Network approach is developed for three-dimensional reconstruction of pseudo-hemispherical geometry of a hyperelastic specimen during a creep bulge test. On the basis of this procedure, the inflated membrane stress and strain states are established to determine the material mechanical properties. A new and economic experimental apparatus, used to conduct the bulge test, is provided with a sliding crossbar for the dome image acquisition in order to detect its strain state. A pressure regulator is used to ensure constant pressure, essential for creep. The artificial neural network was trained by using the position (x, y, z) of the membrane points at different values of the pressure measured in the bulge test chamber. The main characteristics of the system, which consist of the experimental apparatus and the neural post-processing, are accurate characterization of the materials and the ability to analyze materials with moderate anisotropy. This method has been verified on carbon black-reinforced SBR. The hyperelastic parameters that were obtained are in agreement with the values reported in scientific literature.     

Calcium-based coordination polymers from a solvothermal synthesis of HKUST-1 in 3D printed autoclaves

A mixed-metal 1D coordination polymer [CaCu(HBTC)₂(H2O)₈]_n (where H₃BTC – benzene-1,3,5-tric arboxylic acid) was obtained in a solvothermal synthesis of a well-known copper-containing metal–organic framework [Cu₃(BTC)₂(H₂O)₃]_n (HKUST-1) in autoclaves 3D-printed from commercial polypropylene. This material was a source of calcium ions, apparently, leaking from a colorant (calcium carbonate) promoted by glacial acetic acid as a modulator used to produce large single crystals of HKUST-1. This finding was confirmed by elemental analysis and a model experiment that resulted in a new calcium-based 1D coordination polymer [Ca(H₂BTC)₂(H₂O)₅]_n under the same solvothermal conditions with no copper or calcium salts put into a 3D-printed autoclave.     

Mass spectrometry imaging of endogenous metabolites in response to doxorubicin in a novel 3D osteosarcoma cell culture model

Three‐dimensional (3D) cell culture is a rapidly emerging field, which mimics some of the physiological conditions of human tissues. In cancer biology, it is considered a useful tool in predicting in vivo chemotherapy responses, compared with conventional two‐dimensional (2D) cell culture. We have developed a novel 3D cell culture model of osteosarcoma composed of aggregated proliferative tumour spheroids, which shows regions of tumour heterogeneity formed by aggregated spheroids of polyclonal tumour cells. Aggregated spheroids show local necrotic and apoptotic regions and have sizes suitable for the study of spatial distribution of metabolites by mass spectrometry imaging (MSI). We have used this model to perform a proof‐of‐principle study showing a heterogeneous distribution of endogenous metabolites that colocalise with the necrotic core and apoptotic regions in this model. Cytotoxic chemotherapy (doxorubicin) responses were significantly attenuated in our 3D cell culture model compared with those of standard cell culture, as determined by resazurin assay, despite sufficient doxorubicin diffusion demonstrated by localisation throughout the 3D constructs. Finally, changes to the distribution of endogenous metabolites in response to doxorubicin were readily detected by MSI. Principal component analysis identified 50 metabolites which differed most in their abundance between treatment groups, and of these, 10 were identified by both in‐software t test and mixed‐effects analysis of variance (ANOVA). Subsequent independent MSIs of identified species were consistent with principle component analysis findings. This proof‐of‐principle study shows for the first time that chemotherapy‐induced changes in metabolite abundance and distribution may be determined in 3D cell culture by MSI, highlighting this method as a potentially useful tool in the elucidation of chemotherapy responses as an alternative to in vivo testing.     

An LC-MS/MS Method for Analysis of Vitamin D Metabolites and C3 Epimers in Mice Serum: Oral Supplementation Compared to UV Irradiation

Introduction: The most common forms of vitamin D in human and mouse serum are vitamin D3 and vitamin D2 and their metabolites. The aim of this study is to determine whether diet and sunlight directly affect the circulating concentrations of vitamin D metabolites in a mouse model. We investigated the serum concentrations of eight vitamin D metabolites—vitamin D (vitamin D3 + vitamin D2), 25OHD (25OHD3 + 25OHD2), 1α25(OH)₂D (1α25(OH)₂D2, and 1α25(OH)₂D3)—including their epimer, 3-epi-25OHD (3-epi-25OHD3 and 3-epi-25OHD2), and a bile acid precursor 7alpha-hydroxy-4-cholesten-3-one (7αC4), which is known to cause interference in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Method: The LC-MS/MS method was validated according to FDA-US guidelines. The validated method was used for the analysis of mouse serum samples. Forty blood samples from mice were collected and divided into three groups. The first group, the DDD mice, were fed a vitamin D-deficient diet (25 IU VD3/kg of diet) and kept in the dark; the second group, the SDD mice, were maintained on a standard-vitamin D diet (1000 IU VD3) and kept in the dark; and the third group, SDL, were fed a standard-vitamin D diet (1000 IU VD3) but kept on a normal light/dark cycle. LC-MS/MS was used for the efficient separation and quantitation of all the analytes. Results: The validated method showed good linearity and specificity. The intraday and interday precision were both <16%, and the accuracy across the assay range was within 100 ± 15%. The recoveries ranged between 75 and 95%. The stability results showed that vitamin D metabolites are not very stable when exposed to continuous freeze–thaw cycles; the variations in concentrations of vitamin D metabolites ranged between 15 and 60%. The overlapping peaks of vitamin D, its epimers, and its isobar (7αC4) were resolved using chromatographic separation. There were significant differences in the concentrations of all metabolites of vitamin D between the DDD and SDL mice. Between the groups SDD (control) and SDL, a significant difference in the concentrations of 3-epi-25OHD was noted, where C3 epimer was about 30% higher in SDL group while no significant differences were noted in the concentrations of vitamin D, 25OHD, 1α25(OH)₂D, and 7αC4 between SDD and SDL group. Conclusions: A validated method, combined with a simple extraction technique, for the sensitive LC-MS/MS determination of vitamin D metabolites is described here. The method can eliminate the interferences in LC-MS/MS analysis caused by the overlapping epimer and isobar due to them having the same molecular weights as 25OHD. The validated method was applied to mouse serum samples. It was concluded that a standard-vitamin D diet causes an increase in the proportion of all the vitamin D metabolites and C3 epimers and isobar, while UV light has no pronounced effect on the concentrations of the majority of the vitamin D metabolites except 3-epi-25OHD. Further studies are required to confirm this observation in humans and to investigate the biochemical pathways related to vitamin D’s metabolites and their epimers.