222 research outputs found
Bayesian-lopa methodology for risk assessment of an LNG importation terminal
No full textLNG (Liquefied Natural Gas) is one of the fastest growing energy sources in the
U.S. to fulfill the increasing energy demands. In order to meet the LNG demand, many
LNG facilities including LNG importation terminals are operating currently. Therefore,
it is important to estimate the potential risks in LNG terminals to ensure their safety.
One of the best ways to estimate the risk is LOPA (Layer of Protection Analysis)
because it can provide quantified risk results with less time and efforts than other
methods. For LOPA application, failure data are essential to compute risk frequencies.
However, the failure data from the LNG industry are very sparse. Bayesian estimation is
identified as one method to compensate for its weaknesses. It can update the generic data
with plant specific data.
Based on Bayesian estimation, the frequencies of initiating events were obtained
using a conjugate gamma prior distribution such as OREDA (Offshore Reliability Data)
database and Poisson likelihood distribution. If there is no prior information, Jeffreys
noninformative prior may be used. The LNG plant failure database was used as plant
specific likelihood information. The PFDs (Probability of Failure on Demand) of IPLs (Independent Protection
Layers) were estimated with the conjugate beta prior such as EIReDA (European
Industry Reliability Data Bank) database and binomial likelihood distribution. In some
cases EIReDA did not provide failure data, so the newly developed Frequency-PFD
conversion method was used instead. By the combination of Bayesian estimation and
LOPA procedures, the Bayesian-LOPA methodology was developed and was applied to
an LNG importation terminal. The found risk values were compared to the tolerable risk
criteria to make risk decisions. Finally, the risk values of seven incident scenarios were
compared to each other to make a risk ranking.
In conclusion, the newly developed Bayesian-LOPA methodology really does
work well in an LNG importation terminal and it can be applied in other industries
including refineries and petrochemicals. Moreover, it can be used with other frequency
analysis methods such as Fault Tree Analysis (FTA)
Enhancing all-in-one bioreactors by combining interstitial perfusion, electrical stimulation, on-line monitoring and testing within a single chamber for cardiac constructs
No full textTissue engineering strategies have been extensively exploited to generate functional cardiac patches. To maintain cardiac functionality in vitro, bioreactors have been designed to provide perfusion and electrical stimulation, alone or combined. However, due to several design limitations the integration of optical systems to assess cardiac maturation level is still missing within these platforms. Here we present a bioreactor culture chamber that provides 3D cardiac constructs with a bidirectional interstitial perfusion and biomimetic electrical stimulation, allowing direct cellular optical monitoring and contractility test. The chamber design was optimized through finite element models to house an innovative scaffold anchoring system to hold and to release it for the evaluation of tissue maturation and functionality by contractility tests. Neonatal rat cardiac fibroblasts subjected to a combined perfusion and electrical stimulation showed positive cell viability over time. Neonatal rat cardiomyocytes were successfully monitored for the entire culture period to assess their functionality. The combination of perfusion and electrical stimulation enhanced patch maturation, as evidenced by the higher contractility, the enhanced beating properties and the increased level of cardiac protein expression. This new multifunctional bioreactor provides a relevant biomimetic environment allowing for independently culturing, real-time monitoring and testing up to 18 separated patches
Development of an organotypic microfluidic model to reproduce monocyte extravasation process in the osteoarthritic joint
No full textStability of housekeeping genes in human intervertebral disc, endplate and articular cartilage cells in multiple conditions for reliable transcriptional analysis
No full textQuantitative gene expression analysis is widely used to evaluate the expression of specific tissue markers. To obtain reliable data it is essential to select stable housekeeping genes whose expression is not influenced by the anatomical origin of cells or by the culture conditions. No studies have evaluated housekeeping gene stability in intervertebral disc (IVD) cells and only few studies using cartilaginous endplate (CEP) and articular cartilage (AC) cells are present in the literature. We analysed the stability of four candidate housekeeping genes (GAPDH, TBP, YWHAZ and RPL13A) in human cells isolated from nucleus pulposus (NP) and annulus fibrosus (AF), CEP and AC. Cell isolation, expansion, cryoconservation, and differentiation in 3D pellets were tested. GeNorm, NormFinder, BestKeeper tools and the comparative ΔCt method were used to evaluate housekeeping gene stability. In each cell population, TBP alone or combined with YWHAZ was identified as the best normaliser in both monolayer and 3D pellets. GAPDH was the best performer only for AC cells in monolayer. In most culture conditions considering groups of two or more cell types, TBP was the most stable and YWHAZ was the second choice. GAPDH was the best performer only in 3D pellets with factors for AC and AF combined with CEP cells. RPL13A was the most stable only for AF with CEP cells at isolation. Our findings will be useful to properly design the experimental set-up of studies involving IVD, CEP or AC cells in different culture conditions, in order to obtain accurate and high quality data from quantitative gene expression analysis
Isolation and characterization of the human A-myb promoter : regulation by NF-Y and Sp1
No full textThe A-myb transcription factor shows a restricted tissue distribution and is cell cycle regulated. Furthermore its deregulation has profound effects on the growth and/or differentiation of the cells in which it is normally expressed. We have therefore characterized its promoter. A 12 kb genomic clone was isolated that comprises the first exon, part of the first intron as well as upstream regulatory sequences. Multiple transcription start sites have been identified which operate in both B lymphocytes and epithelial cells and the upsteam region was shown to have promoter, activity. The boundaries of the minimal promoter region (-183-14), of a positive upstream (-538-183) and a negative downstream regulatory region (NRE) (+83+374) have been defined. The NRE is promoter- and orientation-independent but position specific. The A-myb minimal promoter is GC-rich, does not contain any TATA box but has a functional CCAAT box. The CCAAT box and minimal promoter is highly conserved in the corresponding murine sequence. The CCAAT box efficiently binds the NF-Y complex and its mutation decreases basal promoter activity by 50%. Two Sp1 binding sites are present upstream from the CCAAT box which can bind Sp1 and contribute to A-myb promoter activity by 70 and 30%, respectively. The two Sp1 sites and CCAAT box together contribute to over 80% of A-myb basal promoter activity and are therefore the major regulatory elements. Finally, we show that the promoter is cell cycle regulated and that the SP1 and CCAAT elements are required for S phase induction
Human umbilical vein endothelial cells (HUVECs) isolation by gravitational fieldflow fractionation: a tag-less method as a starting point for gene expression analysis
No full textOsteogenic differentiated human adipose derived stem cells maintained on chemically modified titanium supports
No full textIn vitro testing of biomaterials osteo-inductive properties by human adipose-derived stem cells
No full textThe clinical success of the implants is related to their early osteo-integration which depends primarily on the properties of the surface. It is known that an increased implant-surface roughness significantly influences the osteoblastic response-, improving their adhesion and proliferation; same results have been described with bone marrow mesenchymal stem cells. Meanwhile, new techniques of surface treatment and coating deposition have been developed to prevent corrosion and debris formation which may causetissue inflammation, osteolysis and finally the loosening of the implant.
In this study either standard hydroxyapatite (HA), or different kind of chemically modified titanium (TIT, TAA ) and silicon carbide obtained by plasma enhanced chemical vapor deposition (SiC-PECVD), a novel and promising biocompatible material for the covering of orthopaedic devices, were analyzed for their properties in supporting cell adhesion, proliferation and osteo-differentiation. Human adipose-derived stem cells (hASCs) were used for all these analyses since they possess a multi-differentiative potential and a reduced immunogenicity,features that make these cells useful and suitable model for these tests.
We have assessed their ability to let hASC cells grow, both in non inductive and in osteo-inductive conditions, in comparison with the one cultured adhered on plastic (PA). Both undifferentiated and osteo-differentiated hASCs well colonized the tested biomaterials without showing any cytotoxic effect. HA, TIT, TAA and SiC-PECVD clearly possessed osteoinductive properties: indeed hASCs cultured on these biomaterials showed a significant (marked) increase of osteogenic markers such asalkaline phosphatase activity and calcified matrix deposition in comparison to cells cultured on plastic.
This study suggest the use of hASC cells to test and to predict in vitro the compatibility and the osteo-inductive properties of biomaterials involved in orthopaedic application
Human adipose-derived stem cells (hASCs) : a useful tool to screen biomaterials for bone Regeneration
No full textMesenchymal stem cells have already been successfully used in association with specific scaffolds to repair tissue damages; in particular, human adipose-derived stem cells posses several features, such as their great multi-differentiative potential and their reduced immunogenicity, that make them suitable candidates for many tissue engineering applications.
We used hASCs to screen several biomaterials which can be used for bone regeneration as titanium (TIT) and two different types of chemically-modified titanium (TAA and TAAK) disks, polyurethane sponges (PU) and silicon carbide-plasma enhanced chemical vapor deposition (SiC-PECVD) to evaluate their influence on growth and osteogenic differentiation of these cells. In order to set the best experimental conditions to use hASCs in future orthopaedic clinical applications, we compared the osteogenic potential of hASCs pre-differentiated for 14 days or directly differentiated on scaffolds.
Both undifferentiated and differentiated hASCs adhere to the tested biomaterials and colonize them without any evidence of cytotoxicity; to evaluate the osteogenic differentiation of hASCs cultured both on plastic and on scaffolds for 14 and 21 days we quantified the Alkaline Phosphatase activity (ALP) and the calcified extracellular matrix deposition, respectively early and late differentiation markers.
TIT and TAA disks possess good osteoinductive properties: indeed undifferentiated hASCs show an ALP activity increased of 32.4% and 68.1% and an extracellular calcium deposition of 54.3% and 82.3% when cultured on TIT and TAA respectively, in comparison to cells maintained for 14 days on monolayer. In contrast, TAAK is not able to promote hASCs osteogenic differentiation.
We also observed an osteo-inductive effect by PU sponges, indeed their presence for 14 and 21 days with undifferentiated hASCs produces an increase in ALP activity of 692.2% and 250.5%, respectively. As expected, TIT, TAA and PU sponges show their osteo-inductive properties also on osteogenic-differentiated hASCs.
SiC-PECVD, a novel and promising biocompatible material, shows an osteo-inductive effect on undifferentiated hASCs: ALP levels and calcium deposition are increased of 72.3% and 24.5%, respectively, in comparison to undifferentiated cells on monolayer; however the presence of SiC-PECVD does not affect differentiated hASCs.
Furthermore, we observed that hASCs, either pre-differentiated and directly differentiated on all the tested scaffolds, do not behave differently suggesting that a pre-differentiation step is not required for future clinical applications, reducing the ex vivo cellular manipulation process.
This study demonstrates that the cytotoxicity and the osteo-inductive properties of several biomaterials can be easily evaluated using hASCs; these cells, with their ability to grow and differentiate towards different cellular lineages such as osteogenic, chondrogenic, myogenic and endothelial, even in the presence of scaffolds, are good candidates for many regenerative medicine applications. Moreover the high availability and the ease of culture of hASCs make them suitable to test drugs and to study their mechanism of action
Stability of housekeeping genes in human intervertebral disc, endplate and articular cartilage cells in multiple conditions for reliable transcriptional analysis
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