Due to the limited number of organ donors, 3D printing of organs is a promising technique. Tissue engineering is increasingly using xenogeneic material for this purpose. This study was aimed at ...assessing the safety of decellularized porcine pancreas, together with the analysis of the risk of an undesirable immune response. We tested eight variants of the decellularization process. We determined the following impacts: rinsing agents (PBS/NH3·H2O), temperature conditions (4 °C/24 °C), and the grinding method of native material (ground/cut). To assess the quality of the extracellular matrix after the completed decellularization process, analyses of the following were performed: DNA concentration, fat content, microscopic evaluation, proteolysis, material cytotoxicity, and most importantly, the Triton X-100 content. Our analyses showed that we obtained a product with an extremely low detergent content with negligible residual DNA content. The obtained results confirmed the performed histological and immuno-fluorescence staining. Moreover, the TEM microscopic analysis proved that the correct collagen structure was preserved after the decellularization process. Based on the obtained results, we chose the most favorable variant in terms of quality and biology. The method we chose is an effective and safe method that gives a chance for the development of transplant and regenerative medicine.
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•Routine TDM of Ciclosporin (CSA) in the pediatric population can be achieved using the Mitra™ device.•The quality of the commercial and in-house calibrators appeared to be equal ...during the validation process.•LC-MS/MS is a suitable reference technique for CSA determination in both whole blood and VAMS samples.•Cross-validation confirmed the interchangeability of the developed methods (WB-LC-MS/MS versus VAMS-LC-MS/MS).•Commercially available external proficiency testing schemes for CSA appear suitable for VAMS-based method standardisation.
Ciclosporin (CSA) is an immunosuppressive agent that requires therapeutic drug monitoring (TDM). High partitioning in erythrocytes indicates that whole blood (WB) is a suitable matrix for CSA determination. Alternative sampling strategies, such as volumetric absorptive microsampling (VAMS), are novel possibilities for blood collection during TDM for various analytes, including immunosuppressants. This technique is attractive for vulnerable pediatric patients, including home-based self-sampling, remote therapy, and adherence control.
This study aimed to develop and validate a new method for CSA determination based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) of WB and VAMS samples. Additionally, these methods were applied for CSA determination in clinical samples from pediatric transplant recipients. A strong point of this study is the assessment of an external proficiency testing scheme.
Both methods were successfully validated within the 1–2000 ng/mL calibration range, with LOD 0.5 and 1 ng/mL for WB and VAMS methods, respectively. All the validation parameters fulfilled the international acceptance criteria for bioanalytical methods. Cross-validation confirmed the interchangeability of the LC-MS/MS method developed in this study.
This study developed and validated novel methods for CSA determination in whole blood and VAMS using LC-MS/MS. Clinical validation and proficiency testing confirmed their utility in routine clinical practice.
The aim of this randomised clinical trial (RCT) was to assess the concentration of fluoride ions in saliva after applying fluoride gel and to examine the extent to which rinsing the mouth with water ...affects the content of fluoride ions in saliva as opposed to no rinsing after brushing teeth with gel.
This single-blind RCT was conducted amongst a group of students (N = 103) and consisted of a questionnaire, clinical examination, saliva collection, and laboratory analysis. After saliva samples were collected, the students brushed their teeth for 2 minutes with Elmex Gelée (Colgate-Palmolive). Next, saliva was collected after 15 and 60 minutes from Group A (spitting out saliva after brushing) and from Group B (spitting out and rinsing with water after brushing). Statistical analysis was performed using a t test to compare the means between the groups.
Fifteen minutes after brushing, the level of fluoride increased in Group A about 15-fold to 15.33 ± 14.73 ppm and in Group B about 6-fold to 6.19 ± 5.97 ppm (P < .001). After 60 minutes, in Group A it decreased to 1.68 ± 0.83 ppm and in Group B to 0.60 ± 0.37 ppm (P < .001).
The use of fluoride gel in the absence of mouth rinsing produced significantly higher level and a concentration of fluoride in saliva. A post-gel brushing without rinsing might be suitable for patients at high risk for caries. Hence fruther research on the effect of a mouth rinse after use of fluoride gels is eesential to confirm our findings, and to promulgate evidence-based recommendations.
Background
Mycophenolic acid (MPA) is widely used in posttransplant pharmacotherapy for pediatric patients after renal transplantation. Volumetric absorptive microsampling (VAMS) is a recent approach ...for sample collection, particularly during therapeutic drug monitoring (TDM). The recommended matrix for MPA determination is plasma (PL), and conversion between capillary-blood VAMS samples and PL concentrations is required for the appropriate interpretation of the results.
Methods
This study aimed to validate and develop a UHPLC-MS/MS method for MPA quantification in whole blood (WB), PL, and VAMS samples, with cross and clinical validation based on regression calculations. Methods were validated in the 0.10–15 µg/mL range for trough MPA concentration measurement according to the European Medicines Agency (EMA) guidelines. Fifty pediatric patients treated with MPA after renal transplantation were included in this study. PL and WB samples were obtained via venipuncture, whereas VAMS samples were collected after the fingerstick. The conversion from VAMS
MPA
to PL
MPA
concentration was performed using formulas based on hematocrit values and a regression model.
Results
LC–MS/MS methods were successfully developed and validated according to EMA guidelines. The cross-correlation between the methods was evaluated using Passing-Bablok regression, Bland–Altman bias plots, and predictive performance calculations. Clinical validation of the developed method was successfully performed, and the formula based on regression was successfully validated for VAMS
MPA
to PL
MPA
concentration and confirmed on an independent group of samples.
Conclusions
This study is the first development of a triple matrix-based LC–MS/MS method for MPA determination in the pediatric population after renal transplantation. For the first time, the developed methods were cross-validated with routinely used HPLC–DAD protocol.
Graphical Abstract
Hydra, as sit‐and‐wait predators with limited food selectivity, could serve as model organisms for the analysis of the effect of a particular dietary component on growth and reproduction. We ...investigated the effect of food quality and of diets enriched with palmitic (PAM) or α‐linolenic acid (ALA) on the life history traits of two hydra species: Hydra oligactis and Hydra vulgaris. We tested the hypothesis that a diet enriched with polyunsaturated fatty acids (PUFA) can stimulate growth and reproduction in simple metazoans with a sit‐and‐wait type of predatory strategy. Our results revealed that a diet based on Artemia nauplii, which are not a natural food for freshwater hydra, stimulated growth, asexual reproduction, and survival in hydra. Artemia nauplii were characterized by the highest lipid content of all used food sources. The analysis of the fatty acid content of hydra indicated the domination the n‐6 fatty acids over n‐3 (eicosapentaenoic acid EPA, docosahexaenoic acid DHA, and ALA). Arachidonic acid appeared to be the dominant PUFA in Hydra, irrespective of diet supplementation with palmitic acid or ALA. The dietary supplementation of ALA negatively affected the survival, asexual reproductive rate, and size of clonal offspring of H. oligactis and had no effect on the life history traits of H. vulgaris. Our results also suggest that the hydras are not able to efficiently convert ALA into other essential fatty acids, such as EPA and DHA. To our knowledge, this is the first report about the adverse effects of n‐3 fatty acid supplementation in primitive metazoans such as hydra.
•novel test and reference sample for calibration and characterization of scanning thermal microscopy (SThM) probes•integration of plasma etching of Pt and chemical-mechanical planarization (CMP) ...processing, enabled manufacturing a nanosize 100 nm thick Pt resistor on SiO2 with topography profile below 10 nm.•active test structure allows to minimize the influence of variable tip-sample thermal resistance during the scanning and to analyze actual performance of the SThM probe.
In this paper we describe the design, technology and application of a test and reference sample for calibration and characterization of scanning thermal microscopy (SThM) probes and systems. In our solution temperature field in thin film structure, which is being contacted with the thermal tip is controlled in the traceable manner. The developed technology, integrating plasma etching of Pt and chemical-mechanical planarization (CMP) processing, enabled manufacturing a nanosize 100 nm thick Pt resistor on SiO2 with topography profile below 10 nm.
Four-point setup makes it possible to generate and measure (in other words control) a defined temperature field of such a structure. The size of the thermally active structure is big enough to enable reliable SThM measurements and small enough to reduce the parasitic heat transport between the surface and the cantilever platform. The proposed solution enables measurement of the output signal of the scanning thermal microscope measurement system when the temperature of the reference sample is varied in the quantitative way. Furthermore, basing on the determined sensitivity the assessment of the resolution capabilities is possible.
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