Decellularized tissue is an important source for biological tissue engineering. Evaluation of the quality of decellularized tissue is performed using scanned images of hematoxylin-eosin stained (H&E) tissue sections and is usually dependent on the observer. The first step in creating a tool for the assessment of the quality of the liver scaffold without observer bias is the automatic segmentation of the whole slide image into three classes: the background, intralobular area, and extralobular area. Such segmentation enables to perform the texture analysis in the intralobular area of the liver scaffold, which is crucial part in the recellularization procedure. Existing semi-automatic methods for general segmentation (i.e., thresholding, watershed, etc.) do not meet the quality requirements. Moreover, there are no methods available to solve this task automatically. Given the low amount of training data, we proposed a two-stage method. The first stage is based on classification of simple hand-crafted descriptors of the pixels and their neighborhoods. This method is trained on partially annotated data. Its outputs are used for training of the second-stage approach, which is based on a convolutional neural network (CNN). Our architecture inspired by U-Net reaches very promising results, despite a very low amount of the training data. We provide qualitative and quantitative data for both stages. With the best training setup, we reach 90.70% recognition accuracy.
Decellularized scaffolds can serve as an excellent three-dimensional environment for cell repopulation. They maintain tissue-specific microarchitecture of extracellular matrix proteins with important spatial cues for cell adhesion, migration, growth, and differentiation. However, criteria for quality assessment of the three-dimensional structure of decellularized scaffolds are rather fragmented, usually study-specific, and mostly semi-quantitative. Thus, we aimed to develop a robust structural assessment system for decellularized porcine liver scaffolds. Five scaffolds of different quality were used to establish the new evaluation system. We combined conventional semi-quantitative scoring criteria with a quantitative scaffold evaluation based on automated image analysis. For the quantitation, we developed a specific open source software tool (ScaffAn) applying algorithms designed for texture analysis, segmentation, and skeletonization. ScaffAn calculates selected parameters characterizing structural features of porcine liver scaffolds such as the sinusoidal network. After evaluating individual scaffolds, the total scores predicted scaffold interaction with cells in terms of cell adhesion. Higher scores corresponded to higher numbers of cells attached to the scaffolds. Moreover, our analysis revealed that the conventional system could not identify fine differences between good quality scaffolds while the additional use of ScaffAn allowed discrimination. This led us to the conclusion that only using the combined score resulted in the best discrimination between different quality scaffolds. Overall, our newly defined evaluation system has the potential to select the liver scaffolds most suitable for recellularization, and can represent a step toward better success in liver tissue engineering.
- Publikační typ
- časopisecké články MeSH
Repopulace decelularizované tkáně buňkami je velmi nadějným směrem, kterým by se mohl řešit nedostatek tkání a orgánů pro transplantace, přičemž jaterní tkáňové inženýrství není výjimkou. Decelularizovaný jaterní skelet slouží jako ideální 3D prostředí pro recelularizaci, neboť je v něm zachována tkáňově specifická mikroarchitektura proteinů extracelulární matrix (ECM) s poziční informací jak pro osídlení novými buňkami, tak pro jejich migraci, růst a diferenciaci. Při použití autologních buněk by navíc nově konstruovaný štěp měl postrádat imunogenicitu v hostitelském organismu, bylo by tedy možné se kompletně vyhnout imunosupresi, která je v současnosti nutnou součástí terapie po transplantaci. Tento přehled uvádí příklady dosud provedených decelularizačních a repopulačních experimentů v játrech, přičemž upozorňuje na pokroky a poukazuje na výzvy, které je třeba řešit.
Repopulation of decellularized tissue with cells is a very promising approach in tissue engineering, with liver tissue engineering not being an exception. Decellularized liver scaffolds can serve as an excellent 3D environment for recellularization as it maintain tissue-specific microarchitecture of ECM proteins with important spatial cues for cell adhesion, migration, growth and differentiation. Moreover, by using autologous cells the newly constructed graft should lack immunogenicity in the host organism and thus eliminate the need for immunosuppressive therapy in the post-transplant period. This review provides an overview of liver decellularization and repopulation experiments done so far while highlighting the advances as well as pin-pointing the challenges that remain to be solved.
- Klíčová slova
- decelularizace jater, depopulace jater,
- MeSH
- játra chemie MeSH
- lidé MeSH
- modely u zvířat MeSH
- prasata MeSH
- regenerativní lékařství metody MeSH
- tkáňové inženýrství * MeSH
- tkáňové podpůrné struktury MeSH
- transplantace jater MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Tento příspěvek se zabývá problematikou jazykové bariéry v prostředí zdravotnických záchranných služeb a možnostmi jejího překonání. U pracovníků zdravotnických záchranných služeb je komunikace mezi dvěma zdravotníky nesmírně důležitá. Projekt „Koncept koordinace a realizace přeshraniční spolupráce zdravotnických záchranných služeb“ (dále Projekt č. 30) se zabývá problematikou přeshraniční spolupráce zdravotnických záchranných služeb České republiky a Spolkové republiky Německo. Na projektu jsme spolupracovali v oblasti komunikace. Dle našich poznatků dosud neexistuje vhodná pomůcka pro překonání jazykové bariéry při komunikaci zdravotníků v česko-německém pohraničí. Tento problém jsme se rozhodli řešit vytvořením komunikační pomůcky. Pro maximální využitelnost v přednemocniční neodkladné péči jsme se rozhodli pomůcku vytvořit pomocí kombinace piktogramů a překladů.
This article deals with the language barrier issue related to ambulance service and possibilities how to overcome it. However, communication between two medical professionals is crucial. The project: „The concept of coordination and realization of crossborder cooperation among emergency medical service workers“ (further referred to as Project no. 30) deals with cross-border cooperation issues between emergency medical services of the Czech Republic and Germany. We have established cooperation with this project in the field of communication. According to our findings, a suitable tool, that would help to overcome the language barrier in the interaction of medical professionals at the Czech-German border doesn´t exist. Therefore, we have decided to create such a tool to help to solve this problem. We have applied a combination of pictograms and translations for maximal utilisation of this tool in the pre-hospital emergency care.
Studie se zaměřuje na hodnocení účinku opakované expozice HBO na mitochondriální respiraci lidských fetálních plicních fibroblastů (HFL1). Mitochondriální spotřeba kyslíku byla hodnocena metodou high-resolution respirometry (HRR), životaschopnost buněk hodnocena PrestoBlue®, morfologie analyzovaná rutinní fluorescenční mikroskopií a fázovým kontrastem a byla zhodnocena aktivita superoxid dismutázy (SOD) a citrát syntázy (CS). Buňky byly vystaveny HBO (3ATA) 2 hodiny denně po dobu 5 po sobě jdoucích dní. Den po poslední HBO expozici vykazovaly buňky podstatně menší plochu a obvod, sníženou životaschopnost a zvýšenou aktivitu SOD. Nebyly zjištěny žádné změny v aktivitě CS ani v kvalitě mitochondriální sítě. HRR odhalila sníženou mitochondriální spotřebu kyslíku, která se projevila snížením aktivity komplexu II a sníženou spotřebou kyslíku při oxidací mastných kyselin. Naše zjištění dokládají, že v podmínkách napodobujících intermitentní expozici HBO, trpí plicní fibroblasty zhoršenou mitochondriální respirací spojenou s komplexem II a poruchou buněčného růstu i při zvýšené antioxidační obraně. Mechanismus této HBO-indukované mitochondriální dysfunkce je nutné hlouběji prozkoumat.
Study aims to evaluate effects of repeated exposure to HBO on mitochondrial respiration assessed by high-resolution respirometry (HRR), cell viability estimated by PrestoBlue® reaction, morphology analyzed by routine phase contrast and fluorescent microscopy, and superoxide dismutase (SOD) and citrate synthase (CS) activities using human lung fibroblasts. The cells were exposed to HBO (3 ATA) for 2 hours per day for 5 consecutive days. One day after the last exposure, HBO cells displayed significantly smaller area and perimeter, compromised viability and elevated SOD activity. No changes were detected in CS activity or quality of mitochondrial network. HRR revealed impaired mitochondrial oxygen consumption manifested by increased leak respiration, decreased activity of complex II and compromised ATP-related oxygen consumption when fatty acids were oxidized. Our findings document that in conditions mimicking chronic intermittent exposure to HBO, lung fibroblasts suffer from compromised mitochondrial respiration linked to complex II and impaired cellular growth in spite of increased antioxidant defense. Underlying mechanism of this HBO-induced mitochondrial dysfunction should be further explored.
- Klíčová slova
- respirometrie,
- MeSH
- buněčné dýchání MeSH
- fibroblasty * enzymologie metabolismus MeSH
- fluorescenční mikroskopie MeSH
- hyperbarická oxygenace * škodlivé účinky MeSH
- hyperoxie MeSH
- lidé MeSH
- mitochondrie MeSH
- plíce anatomie a histologie MeSH
- plod MeSH
- superoxiddismutasa MeSH
- techniky in vitro MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Hyperbaric oxygen (HBO) therapy, i.e. breathing pure oxygen under increased environmental pressures serves as a treatment for diverse medical conditions. However, elevated oxygen concentration can be detrimental to central nervous system or lungs. Our study aimed to evaluate the effects of repeated exposure to HBO on mitochondrial respiration assessed by high-resolution respirometry (HRR), cell viability estimated by PrestoBlue® reaction, morphology analyzed by routine phase contrast and fluorescent microscopy, and superoxide dismutase (SOD) and citrate synthase (CS) activities using human lung fibroblasts. The cells were exposed to HBO for 2 h per day for 5 consecutive days. One day after the last exposure, HBO cells displayed significantly smaller area and perimeter, compromised viability and elevated SOD activity. No changes were detected in CS activity or quality of mitochondrial network. HRR revealed impaired mitochondrial oxygen consumption manifested by increased leak respiration, decreased activity of complex II and compromised ATP-related oxygen consumption when fatty acids were oxidized. Our findings document that in conditions mimicking chronic intermittent exposure to HBO, lung fibroblasts suffer from compromised mitochondrial respiration linked to complex II and impaired cellular growth in spite of increased antioxidant defense. Underlying mechanism of this HBO-induced mitochondrial dysfunction should be further explored.
- MeSH
- buněčné dýchání fyziologie MeSH
- buněčné linie MeSH
- fibroblasty metabolismus MeSH
- hyperbarická oxygenace škodlivé účinky MeSH
- lidé MeSH
- mitochondrie fyziologie MeSH
- oxidační stres fyziologie MeSH
- plíce cytologie metabolismus MeSH
- spotřeba kyslíku fyziologie MeSH
- viabilita buněk fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- 3D tisk * trendy MeSH
- fraktury očnice chirurgie MeSH
- lidé MeSH
- modely anatomické * MeSH
- ortognátní chirurgie * trendy MeSH
- zákroky plastické chirurgie trendy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- kazuistiky MeSH
- práce podpořená grantem MeSH
BACKGROUND: Photodynamic therapy (PDT) is linked with oxidative damage of biomolecules causing significant impairment of essential cellular functions that lead to cell death. It is the reason why photodynamic therapy has found application in treatment of different oncological, cardiovascular, skin and eye diseases. Efficacy of PDT depends on combined action of three components; sensitizer, light and oxygen. In the present study, we examined whether higher partial pressure of oxygen increases lethality in HeLa cell lines exposed to light in the presence of chloraluminium phthalocyanine disulfonate (ClAlPcS2). METHODS: ClAlPcS2- sensitized HeLa cells incubated under different oxygen conditions were exposed to PDT. Production of singlet oxygen ((1)O2) and other forms of reactive oxygen species (ROS) as well as changes in mitochondrial membrane potential were determined by appropriately sensitive fluorescence probes. The effect of PDT on HeLa cell viability under different oxygen conditions was quantified using the standard methylthiazol tetrazolium (MTT) test. RESULTS: At the highest oxygen concentration of 28 ± 2 mg/l HeLa cells were significantly more sensitive to light-activated ClAlPcS2 (EC50=0.29 ± 0.05 μM) in comparison to cells incubated at lower oxygen concentrations of 8 ± 0.5 and 0.5 ± 0.1 mg/l, where the half maximal effective concentration was 0.42 ± 0.06 μM and 0.94 ± 0.14 μM, respectively. Moreover, we found that the higher presence of oxygen is accompanied with higher production of singlet oxygen, a higher rate of type II photodynamic reactions, and a significant drop in the mitochondrial membrane potential. CONCLUSION: These results demonstrate that the photodynamic effect in cervical cancer cells utilizing ClAlPcS2 significantly depends on oxygen level.
- MeSH
- fotochemoterapie * MeSH
- fotosenzibilizující látky farmakologie MeSH
- HeLa buňky MeSH
- indoly farmakologie MeSH
- kyslík analýza farmakologie MeSH
- lidé MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- parciální tlak MeSH
- reaktivní formy kyslíku metabolismus MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
