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Instruments and Methods for Biology and Medicine 2020
Sítná Sq. 3105 272 01 Kladno
October 8, 2020
Conference proceedings
Romana Široká Taťána Jarošíková (Editors)
INSTRUMENTS AND METHODS FOR BIOLOGY AND MEDICINE 2020 Conference proceedings
Edited by Romana Široká, Taťána Jarošíková
Published by Czech Technical University in Prague Compiled by Faculty of Biomedical Engineering Sítná sq. 3105, 272 01 Kladno Tel.: +420 224 359 967 Printed by Powerprint s.r.o. Brandejsovo nám. 1219/1, Praha - Suchdol, 16500 86 pages First Edition ISBN 978-80-01-06796-3 Preface The student conference Instruments and Methods for Biology and Medicine (IMBM) took place at the Faculty of Biomedical Engineering (FBME) of the Czech Technical University in Prague on the 8th October 2020 in its tenth anniversary. The conference date was moved from the originally planned May 23, 2020, because of the COVID-19 pandemic. The conference was intended for master’s and doctoral students whose theses and research projects focus on: Biophysical Methods and Imaging, Advanced Methods for Biomedicine a Data analyzing and Processing. The aim of the conference was to provide a forum for students, who were enrolled in the two-year research project seminar where they honed their scholarly presentational skills in English, to demonstrate their accomplishments before a committee of senior members of the department. For this reason we did not hold the conference fully online, a format used nowadays by the majority of international conference at home and abroad. Instead, the conference was organized in a hybrid format, just as the courses taught at CTU. As the limited number of persons allowed in a room, the active conference participants were divided into two lecture halls and were connected via MS Teams, which also allowed the invited speakers and the audience from our faculty to access the conference. This year’s evaluation committee consisted of Prof. Blanka Brůnová, Prof. Miroslava Vrbová, Discipline guarantor of the conference, Assoc. Prof. Marie Pospíšilová, Assoc. Prof. Ján Lešták, and Assoc. Prof. Vlastimil Fidler, who participated online from Brown University, USA, and Ing. Otáhal Martin Ph.D. The committee selected and awarded the best papers. The conference was attended physically by 26 participants and virtually by 48 participants via MS Teams. It was enriched by two invited lectures. Prof. Ing. Homola, DrSc. an excellent physicist and the third most cited author in the Czech Republic, introduced the morning session with his talk on the topic: “Optical biosensor and medical application”. The afternoon session was introduced by Ing. Vladimíra Petráková, PhD. who lectured on „Plasmon Enhanced Fluorophores for Superresolution Microscopy“. I would like to express my sincere appreciation to my colleagues of the organizational committee who ensured smooth operation of the conference and compiled this volume:
Prof. Ing. Vrbová Miroslava, CSc., who served as the discipline guarantor of the conference. Doc. Ing. Pospíšilová Marie, CSc., who was responsible for refereeing the papers. Mgr. Široká Romana, PhD., who prepared the Proceedings. Ing. Hana Kalábová, who prepared the Book of abstracts and created the conference website. Ing. Ida Skopalová, who was responsible for conference public relations. Ing. Tomáš Parkman, who successfully coordinated the technical aspects of the conference in both hybrid and online formats. I would like to thank the CTU, because the conference was made possible by the general financial support by the Czech Technical University in Prague student’s conference – CTU grant No SVK 48/20/ F7. I also thank FBME for providing environment for organizing IMBM 2020. Many thanks also to all the student participants of the conference for their active role and preparation of their papers for this volume.
In Kladno, November 18, 2020
Taťána Jarošíková Conference coordinator
Table of Contents
Invited Lectures ...... 7
Optical biosensors and their medical applications ...... 9 Plasmon enhanced fluorophores for superresolution microscopy ...... 10
Biophysical Methods and Imaging ...... 11
Whole-cell detectors of aromatic hydrocarbon contaminants constructed by immobilization of bioreporters on special optical fiber elements ...... 13 Functionalized nanofibers as a specific bionanosensor for detection of Staphylococcus aureus ...... 19 Table-top water-window microscope using a capillary discharge plasma source with spatial resolution 75 nm ...... 23 Fluorescence properties of gold and silver clusters and nanoparticles ...... 24
Advanced Methods for Biomedicine ...... 29
Molecular genetics methods in use ...... 31 Experimental study of model hepatocellular carcinoma cell lines ...... 36 Improving properties of titanium alloy used for modern hip prosthesis ...... 40 Effects of a change in the direction of view to near uncorrected visual acuity following implantation of monofocal intraocular lens ...... 44 Comparison of multipotent mesenchymal stromal cells from human adipose tissue and Wharton's jelly for soft tissue reconstruction ...... 52 The application of contact lenses as carriers of multipotent mesenchymal stromal cells for regenerative medicine applications ...... 57 Tribological properties of contact lenses ...... 61
Data Analysing and Processing ...... 65
Functional differences of the brain in patients in the presymtomatic and manifestic phases of Parkinson´s disease ...... 67 Comparison of brain iron levels in basal ganglia and thalamus in patients with REM sleep disorder and Parkinson's disease ...... 71 Summary of the substantia nigra atlases ...... 76 Reconstruction and properties of T2* maps based on the number of iterations and their correlation with QSM in Parkinson's disease patients ...... 80
Proc. of IMBM 2020
Proc. of IMBM 2020 Invited Lectures
7
8 Optical biosensors and their medical applications
Jiří Homola
Institute of Photonics and Electronics, Academy of Sciences of the Czech Republic Chaberska 57, 182 51 Prague, Czech Republic
Optical biosensors are a modern analytical technology with numerous potential applications in medicine – from unraveling molecular foundations of diseases to their diagnosis and treatment monitoring. Affinity biosensors based on surface plasmons (sometimes referred to as plasmonic biosensors) represent the most advanced and mature optical label-free biosensor technology. In this lecture, we discuss the main challenges in the development of plasmonic biosensors for medical diagnostics and present selected advances in plasmonic biosensor research that aim to address some of these challenges. Specifically, we present new approaches in plasmonic sensor instrumentation for parallelized detection of multiple analytes, discuss transport of target molecules in microfluidic systems of plasmonic biosensors and routes to improving performance of nanoplasmonic biosensors, report on advances in the development of functional coatings based on low-fouling polymer brushes and present new assays enabling detection of low levels of biomolecules in complex biological media. Moreover, we discuss three examples of medical applications of plasmonic biosensors, including the monitoring of progression of Myelodysplastic syndromes based on the analysis of protein interactions, study of prognostic role of pregnancy associated plasma protein A2 in hemodialysis patients and investigation of molecular interactions related to Alzheimer’s disease.
9
Plasmon enhanced fluorophores for superresolution microscopy
Vladimíra Petráková
J. Heyrovsky Institute of Physical Chemistry, Dolejškova 2155, 182 00 Prague 8, Czech Republic
In this paper, I described possible routes to advance the resolution of optical imaging by enhancing the spatial separation and intensity of fluorescent probes using plasmonic nanoparticles. Our ability to understand how the structural organization of protein units translates into their biological function is limited by the techniques for observing protein structures and their dynamics in their natural environment. Protein assembly in higher-order complexes is fundamental to the diverse functions of proteins in cells. A most promising new technique for such studies is single molecule localization microscopy that overcomes the diffraction limit of optical imaging. But its current limit in resolution (10-20 nm) prevents the observation of single protein units with sufficient accuracy. The key to higher resolution is in brighter fluorophores and higher spatial separation of fluorophores. I introduced plasmonic nanoparticles as tools to achieve both. By effectively focusing light into nanoscale volumes through their electromagnetic near-field, plasmonic nanoparticles enhance the absorption and emission of a molecule. Additionally, plasmonic enhancement leads to the shift in the projected position of the photon emitted by the fluorophore, creating an image that no longer corresponds to the fluorophore original position. I talked about a system based on DNA origami to exploit this shift in detected position as a way to expand the projection of the sample structure and further increase resolution.
10
Biophysical Methods and Imaging
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12
Whole-cell detectors of aromatic hydrocarbon contaminants constructed by immobilization of bioreporters on special optical fiber elements
Jakub Zajíc1,2,3*, Steven Ripp3, Gabriela Kuncová4, Josef Trögl4, Marie Pospíšilová2, Jan Morava1
1Department of Cardiology, Regional Hospital Liberec, 46063 Liberec, Czech Republic 2Czech Technical University in Prague, Faculty of Biomedical Engineering, Prague, Czech Republic 3Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN 37996, USA 4Faculty of Environment, Jan Evangelista Purkyně University in Ústí n.L., 40096 Ústí nad Labem, Czech Republic
Abstract: Whole-cell fiber-optic sensor of toluene was prepared by adsorption of Pseudomonas putida TVA8 bioluminescent bioreporter onto a tapered optical fiber element (OFE). Bioreporter immobilization was facilitated by (3-Aminopropyl)triethoxysilane treatment of the OFE. Repeatability of preparation and response of the active part of biosensor was demonstrated in five trials with different OFE geometries, possessing transmittances up to 5.0%, in which the bioluminescence was induced with toluene solution (26.5 mg L-1) for the period of 2 weeks. Longevity of inductions of the biosensor was tested in over 135 days trial, in which the biosensor was induced 68 times with toluene solution (26.5 mg L-1). The intensities of bioluminescence gradually decreased due to release of the adsorbed cells but were significantly higher than background noise of the detector. The intensities of bioluminescence induced with contaminated ground water were lower than in mineral medium with the same content of toluene. Ideal shape of OFE, which maximizes the detected signal, was calculated to be a Frustrum cone. The developed sensor layered with positively induced P. putida TVA8 bioreporters was a reliable detector of toluene. Further experiments also immobilized constitutively bioluminescent cells of Escherichia coli 652T7 on OFEs with polyethyleneimine, repetitively induced with Lauria-Bertani medium.
Introduction demonstrated numerous times [5-7]. The past decade experienced a growth of whole cell Chemicals with adverse health effects, recognized bioreporters applications in laboratory and under by multiple authorities (e.g. World Health controlled conditions [8-10]. The first genetically Organization or United Nations Environment engineered microorganism allowed to be released Programme), are selected due to their toxicity in in the field to monitor bioremediation potential was low concentrations, bioaccumulativity, Pseudomonas fluorescens HK44 in 2001 [11]. persistency, or carcinogenicity [1,2]. Some of the Recently, smartphones [12] and drones [13] have most frequently found pollutants, which pose a been used as whole cell biosensor devices serious environmental concern, are benzene, encompassing bioluminescent bioreporters. toluene, ethylbenzene and the xylenes (BTEX), Nevertheless, the main reason for limited use of widely found in landfill leachates or petroleum engineered bioreporters is legislative regulation products leakage sites [3]. against the environmental release of genetically Conventional chromatographic and mass manipulated organisms [14]. spectrometry analyses of pollutants in aquatic Continuous monitoring with whole cell environments are costly and time consuming. biosensors requires repeated inoculation or Whole-cell biosensors, which produce light immobilization of cells on the sensing element selectively in the presence of a specific analyte, [13,15]. Formation of a biofilm layer tightly represent a low-cost and fast in-situ alternative for attached to an OFE surface, without an established BTEX detection. Biological sensing immobilization matrix, does not involve any elements are whole cells which have been dropping or printing machines, and minimize loss constructed to produce light signal in response to a of the detected bioluminescence signal in compound of interest in their environment. Such comparison to entrapment of cells in polymers, bioreporters have been engineered since 1992 [4], silica gel or plasma deposited films [16,17]. The and the proof-of-principle of detection has been
13 Zajíc et al.: Whole-Cell Detectors of Aromatic Hydrocarbon Contaminants Constructed by Immobilization of Bioreporters on Special Optical Fiber Elements adhesion of living cells to solid surfaces is influenced by the surface properties of both interacting entities, their motility and properties of the surrounding environment. Targeted modification of the surface properties of solid materials can significantly enhance the adsorption of cells [18]. Bioreporter cells immobilized on the tip of an optical fiber might serve for continuous long-term Figure 1: Diagrammatic representation and photograph of a measurements in small volume samples and remote tapered optical fiber element. localities and, unlike electrical sensors, might be applied in places with an explosive atmosphere. pressure. The microbial lawns were then deposited Small dimensions of an optical fiber allow to on agar plates to stabilize the moisture content, immobilize only a small number of living cells, then were fixed on a microscopic glass slide and which provide low bioluminescence intensity, allowed to dry for 30 min. The CA measurements resulting in low biosensor sensitivity. Optical fiber of both the algal lawns and glass slides were element was developed to increase the signal performed by the sessile drop technique (volume of intensity by increasing the number of light sources 3 μL) using the CAM 200 goniometer (KSV (cells) on its wider end (Ø 1 mm–1 cm), where the Instruments, Finland). The measurements were light coupling efficiency is maximized. [19,20] performed at 25 °C with three test liquids (water, This work demonstrates the adhesion of P. formamide, 1-bromnaphtalene), readings were Putida TVA8, microorganism producing taken after 0.5 s of deposition, and each sample was bioluminescence upon metabolizing aromatic tested ten times. The total surface tension and its hydrocarbons, to an OFE surface after its treatment components, and the values of the free energy of with (3-Aminopropyl)triethoxysilane (APTES). interaction between cells and carrier in water were This active part of biosensor was immersed in calculated in accordance with [23]. The zeta toluene solution, positively induced potential (ZP) of the bacteria was measured using bioluminescence was than measured daily in a Zetasizer Nano-ZS (Malvern, UK) in LB at pH 7. light-tight box. Repetitive long-term use of the The surface charge of APTES-modified quartz was whole-cell sensor with the biorecognition layer determined in an adjustable gap cell on SurPASS was tested in over 135 days trial. Bioluminescence (Anton Paar, Austria) in contact with 170 mM KCl of the biorecognition layer was also induced in a (ionic strength of LB medium) at pH 7. For the zeta real polluted water sample. Repeatability of potential determination, a streaming current preparation of the biorecognition layer was tested approach and the Helmholtz–Smoluchowski in a trial with five OFEs with different geometries, equation were used. and an ideal shape of an OFE was calculated. Author also immobilized constitutively Tapered OFE and its surface modification bioluminescent bioreporter E. coli 652T7 on OFE The OFE is a residue of quartz preform (pure treated with polyethyleneimine and induced the SiO2) from the drawing of polymer-cladded silica microorganism in Laurea-Bertani medium (LB) fibers. The ends of OFE were polished. The medium. This conference paper is a brief summary element is characterized by diameters (Dx) of author’s published experimental works [21,22]. measured in 10 mm distances along its length (Figure 1). OFE shapes were characterized by a bi- Experimental procedures exponential equation. Prior to an experiment, the elements were Contact angles and zeta potential washed in acetone and rinsed with deionized water. With the aim of proposing a surface The wider end of the OFE was immersed in Piranha modification of quartz glass to ameliorate cell solution (H2SO4 and 30% H2O2 in volume ratio adsorption, zeta potentials and contact angles of 7:3) at 70 °C for 30 min, washed again in deionized both the cells and the quartz glass were measured. water and dried at 110 °C for 1 h. Subsequently the The surface properties of P. putida TVA8 cells, in wider end of the OFE was immersed in a solution the form of algal layers on membranes filters, and of APTES (5 mass %) in dry toluene at ambient the APTES-modified quartz were characterized by temperature for 24 h. In the end, the OFE was contact angles (CA). Bacterial cells were deposited rinsed with toluene and acetone and dried at 110 °C on a filter (0.45μm nitrate cellulose) under negative for 1h.
14 Zajíc et al.: Whole-Cell Detectors of Aromatic Hydrocarbon Contaminants Constructed by Immobilization of Bioreporters on Special Optical Fiber Elements
(photon counter Perkin–Elmer 3954-P-087, USA). The wider end with the adsorbed TVA8 cells was immersed in the induction solution, containing 26.5 mgL-1 of toluene in MSM (Figure 2, left). The intensity of the bioluminescence produced by the cells was recorded for 18 h. The cells adsorbed on the element were also induced by immersion in a real polluted water sample on days 69–70. For the experiment which tested the repeatability of P. putida TVA8 sensor preparation and response, five different OFE geometries were Figure 2: Experimental set-ups for monitoring bioluminescence from P. putida TVA8 and E. coli 652T7 connected to water tube that was connected to the bioreporters adhered to surface modified OFEs. Light tight Oriel 70680 photon multiplier. Generated current, box contained: 1,E-photon counter; 2-SMA connector; D- proportional to the intensity of bioluminescence, water tube; 3,C-OFE; 4,B-induction solution; A,5-adjustable was manually read every 30-60 min from the Oriel stand 7070 detection system in nano-Amperes (Figure 2, Cell adsorption right). Each OFE was induced 10-17 times over the The APTES-modified end of the OFE was fixed period of 14-20 days. in an Erlenmeyer flask filled with 150 mL of LB, 1 Bioluminescence of PEI treated E. coli 652T7 ml of overnight culture of P. putida TVA8 and was induced 10 times over the period of 11 days kanamycin for selective growth. The P. putida and was also recorded by the Oriel 7070 detection TVA8 cells were left to grow and to be adsorbed system. on the element end in shaker at 25 rpm, 28 °C for 4 days. Results and discussion Due to inadequate growth of E. coli 652T7 on the APTES modified OFE surface, the PEI Modification of quartz surface, comparison to treatment of cells was attempted as an alternative. model prediction A 20 mL aliquot of an overnight culture of E. coli Experiments verified the hydrophilic properties 652T7 in LBkan medium was centrifuged at 3000 of quartz surface. It was also verified that no g for 5 min. The pellet was resuspended in 20 mL spontaneous adhesion of P. Putida TVA8 would MSM and centrifuged again at 3000 g for 5 min. occur on unmodified OFE surface. APTES surface The pellet was then resuspended in 20 mL of 0.2% modification with aminopropyl functional groups PEI in MSM and left in a shaker for 30 min at 100 increased the surface hydrophobicity (CA of water rpm and 28 °C. Finally, the culture was centrifuged drops increased from 25 to 72 degrees). The initial at 3000 g for 5 min and the pellet resuspended in negative charge was mitigated from -21 mV to 20 mL of MSM. The wider end of the OFE was -3 mV. then immersed in the 20 mL suspension of E. coli The extended DLVO (XDLVO) theory was 652T7 and shaken at 50 rpm for 30 min and 28 °C. used subsequently. This combines the conventional noncovalent Liftshitz–vanderWaals (LW) and Measurement of induced bioluminescence electrostatic interactions with the Lewis acid–base Experiments were performed in a light tight box (AB) interactions [24]. Given the od-shaped cells, (Figure 2). Suspended arrangement of OFE the simulations in accordance with the XDLVO prevented formation of a sediment on the wider end theory were made for the cylinder-flat plate of the OFE. Standard laboratory temperature was interactions, the Hamaker constant [25] was 25 °C. The end of OFE with immobilized cells was estimated from the ΔG LW value and the daily washed with MSM and immersed in a fresh characteristic decay length of 0.6 nm for AB induction solution (toluene for P. putida TVA8 or interactions in water was used [26]. The profile of LB/MSM for E. coli 652T7). Exceptions were 1–3 total interaction-free energy (GTOT) vs. the day pauses for weekends and holidays when the separation distance predicted favorable energy element was immersed in the induction solution for balances for adhesion of the P. putida TVA8 to the more than 24 h. APTES quartz with a total absence of potential For the experiment which tested longevity of P. energy barriers. This XDLVO model prediction putida TVA8 inductions, and which lasted 135 supports the experimental observations of rapid days, the narrow end of the element was connected bioreporter cell adsorption onto APTES quartz. At by an SMA optical fiber connector to a detector the same time, the XDLVO model prediction for P.
15 Zajíc et al.: Whole-Cell Detectors of Aromatic Hydrocarbon Contaminants Constructed by Immobilization of Bioreporters on Special Optical Fiber Elements
Figure 4: Typical time records of bioluminescence. Six 18h records recorded between the day number 7 and 17.
theoretical OFE geometries with the same length, Dmin, and Dmax, but a different bent. Increasing T of OFE shapes from the most curved to nearly linear (from 2 % to 42 %) confirmed the assumption that the best shape of an OFE is a frustum cone. The limits to this construction are the numerical Figure 3: Scanning Electron Microscopy image of P. putida aperture of an OFE, aperture of a sensor or TVA8 on an APTES modified OFE. Sample 3 days (top), diameter of an optical fiber to which s the OFE and 3 months (bottom) after immobilization. connected, and maximum possible length that would maximize the size of the OFE face and thus putida TVA8 adhesion to unmodified quartz is the number of effective light sources. characterized by the presence of a high energy barrier (6911 kT at 0.5 nm). In the frame of the Repetitive inductions of P. putida TVA8 colloidal interaction model (XDLVO), this bioluminescence energetically unfavorable barrier prevents close The result of a daily induction was a time- contact between the bacteria and the quartz surface. record of the intensities of the induced This model prediction was confirmed by the bioluminescence. Each line in Figure 4 is the daily absence of P. putida TVA8 adhesion to unmodified time record of the bioluminescence. quartz. The scatter of results among the repeats of the OFE modification with APTES leads to trial was large; nevertheless, the following successful formation of P. putida TVA8 biofilm common features were observed: (i) Two layer on its surface. Visible P. putida TVA8 bioluminescence maxima were observed on the vegetation formed on all five available OFEs. Two diel records of induced bioluminescence (Figure days after beginning of the bioluminescence 4). The first peaks were caused by adsorbed cells. inductions, lumps of cell colonies (100-1000 μm The second peaks, which appeared 8–12 h after the apart) between much smaller scattered colonies or first peak, were the results of cell growth because single cells were observed on the glass surface TVA8 used toluene as a carbon source. Thus the under scanning electron microscope (SEM), which second peak is an insignificant element for the corresponds to standard biofilm establishment purpose of toluene detection. (ii) In the first week [27]. The cell layer kept developing until it covered of the experiment, the bioluminescence maxima the entire OFE face surface, as it could be seen were gradually developing and were achieved in from the Figure 3. shorter times (Figure 5). (iii) The intensities of the detected light were low during the first few days Tapered optical fiber element after induction and then gradually increased Using the previously developed software [19], (Figure 6). This might be ascribed to an advanced light transmittance (T), which represents the covering of the base of the OFE with cells. After fraction of bioluminescence transmitted from the one month there was an increased leak of cells light source to a detector, was calculated for several which resulted in gradual decrease in light
16 Zajíc et al.: Whole-Cell Detectors of Aromatic Hydrocarbon Contaminants Constructed by Immobilization of Bioreporters on Special Optical Fiber Elements
Figure 5: Time of the first bioluminescence maxima. Aggregated data from six OFEs. First 20 days are shown. Figure 7: Time records of bioluminescence of E. coli 652T7 immobilized on an OFE in polyethyleneimine. Legend intensities measured with the OFE (Figure 6). Cells denotes the days after the immobilization. across a large range, the times of appearance of the first bioluminescence maxima were between 2 h and 5 h (Figure 5). Nevertheless, any signal above twice the amount of the background noise of the detector could reliably confirm the presence of toluene in the sample. Such an increase in bioluminescence appeared within 0.5 h from the induction. The repeatability of biofilm preparation was shown on five different OFEs. The reproducibility of all the analytical responses tested remains so low that the element with adsorbed cells is plausible only for identification of the presence of Figure 6: Daily bioluminescence maxima normalized to 1. bioavailable BTEX. The fiber biosensor with Aggregated data from six OFEs. First 20 days are shown. adsorbed bioluminescent bioreporters was repetitively induced with toluene for more than 2 adsorbed on the OFE were induced 68 times with months, which is the longest time-interval reported toluene and 4 times with contaminated water over for the service of such a biosensor. However, the the course of 135 days. technique of cell adsorption and the conditions of The biofilm layer was induced with real bioluminescence inductions require optimization polluted water sample 60 days after to achieve better reproducibility. immobilization. At that time, the bioluminescence responses to the induction solution were lower than Induction of Bioluminescence from E. coli during the first 30 days. Nevertheless, the 652T7 intensities of bioluminescence induced with this E. coli 652T7 was immobilized on the base of contaminated water were much lower than an OFE with PEI. The time records of daily expected based on the content of toluene (172 inductions with LB medium is presented in Figure mgL−1) and were also lower than the intensities 7. Besides the first induction intensities, induced with the induction solution (26.5 mgL−1). bioluminescence increased within 15 min after This decrease in induced bioluminescence immersing the OFE into the LB solution. The probably results from a toxic effect of high intensities remained stable for 18 h on the first day, concentrations of toluene and xylenes. Increased 6–9 h on all other days, and then sharply decreased temperature could also have had an influence on P. due to a depletion of nutrients. To test the OFE with putida TVA8 cells. immobilized E. coli 652T7 as a biosensor for The bioluminescence maxima and the times at biotoxicity, HCl was added to the induction which they were achieved are shown in Figure 5. solution on the eighth day. This caused pH While the bioluminescence maxima fluctuated lowering to pH=6 and decreased the
17 Zajíc et al.: Whole-Cell Detectors of Aromatic Hydrocarbon Contaminants Constructed by Immobilization of Bioreporters on Special Optical Fiber Elements bioluminescence, which did not recover after the [3] Mitra S. et al., BTEX: A Serious Ground-Water following two inductions. These results imply that Contaminant. Res. J. Environ. Sci. 5(5), 394-398 (2011). doi: 10.3923/rjes.2011.394.398 an OFE immobilized with E. coli 652T7 is sensitive to influences that affect cell viability but [4] Heitzer, A. et al., Specific and Quantitative Assessment cannot be repetitively used as a biosensor since the of Naphthalene and Salicylate Bioavailability by Using a Bioluminescent Catabolic Reporter Bacterium. Appl. cells are dying and not recovering. Environ. Microbiol. 58, 1839–1846 (1992). doi: 10.1128/AEM.58.6.1839 Conclusion [5] Belkin S., Microbial Whole-Cell Sensing Systems of Environmental Pollutants. Curr. Opin. Microbiol., In the study we have developed a biosensor for 6(3):206-12 (2003). doi: 10.1016/s1369- the detection of liquid toluene. We used physico- 5274(03)00059-6 chemical models, using contact angles and zeta [6] Diplock E. et al., Application of Microbial Bioreporters potential, to facilitate the attachment of in Environmental Microbiology and Bioremediation. bioluminescent bioreporter P. putida TVA8 to Adv. Biochem. Engin./Biotechnol. 118:189-209 (2009). quartz surfaces of tapered OFEs after its treatment doi: 10.1007/10_2009_3 with APTES. We proved the best shape of an OFE [7] Trögl J. et al., Response of the Bioluminescent is Frustum cone. The biofilm development of P. Bioreporter Pseudomonas Fluorescens HK44 to Analogs putida TVA8 with time was quantified and the of Naphthalene and Salicylic Acid. Folia microbiol. repeatability of the biofilm preparation and the 52(1):3-14 (2007). doi: 10.1007/BF02932131 repeatability of bioluminescence detection was [8] Diplock E. E. et al., Commercial Application of determined. Other than a short maturation period Bioluminescence Full Cell Bioreporters for Environmental Diagnostics. Handbook of Hydrocarbon (~5 days), the OFEs exhibited a bioluminescent and Lipid Microbiology (Springer, Heidelberg, 4445– response for the period of 135 days. We 4458, 2010). doi: 10.1007/978-3-540-77587-4_347 additionally immobilized the constitutively [9] Close D. M. et al. Reporter Proteins in Whole-Cell bioluminescent toxicity bioreporter E. coli 652T7 Optical Bioreporter Detection Systems, Biosensor treated in PEI on a modified OFE and Integrations, and Biosensing Applications. Sensors 9, demonstrated its potential use as a biosensor for 9147–9174 (2009). doi: 10.3390/s91109147 cytotoxicity. Additionally, the immobilization [10] Gutiérrez, J. C. et al. Heavy metal whole-cell biosensors process that we used, without any bulky matrix using eukaryotic microorganisms: An updated critical requirements, could be applied towards many other review. Front. Microbiol. 6 (2015). doi: microbial bioreporters for the biosensing of a 10.3389/fmicb.2015.00048 variety of different analytes. However, since the [11] Ripp S. A. et al., Controlled Field Release of a reproducibility of the bioreporter responses Bioluminescent Genetically Engineered Microorganism remains low, the developed biosensor can be used for Bioremediation Process Monitoring and Control. Environ. Sci. Technol. 34, 5, 846–853 (2000). doi: for online, rapid and multiplexed monitoring of the 10.1021/es9908319 presence of a pollutant, but not its concentration. [12] Kim H. et al. Smartphone-based low light detection for bioluminescence application. Sci. Rep. 7, 40203 (2017). Acknowledgements: Research was supported by the internal doi: 10.1038/srep40203 Student Grant Competition of the Czech Technical University in Prague, CZ, grant numbers SGS17/110/OHK4/1T/17 and [13] Lu Y. et al. A UAV-Mounted Whole Cell Biosensor SGS18/097/OHK4/1T/17. Workplace, material support, and System for Environmental Monitoring Applications. microorganisms were kindly provided by the University of IEEE Trans Nanobioscience. 14(8):811-817 (2015). doi: Tennessee, Knoxville, TN, USA; and the Institute of Chemical 10.1109/TNB.2015.2478481 Process Fundamentals of the CAS, Czech Republic. [14] European Union. Chemicals and GMO Legislation - Section 8. Handbook on the Implementation of EC References Environmental Legislation (Regional Environmental Center and Umweltbundesamt GmbH, Szentendre, [1] Harrison R. M. et al. Pollution: Causes, Effects and Hungary, 2014) Control. (Cambridge: Royal Society of Chemistry, 2001). ISBN 1591249279. [15] Lobsiger N. et al. Strategies of immobilizing cells in whole-cell microbial biosensor devices targeted for [2] Hill M. K. Understanding Environment Pollution. analytical field applications. Anal. Sci.35, p839–847 93– (Cambridge. Cambridge University Press, 2004). ISBN 126 (2019). doi: 10.2116/analsci.19R004 978-0-521-73669-5.
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Functionalized nanofibers as a specific bionanosensor for detection of Staphylococcus aureus
Leontýna Varvařovská1*, Taťána Jarošíková1, Evžen Amler2
1Czech Technical University in Prague, Faculty of Biomedical Engineering, Kladno, Czech Republic 2University Centre for Energy Efficient Buildings of Czech Technical University in Prague, Buštěhrad, Czech Republic Second Faculty of Medicine (2. LF UK), Charles University, Prague 5, Czech Republic
Abstract: Nanofibers are fibres with diameters in nanometre range and one of their biggest advantages are high porosity and high surface area to volume ratio. Nanofibers are prepared from a large number of materials, such as natural polymers, synthetic polymers, carbon-based materials and composite nanomaterials. Nowadays nanofibers have a great potential in various disciplines. Main use can be found, for example, in energy generation and storage, water and environment treatment or biomedical engineering. Wound dressing, drug delivery and biological sensing are main applications of nanofibers in biomedical engineering and healthcare. The aim of the work was to approach the fabrication of nanofibers and their use for production of bionanosensors. These specific bionanosensors will be used for fast and easy detection of bacteria such as Staphylococcus aureus.
Introduction made of cellulose nanofibers and nanofibers with metal nanoparticles (Au, Ag, Zn, Cu etc.). Nanofibers are fibers whose diameter is in the nanometer range. They are made of various Nanofiber fabrication materials – natural and artificial polymers. It is also Most common method of nanofiber fabrication is possible to add other components to the used the electrospinning. This method has been polymers, such as metal nanoparticles. Such added introduced at the beginning of the 20th century. In components further affect the properties of the these days it is the most established method. [1] prepared fibers. The great advantage of nanofibers The electrospinning set-up consist of a syringe is their high porosity and high surface area to with nozzle, electric field source, pump and volume ratio. [1] counter electrode (grounded target). [1] Nowadays nanofibers are used in various High voltage source generates an electrostatic industries. The field of biomedicine uses field which is applied at the nozzle. This field nanofibers in various ways. The main uses are forms the charged jet of the polymer. The droplet wound dressing, drug delivery and biological of solution (polymer) at the nozzle has a conical sensing. [1] shape (Taylor’s cone). The charged jet accelerates Staphylococcus aureus is a gram positive to counter electrode and the solvent in solution bacteria which causes various diseases such as evaporates. The polymer is formed to solid meningitis, pneumonia, pyogenic infections, nanofibers by evaporation. [1,3,4] endocarditis etc. Most commonly it is diagnosed There are several nozzles which can be used from urine and blood. Currently PCR, ELISA and for electrospinning. Most commonly used are a metabolic tests are the most used methods of classical spinneret needle and a needle terminated diagnostics. However, these methods are with an edge. Depending on the shape and size of expensive, time-consuming and equipment the needle, nanofibers with different quality is intensive. [2] formed. [4] Recently, biosensors have been introduced as Emergent layer of nanofibers is laid on a an effective device for diagnosis. Various collector. The orientation of nanofibers depends on biosensors have been tested for the detection of S. electric field. Nanofibers with arranged fibers are aureus and other pathogens. These biosensors were formed on collectors in the form of a metal grid. The size of the grid affects the density distribution
19 Varvřovská et al.: Functionalized nanofibers as a specific bionanosensor for detection of Staphylococcus aureus
of nanofibers. Aligned nanofibers can be also The function of biosensor is to bind the formed on a rotating collector. biomolecule to the bioactive sensing layer, which creates a primary signal (chemical or physical).
Figure 1: Scheme of nanofiber fabrication – electrospinning. [5]
Arrangement of the fibers is a direct function of the velocity of the collector. [4] Polymers from which nanofibers are made can be divided to two main types – water soluble polymers (such as PVP and PVA [6]) and water insoluble polymers. Other important properties of polymers are viscosity, elasticity, electric potential, surface tension of polymer, Figure 2: Configuration of a biosensor. [8] hydrostatic pressure and some of the ambient influences. The concentration of polymer depends The primary signal is converted by the on volatile solvent which is also a carrier of the transducer into a secondary signal (electrical, polymer. [4] electrochemical, thermal etc.). The nature of the Nanofibers can be formed of pure polymer but secondary signal depends on the type of the also of polymers with nanoparticles and a transducer. [8] combination of polymers. It is nanofibers with The specificity of the biosensors is ensured by nanoparticles that are important in the field of the sensitive molecular recognition of biomedicine. [4] biomolecules. This recognition can be achieved Other methods of nanofiber fabrication are with various affinity systems, such as enzyme- Self-assembly in solution, CO2 laser supersonic substrate, antibody-antigen, nucleic acid- drawing, Solution blow spinning, Plasma- complementary sequence etc. [8] induced synthesis, Centrifugal jet spinning and Biosensors are very sensitive and specific, Electrohydrodynamic direct writing. [1] which also makes them very fragile. Because of After electrospinning, newly formed that, it is important to use them under the defined nanofibers are checked by SEM. [7] conditions (strictly defined pH zone, temperature, humidity or lighting level). [8] Biosensors Biosensors can be used for sensitive detection of Materials and methods pathogens. The biosensor is an analytical device with a selective response. This device responds to In recent years, various experiments have been a change of concentration or activity of performed to capture cells using nanofibers. The biomolecules. [8] group of doc. Gášková dealt directly with uptake of This type of sensor is made by a combination Staphylococcus aureus. In this work, they tested of biological material and transducer. Biological the uptake of S. aureus on pure nanofibers and material in this case means a bioactive sensing antibody-bound nanofibers. After cultivation of layer which is able to detect the analytes of our bacterial colony formed from cells trapped on interest (metabolites, toxins etc.). [8] nanofibers, the group of doc. Gášková measured the optical density of these colonies
20 Varvřovská et al.: Functionalized nanofibers as a specific bionanosensor for detection of Staphylococcus aureus
(spectroscopy). From their results it is evident that distilled water which is fed to the nanofibers nanofibers with antibody are more effective in through a PVC tube. capturing the respective cells (these nanofibers are The nanofibers with antibody will be applied able to retain 10 times more cells than pure to a target that will be placed in a filter vessel. This nanofibers without antibody). vessel is part of the pump system.
Figure 3: The air transfer system. Figure 5: Biological box (biohazard II).
The whole system for driving the air, in which Staphylococcus aureus is present, consists of a sealed container with S. aureus, the pump system (the filter vessel with antibody-bound nanofiber and the pump) and the nebulizer (Figure 3 and figure 4). Due to the possible threat of S. aureus, most of the mentioned system is placed in a biological box (biohazard II). Testing of such a system was performed using fluorescein, which replaced the bacteria for safety reasons, and pure PVA nanofibers without Figure 4: Layout diagram of the air transfer system. The air antibody. We also used fluorescein because it can with S. aureus is sucked by the pump from the sealed container be detected in a very small amount. and goes into the filter vessel. In the filter vessel it passes The PVA nanofibers on which fluorescein through the target with nanofiber where S. aureus is uptaken molecules were attached will be dissolved in water by the antibody. The filter vessel is also connected to the nebulizer which maintains sufficient humidity for the and analyzed (spectroscopy). Thanks to this nanofiber. method, it is possible to define the amount of the fluorescein, which could be detected. The same In this project, it was further found that for the procedure will be applied later in the further provability of the results, it is necessary to keep the experiments for the detection of Staphylococcus antibody-bound nanofibers in sufficient humidity. aureus. [8] Our goal is to create effective bionanosensor Conclusion for detection of S. aureus. In that case we will use antibody-bound PVA nanofibers (2 g/m2). Such As can be seen from various previous experiments, nanofibers represent the biosensor. Therefore, it is the use of nanofibers in biomedicine is a rapidly very important to keep these nanofibers under the evolving industry. Not only can they be used for defined conditions. As already found in the work wound dressing and drug delivery, but they are also of doc. Gášková, it is necessary to keep these suitable for creating sensors. Such sensor can be, nanofibers in sufficient humidity for the proper for example, an antibody-bound nanofiber. The function of the antibody. For this purpose, we will advantage of such sensors is small size, high use a nebulizer (Philips Respironics InnoSpire specificity and high sensitivity. Essence). The nebulizer creates a very fine mist of
21 Varvřovská et al.: Functionalized nanofibers as a specific bionanosensor for detection of Staphylococcus aureus
The primary task of our work was to devise a nanoparticles/carbon nanoparticles/cellulose nanofibers prototype of a system which will be able to capture nanocomposite for rapid and sensitive detection of Staphylococcus aureus (2018). Bioelectrochemistry. the bacteria Staphylococcus aureus on a prepared Elsevier, 2018, (123), 70-76. DOI: sensor. The system was designed so that air with 10.1016/j.bioelechem.2018.04. the bacteria from the sealed container passed [3] Nanopharma. Nanopharma [online]. Praha, 2015 [cit. through the biosensor (i.e. so that the bacteria could 2020-09-25]. Available from: be detected directly from the air). To ensure proper https://www.nanopharma.cz/cs/produkty-a- function of the antibody it was necessary to keep technologie/technologie sufficient humidity for the biosensor. A connected [4] ABD RAZAK, Saiful Izwan, Izzati Fatimah WAHAB, nebulizer is used for this purpose in our system. Fatirah FADIL, Farah Nuruljannah DAHLI, Ahmad This system will be further used to capture the Zahran MD KHUDZARI and Hassan ADELI. A Review bacteria from the air and to determine the lowest of Electrospun Conductive Polyaniline Based Nanofiber Composites and Blends: Processing Features, possible amount of the bacteria needed to detect it. Applications, and Future Directions. Advances in Materials Science and Engineering [online]. 2015 [cit. Further work 2020-09-25]. DOI: 10.1155/2015/356286 ISSN 1687- The next step in our study will be to define the 8434. Available from: minimal amount of Staphylococcus aureus needed https://www.hindawi.com/journals/amse/2015/356286/ for its demonstrable detection. This finding will [5] BHATTARAI, R. S., Xin LI, R. D. BACHU, S. H. S. help us in further study and subsequently in the BODDU a S. BHADURI. Biomedical Applications of Electrospun Nanofibers: Drug and Nanoparticle Delivery. preparation of the bionanosensor, which could be Pharmaceutics. MDPI, 2019, 11(1). DOI: used commercially in the near future. 10.3390/pharmaceutics11010005. [6] KADAJJI, Veeran Gowda and Guru V. BETAGERI. Acknowledgements: I would like to thank my supervisor Water Soluble Polymers for Pharmaceutical Taťána Jarošíková and consultant Evžen Amler for all the time Applications. Polymers[online]. 2011, 2011, 3(4) [cit. they have given me. I would also like to thank UCEEB, from 2020-09-25]. DOI: 10.3390/polym3041972. Available whom we obtain the necessary nanofibers. Finally, I would from: https://www.mdpi.com/2073-4360/3/4/1972/htm like to thank Dana Gášková and her student Tomáš Bartl for the opportunity to use their laboratory and for their help with [7] ZENDEHDEL, R., F. GOLI and M. HAJIBABAEI. experiments. Comparing the microbial inhibition of nanofibres with multi-metal ion exchanged nano-zeolite Y in air sampling (2019). Journal of Applied Microbiology. Society for References applied microbiology, 2019, 1-7. DOI: [1] KENRY and Chwee Teck LIM. Nanofiber technology: 10.1111/jam.14455. ISSN 1364-5072. current status and emerging developments [8] COULET, Pierre R. What is a Biosensor?, COULET, (2017). Progress in Polymer Science. Elsevier, 2017, (70), Pierre R. and Loic J. BLUM. Biosensor principles and 1-17. DOI: 10.1016/j.progpolymsci.2017.03.002. applications. New York: Marcel Dekker, 1991. ISBN 0- [2] RANJBAR, Saba and Saeed SHAHROKHIAN. Design 8247-8546-0 and fabrication of an electrochemical aptasensor using Au
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Table-top water-window microscope using a capillary discharge plasma source with spatial resolution 75 nm
Tomáš Parkman1,*, Michal Nevrkla2, Alexandr Jančárek2, Jana Turňová1, Dalibor Pánek1, Miroslava Vrbová1
1Department of Natural Sciences, Faculty of Biomedical Engineering, Czech Technical University in Prague, Kladno, Czech Republic 2Department of Physical Electronics, Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Prague 1, Czech Republic
Introduction One of the emerging imaging methods for cell biology is soft X-ray microcopy with its spatial resolution in the order of tens nm covering the gap between transmission electron microscopy and visible light microscopy. Soft X-ray (SXR) microscopes typically operate within the “water-window” spectral region, between the K-absorption edges of oxygen (2.34 nm; 530 eV) and carbon (4.4 nm; 280 eV) and have three advantages over light and electron microscopes. Firstly, SXR radiation penetrates biological materials much more easily than electrons, which allows specimens up to 10 µm thick to be imaged. Unlike electron microscopy, there is no need for sectioning eukaryotic cells with an ultramicrotome before imaging. Secondly, the short-wavelength allows image cells with higher spatial resolution to be compared to light sources. Finally, image contrast is obtained from the absorption of X-rays by the specimen; thus, there is no need to insert any fluorescence markers or other substances into the sample. Methods The compact transmission water-window microscope based on the Z-pinching capillary discharge nitrogen plasma source was developed at CTU in Prague, and operates at wavelength of 2.88 nm (430 eV). The emitted soft X-ray radiation is filtered by a titanium foil and focused by an ellipsoidal condenser mirror into the sample plane. A Fresnel zone plate was used to create a transmission image of the sample onto a charge-coupled device camera. Results To assess the resolution of the microscope, we imaged a standard sample-copper mesh (1000 lines/inch). The spatial resolution of the microscope is 75 nm at half-pitch, calculated via a 10–90% intensity knife-edge test. The green algae-Desmodesmus communis deposited onto a Si3N4 membrane was imaged. The SXR images of the algae exhibit a superior spatial resolution compared to visible light microscopy, owing to a much shorter wavelength beyond the capabilities of diffraction-limited visible light microscopes. Small features in the order of hundreds of nanometers can be easily observed. Conclusion The microscope allows the capture of images of a specimen with a magnification between 190× and 400× with a 75 nm half-pitch spatial resolution. This microscopic system is easy to operate, affordable, and accessible providing an alternative to large-scale facilities. The compact SXR microscopic systems have already found applications, not only in biomedicine, but also in material science, nanotechnology, and microelectronics, among others. These cost-effective table-top sources allow access to this imaging technique for the broader scientific community, and the future development of compact, table-top sources promises to broaden access to soft X-ray imaging in other laboratories. Acknowledgements: Supported by grants LTT17015, CAAS grant CZ.02.1.01/0.0/0.0/16-019/0000778 and SGS19/084/OHK4/1T/17. The full-text article is available online at: https://www.mdpi.com/2076-3417/10/18/6373/htm
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Fluorescence properties of gold and silver clusters and nanoparticles
Jan Svoboda1*, Peter Kapusta2, Stefan Vajda2, Vlastimil Fidler1,3,
1Czech Technical University in Prague, Faculty of Biomedical Engineering, Kladno, Czech Republic 2J. Heyrovský Institute of Physical Chemistry, Czech Academy of Sciences, Prague, Czech Republic 3Brown University, Department of Chemistry, Providence, Rhode Island, USA
*[email protected] Abstract: Properties of nanoparticles can be characterized either in bulk using spectroscopic methods to measure their emission or absorption spectra, or using amicroscope to visualize single particles or clusters of particles. While there is not a possibility to picture such small particles directly under a regular optical microscope, there is an option of catching their fluorescence using a confocal fluorescence microscope. At the first step, we purchased commercially available samples of 2 nm and 4 nm gold nanoparticles coated with a protective layer of dodecanethiol to prove feasibilty. We prepared solutions of these particles in toluene. These solutions were then deposited on a microscope cover glass to create a monolayer of nanoparticles. Using MicroTime 200 system (PicoQuant), we pictured the layer of nanoparticles by tracing their fluorescence upon excitation at 640 nm from a diode laser on a photodetector equipped with an 840 nm band pass filter. Resulting images reveal areas of dispersed light with FWHM of or less than 400 nm, which indicate a dispersed signal from single nanoparticles. There are also results from 4x2 nm hemispheric nanoparticles prepared by cluster deposition system excited by 405 nm filtered Hg lamp and using 550 nm band pass filter. Their images show similar results as previous measurements, albeit with higher FWHM.
properties of these nanoparticles, not taking into Introduction account the potential differences of each nanoparticle or their impurities. Nanoparticles or clusters are particles with dimensions below 100 nanometres. These particles can be either in pure state or coated with stabilizing Table 1. Nanoparticles’ properties 1 – size and coating - ligands and can be dry or immersed in solvent. Nanoparticles are organized by their study ([1] to [5]) and their Their small size causes them to have properties element, number of study and number in study (for example different from those of bulks of the same material Au 1.1 meaning first gold sample in firststudy). Exception lies in study [5], where samples are named by their elements’ ratio. and these properties can further change depending Symbol (-) means absent. on their coating with ligands or whether exposed to solvent molecules. Size±size distribution, Due to these properties nanoparticles are Nanoparticle Ligand/shell subjects in numerous research fields, but their size FWHM does not permit the use of ordinary optical methods (nm) for their imaging or direct measurements of their [1] Au 1.1 9.45±1.33,- - size-dependent properties. That is due to them Au 1.2 20.18±1.80, - - Au 1.3 50.73±3.58, - - being of sizes smaller than diffraction limit of the [2] Au 2.1 2.6±0.3 , - glutathione visible light. One of the options to picture Au 2.2 2.5±0.3 , - glutathione individual nanoparticles is using an electron Au 2.3 2.5±0.3 , - glutathione microscope. Spectroscopic methods are used to [3] Au 3.1 3 , 1 - measure fluorescence properties of nanoparticles in Au 3.2 4 , 1 - bulk or from single particles. Bulk measurements Au 3.3 6 , 1 - consist of measuring the absorption and emission Au 3.4 9 , 1 - dodecane- spectra and emission lifetime of the nanoparticles [4] Au 4.1 3.5±1.2, - in solvent or deposited on a substrate to find their thiole material composition or ligands. [5] Ag 5.1 30 , - SiO2 Au:Ag (1:3) 30 , - SiO2 Measuring using bulk spectroscopic Au:Ag (1:1) 30 , - SiO2 methods can thus only be used to measure average Au:Ag (3:1) 30 , - SiO2
Au 5.1 30 , - SiO2
24 Svoboda et al.: Fluorescence properties of gold and silver clusters and nanoparticles
Tables 1, 2 and 3 contain information about absorption peak, excitation wavelength and emission peak of gold and silver nanoparticles from several studies. These nanoparticles vary in their size from 2.5 to 50 nanometers with different or no coating.
Table 2. Nanoparticles’ properties 2 – solution and absorption peak - Nanoparticles are organized by their study ([1] to [5]) and their element, number of study and number in study (for example Au 1.1 meaning first gold sample in first study). Exception lies in study [5], where samples are named by their elements’ ratio. Symbol (-) means absent. Figure 1. MicroTime 200 system Absorption Max Nanoparticle Solution λA , FWHM (nm) [1] Au 1.1 water 517, 20 Au 1.2 water 524 , 20 Au 1.3 water 532 , 20 [2] Au 2.1 - - Au 2.2 - - Au 2.3 - - [3] Au 3.1 colloid 508 , 30 Au 3.2 colloid 514 , 30 Au 3.3 colloid 512 , 30 Au 3.4 colloid 520 , 30 [4] Au 4.1 toluene 511 , 50 [5] Ag - 440 , 30 Au:Ag (1:3) - 460 , 30 Au:Ag (1:1) - 490 , 30 Figure 2. Scheme of MicroTime 200 system.
Au:Ag (3:1) - 510 , 30 There are dry or solved samples in both polar and Au 5.1 - 530 , 30 organic solvents. As seen in the tables 2 and 3 the absorption or Table 3. Nanoparticles’ properties 3 – excitation peak and emission peak - Nanoparticles are organized by their study ([1] emission spectra change depending on size of the to [5]) and their element, number of study and number in study nanoparticles, whether they are in solvent or dry (for example Au 1.1 meaning first gold sample in first study).. and on their stabilizing ligands. Average Excitation Emission absorption peak of gold nanoparticles is around Max λexc λF , 510 nm and peak for silver nanoparticles is around Nanoparticle (nm) FWHM 440 nm. All gold nanoparticles were excited in (nm) ultraviolet range (300 to 410 nm) and had [1] Au 1.1 308 423, 40 emissions around 420 nm, except for nanoparticles Au 1.2 308 423, 40 Au 2.1, Au 2.2, and Au 2.3, which had emission at Au 1.3 308 423, 40 600 or 810 nm. [1, 2, 3, 4, 5] [2] Au 2.1 396 600, 70 The aim of this study is to use the fluorescence Au 2.2 350 810 , 50 properties of gold or silver nanoparticles on very 600 , 70 / Au 2.3 410 small particles of only few nanometers. 810 , 50 [3] Au 3.1 300 408, 30 Au 3.2 300 408, 30 Materials, instrumentation and methods Au 3.3 345 456, 40 We chose to use commercially available Au 3.4 300 408, 30 nanoparticles from PlasmaChem for the first [4] Au 4.1 - - experiment, specifically dry hydrophobic Au [5] Ag - - nanopowder of 2 nm and 4 nm sizes coated in Au:Ag (1:3) - - dodecanethiol. [6,7] This nanopowder was Au:Ag (1:1) - 560 , 30 dissolved in toluene and deposited on microscope Au:Ag (3:1) - - glass cover slide. The amount of used nanopowder Au 5.1 - -
25 Svoboda et al.: Fluorescence properties of gold and silver clusters and nanoparticles
Figure 6. Fluorescence image of 4 wide and 2 nm tall Ag Figure 4. Fluorescence image of a sample made of 2 nm Au nanoparticles. Area of image is 40 x 40 μm nanoparticles. Measuring of smaller signal spot. Area of image is 10 x 10 μm. computer. This mode is used for viewing emission of sample after excitation. was changed after each measurement to create a We have first used 405 nm laser diode. This monolayer of nanoparticles. was used to picture Figure 3 along with 140 nm For measurement and picturing we used wide 730 nm center emissio n filter. MicroTime 200 system (PicoQuant). This system Later wehave found higher photon counts allows two modes of operation. First one uses with 640 nm laser diode. Emission detector was microscope’s optics to directly view samples, used along with band pass filter of center at 840 nm which is mostly used for locating the sample’s and width of 270 nm. This second setup was used pocition and focusing. Second mode links the to picture Figure 4. and Figure 5. In both cases we optics with emission detection optics connected to used 60x magnification water immersion objective.
26 Svoboda et al.: Fluorescence properties of gold and silver clusters and nanoparticles
After measuring these commercial nanoparticles or clusters. These signals have nanoparticles we used custom made 4 x 2 Ag FWHM dependent on size of the particle or cluster, nanoparticles provided to us by Dr. Stefan Vajda. which could be used to distinguish between Those were created by a cluster deposition system nanoparticles of different sizes or their clusters. on TiO2 substrate. [8] These fluorescence pictures also show distribution For measuring we used MicroTime 200 of nanoparticles on substrate. system with 60x water immersion objective.This This method could in the future be used to time the objective was used dry, as the substrate, measure sizes of nanoparticles, their distribution on unlike glass cover slide, wasn’t transparent. That the support and potentially even their material caused lower resolution of pictures(Figure 6).For composition, via their emission peak, all at once. excitation we used Hg bulb with 405 nm excitation filter. Emission detector was used with with 550 Acknowledgements: I am thankful to J. Heyrovský Institute of Physical Chemistry for being able to participate in these nm emission filter. measurements. Results References In Figure 3 we can see large Au particles of [1] Abdelhalim, Mohamed Anwar K, and Mohsen M. uneven sizes. Given the area of the image these are Mady. 2012. “Physical Properties Of Different Gold most definitely conglomerates and / or large Nanoparticles: Ultraviolet-Visible And Fluorescence clusters of nanoparticles. That is caused by high Measurements” 03 (03). https://doi.org/10.4172/2157- 7439.1000133. concentration of nanoparticles in the sample. [2] Liu, Jinbin, Paul N. Duchesne, Mengxiao Yu, Xingya Concentration was changed based on previous Jiang, Xuhui Ning, Rodrigo D. Vinluan, Peng Zhang, measurements. Low signal intensity while and Jie Zheng. 2016. “Luminescent Gold Nanoparticles measuring with low concentrations of With Size-Independent Emission”. Angewandte Chemie International Edition 55 (31): 8894-8898. nanoparticles while using 405 nm excitation laser https://doi.org/10.1002/anie.201602795. forced us to highly increase concentration of [3] Philip, Daizy. 2008. “Synthesis And Spectroscopic nanoparticles to the point of not being able to find Characterization Of Gold Nanoparticles”. single particles anymore. Spectrochimica Acta Part A: Molecular And In Figures 4 and 5 we already found signal of Biomolecular Spectroscopy 71 (1): 80-85. https://doi.org/10.1016/j.saa.2007.11.012. Au nanoparticles, and using 640 nm excitation and [4] Soliwoda, Katarzyna, Emilia Tomaszewska, Beata appropriate emission filter we recorded images of Tkacz-Szczesna, Marcin Rosowski, Grzegorz single particles. Smaller darker spots have FWHM Celichowski, and Jaroslaw Grobelny. 2014. “The of their size below 400 nm (measured one has red Influence Of The Chain Length And The Functional Group Steric Accessibility Of Thiols On The Phase arrow over it), which means they are likely single Transfer Efficiency Of Gold Nanoparticles From Water nanoparticles. Larger brighter spots have FWHM To Toluene”. Polish Journal Of Chemical Technology of their sizes above 600 nm, which means they are 16 (1): 86-91. https://doi.org/10.2478/pjct-2014-0015. likely small clusters. [5] Yang, Jin-Sen, Miao-Miao Zhang, Zhen Han, Hai-Yang In Figure 6, we see an image of Ag Li, Lin-Ke Li, Xi-Yan Dong, Shuang-Quan Zang, and Thomas C. W. Mak. 2020. “A New Silver Cluster That nanoparticles excited by 405 nm. This wavelength Emits Bright-Blue Phosphorescence”. Chemical proved the best for these Ag nanoparticles. While Communications 56 (16): 2451-2454. its sizes’ FWHM is above 700 nm, we assume these https://doi.org/10.1039/C9CC09439C. are single nanoparticles as confirmed form earlier [6] “Au - Dry Nanopowder, Hydrophobic - Average Particle Size: Ca. 4 Nm”. Online. Plasmachem. X-ray characterization. Thus, the increased FWHM http://www.plasmachem.com/shop/en/average-particle- arises mostly because of worsened resolution size-ca-4-nm/268-pl-au-hpb2.html. caused by dry use of 60x immersion objective in [7] “Au - Dry Nanopowder, Hydrophobic - Average Particle combination with a non-transparent support. Size: Ca. 2 Nm”. Online. Plasmachem. http://www.plasmachem.com/shop/en/192-average- particle-ca-2nm. Conclusion [8] Yin, Chen., Eric Tyo, Kerstin Kuchta, Bernd von Issendorff and Stefan Vajda, 2014. “Atomically precise Images of nanoparticles from the fluorescence (catalytic) particles synthesized by a novel cluster microscope show a possibility of visualization of deposition instrument“. The Journal of Chemical bright spots caused by stimulated emission of Physics. 140 (17). Available from: doi:10.1063/1.48717
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Advanced Methods for Biomedicine
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Molecular genetics methods in use
Lucie Mrázková1,2*, Adéla Štěpánová1, Barbora Zavoloková1, Taťána Jarošíková1, Imtissal Krayem2, Jakub Mrázek3, Chahrazed Mekadim3, Martina Slapničková2,4, Peter Demant5 and Marie Lipoldová1,2
1Czech Technical University in Prague, Faculty of Biomedical Engineering, Prague, Czech Republic 2Laboratory of Molecular and Cellular Immunology, Institute of Molecular Genetics of the CAS, Prague, Czech Republic 3Institute of Animal Physiology and Genetics of the CAS, Prague, Czech Republic 4Laboratory of Functional Biology Protist, Institute of Parasitology, Biological Centre of the CAS, České Budějovice, Czech Republic 5Roswell Park Comprehensive Cancer Center, Buffalo, New York, United States of America
Abstract: The importance of studying microbial populations of the digestive tract in connection with various diseases is gaining relevance. Knowledge of the effect of leishmaniasis on the microbiome is limited. Genetically different individuals infected with the same species of parasite show a different composition of microbial populations of the digestive tract, which points to the importance of studying intestinal microbiota in relation to the host genotype. Aim of this study is a comparison of the composition of microbial populations of the digestive tract in health and disease depending on the host genotype. In the experimental model, we were able to define the effect of genetic background using several inbred strains in the chronic phase of infection and thus determine the effect of the disease itself. This work presents the molecular biology methods used for detailed analysis of immunopathological manifestations of Leishmania infection (PCR-ELISA) in combination with study of gut microbiome diversity (DGGE, NGS) in one experiment. These molecular genetics methods gave us a tool to prepare a more complex model of disease effects.
Introduction southern Europe [2]. Leishmaniasis has three main clinical manifestations: dermal (see fig. 1), Mammals gut microbiome is an important part of mucocutaneous and visceral. The main factors natural development of human and animals. Any influencing the type of pathology include the type disbalance (dysbiosis) has significant impact on of parasite and the genotype of the host [3]. health and well-being of its host. Presented project aims to describe the influence of Leishmania major infection on composition and diversity of microbiome in various parts of animals. We used modern tools of molecular microbiology (denaturating gradient gel electrophoresis, sequencing analyses of DNA) to distinguish differences between healthy and infected animals and between healthy individuals from different basic mice strains. The studies of the influence of Leishmania major on gut microbiome are limited Figure 1: Dermal manifestation of leishmaniasis in mouse (the lesion is formed around the site of inoculation) [Authors [1], so presented study can significantly contribute photo] to description of this severe infection. Leishmaniasis is a current problem in more The composition of microbial populations in than 98 countries, threatens the life, health and the gastrointestinal tract is studied in connection quality of life of more than 12 million patients and with many different diseases (autism, diabetes, is one of the world's most common parasitic Alzheimer's disease, obesity, Crohn's disease and diseases. This disease causes huge problems in the other), where the influence of the microbiome of tropical and subtropical part of the world, but its patients on health and disease is studied. The basic occurrence has also been noted in the states of functions of microbial populations include the
31 Mrázková et al.: Molecular genetics methods in use development of the immune system, protection strains, where on the genetic background BALB/c from pathogens, nutrient breakdown, synthesis of (87.5%) short donor sections of the strain STS vitamins and amino acids, metabolism of various (12.5%) are studied. Individual CcS strains vary substances and drugs, influence of the CNS and widely and even higher resistance has been many others [4]. Clarifying the role of the observed in some of these strains than in the STS microbiome in relation to parasitic infection brings strain. new insight into this issue. The results of such In second part of our study, the effect of studies could be a useful tool in the preparation of leishmaniasis disease on bacterial populations in new types of medicines, therapies and thus the small and large intestines was investigated on improvements in the care of the sick. 98 experimental animals from 6 basic mouse The originality of the proposed project lies in strains (B10, O20, B10.O20, PWD, PWK, the analysis of microbial populations in healthy C3H/eNpde). These strains are also quite individuals and in individuals infected with interesting due to the various reactions to infection Leishmania major protozoa. In our study, the effect caused by Leishmania major. Both strains B10 and of leishmaniasis on bacterial populations in the O20 are resistant to manifestations of small and large intestines was investigated on 211 leishmaniasis, that make susceptible strain experimental animals from 14 mouse strains, with B10.O20 even more interesting. B10.O20 is inbred all experimental animal being kept on the same strain formed by crossing B10 and O20 strains, diet. where on the genetic background B10 (96%) short donor sections of the O20 strain (4%) are studied Materials and methods [7].
Parasite Methods In our study we used protozoan parasites PCR ELISA is a molecular genetic method used for Leishmania major LV 561 (MHOM/IL/67/LRC- qualitative and quantitative estimation parasite L137 JERICHO II) was used for mouse infection. number in organs. In our study we measured organs Amastigotes were transformed to promastigotes (livers, spleens) from infected individuals. PCR using SNB-9, 16107 promastigotes from 6 days old ELISA was realized according to protocol [8]. subculture 2 were inoculated in 50 μl of sterile Next-Generation Sequencing (NGS) is a saline s.c. into the tail base. Inoculum was prepared molecular genetic tool that uses high-throughput according to the protocol for the preparation of parallel sequencing of immobilized template, to highly infective Leishmania promastigotes [5]. generate large quantities of data. In our study we have used a semiconductor sequencing developed Mice by Life Technologies on Ion Torrent PGM Our experiment was divided into two parts due to (ThermoScientific) platform to obtain detailed the large number of participating basic strains and composition of microbial communities. The Ion thus experimental animals. For inoculation were torrent platform was used in collaboration with used only female mice aged 8-16 weeks (sexual Institute of Animal Physiology and Genetics and immune maturity), the infection lasted 8 weeks according to protocol [10]. and than were experimental animals weighted, euthanized and dissected. A higher number of Measurements individuals is needed for the statistical weight of The lesion development was verified once a week the whole study. since the second week after inoculation. The First part of experiment contained 113 lesions were measured with digital vernier caliper experimental animals from 8 basic mouse strains and the area of the lesion was estimated as an (BALB/C, CcS-1, CcS-4, CcS-5, CcS-12, CcS-18, eclipse with two measured diameters equation (1). CcS-20, STS) for detailed analysis of genetic We measured these lesion areas weekly for a effects on the composition of the microbiota in follow-up estimate of growth dynamics. health and disease. These strains are particularly w h A (1) interesting due to the various reactions to infection 2 2 caused by Leishmania major [6]. The BALB/c strain is susceptible to immunopathological where A is lesion area, w is width, h is height and manifestations of leishmaniasis and the STS strain π is Archimedes constant (3.14). is resistant to these manifestations. CcS strains are inbred strains formed by crossing BALB/c and STS
32 Mrázková et al.: Molecular genetics methods in use
Hepatomegaly and Splenomegaly Ileum Hepatomegaly is an increase in the liver and splenomegaly is an increase in spleen, often caused by ongoing infection. These signs are among the basic pathophysiological manifestations of leishmaniasis. Values for statistical evaluation of splenomegaly and hepatomegaly were estimated by the experimental equation (2), these values must be optimized to weight of the mouse, because mice strains differ also in size and weight. To calculate these values, the spleens and livers were weighed immediately after dissection, and the weight of the mouse was determined the day before the end of the experiment.
mo Vo 1000 (2) m where Vo is representative value (for splenomegaly/hepatomegaly), mo is weight of organ (spleen/liver), m is weight of mouse.
Statistical Analyses Statistical analyses will be performed in Statistica software (Statsoft), BioNumerics (AppliedMaths) Figure 2: Dendrogram of microbial profiles from the ileum of and Qiime software [11]. mouse strains B10.O20 and O20. “E” stands for infected and “K” is a control group. Results This study was performed by collaboration CTU, Institute of Molecular Genetics of the CAS and Institute of Animal Physiology and Genetics of the CAS, where the experimental part was realized in laboratories of the CAS. We conducted a comparative study for 14 mouse strains, where common pathophysiological manifestations of leishmaniasis were examined and, in addition, the ileum and colon microbiome was compared for all participating mouse strains. For each of the basic mouse strains that participated in the study, a comparison was made between healthy and infected individuals. Dendrograms of microbial profiles from ileum and colon were performed (fig. 2, 4). Selected bands Figure 3: PCoA analysis of microbial profiles from the ileum were identified by Sanger sequencing as: 1. of mouse strains B10.O20 and O20. “E” stands for infected and “K” is a control group. Comparison of dendrogram data Bacteroides oleiciplenus (with an accuracy of was performed in BioNumerics (AppliedMaths). 89.23%), 2. Lactobacillus johnsonii (with an accuracy of 94.21%) 3. Lactobacillus animalis (with an accuracy of 97.06%), 4. Mucispirillum schaedleri (with an accuracy of 98.44%) and 5. Anaerocolumna xylanovorans (with an accuracy of 89.23%). Preliminary results of CcS mice strains were published on conference IMBM 2018 and could be found in the proceedings of this conference.
33 Mrázková et al.: Molecular genetics methods in use
Colon This data proves influence of parasite infection on host gut microbiota, depending on host genome.
Acknowledgements: The work was supported by SGS15/173/OHK4/2T/17, GACR14-0186S, GACR16- 22346S and SGS18/202/OHK4/3T/17. The ethics committee's consent to the project's request for experiments was given (File number: 16OZ30963/2015-17214, 66866/2015-MZE-17214 - valid until 2. 1. 2021). References [1] Lamour SD, Veselkov KA, Posma JM, Giraud E, Rogers ME, Croft S, Marchesi JR, Holmes E, Seifert K, Saric J. Metabolic, immune, and gut microbial signals mount a systems response to Leishmania major infection. J Proteome Res. 2015; 14(1):318-29. [2] Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M; WHO Leishmaniasis Control Team. Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 2012; 7(5):e35671. [3] Lipoldová M, Demant P. Genetic susceptibility to infectious disease: lessons from mouse models of leishmaniasis. Nat Rev Genet. 2006; 7(4):294-305. Figure 4: Dendrogram of microbial profiles from the colon of [4] Hand TW, Vujkovic-Cvijin I, Ridaura VK, Belkaid Y. mouse strains B10.O20 and O20. “E” stands for infected and Linking the microbiota, chronic Disease, and the “K” is a control group. immune system. Trends Endocrinol Metab. 2016; 27(12):831-843. [5] Grekov I, Svobodová M, Nohýnková E, Lipoldová M. Preparation of highly infective Leishmania promastigotes by cultivation on SNB-9 biphasic medium. Journal of Microbiological Methods 87 (2011): 273–277. [6] Lipoldová M, Svobodová M, Havelková H, Krulová M, Badalová J, Nohýnková E, Hart AA, Schlegel D, Volf P, Demant P. Mouse genetic model for clinical and immunological heterogeneity of leishmaniasis. Immunogenetics. 2002; 54(3):174-83. [7] Sohrabi Y, Volkova V, Kobets T, Havelková H, Krayem I, Slapničková M, Demant P, Lipoldová M. Genetic regulation of guanylate-binding proteins 2b and 5 during leishmaniasis in mice. Front Immunol. 2018 Feb 7;9:130. doi: 10.3389/fimmu.2018.00130. [8] Kobets T, Badalová J, Grekov I, Havelková H, Figure 5: PCoA analysis of microbial profiles from the colon Svobodová M, Lipoldová M. Leishmania parasite of mouse strains B10.O20 and O20. “E” stands for infected detection and quantification using PCR-ELISA. Nature and “K” is a control group. Comparison of dendrogram data Protocol 5, 1074–1080 (20 May 2010). was performed in BioNumerics (AppliedMaths). https://doi.org/10.1038/nprot.2010.68. [9] Muyzer G, de Waal EC, Uitterlinden a G (1993) Conclusion Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of Identification microbiome composition of the polymerase chain reaction-amplified genes coding for gastrointestinal tract gave us a comparison between 16S rRNA. Applied and Environmental Microbiology, 59(3): 695–700. healthy and infected individuals in 14 mouse strains that differ in susceptibility to the [10] Milani Ch, Hevia A, Foroni E, Duranti S, Turroni F, leishmaniasis. The PCoA analysis of preliminary Lugli GA, Sanchez B, Martin R, Gueimonde M, van Sinderen D, Margolles A, Ventura M (2013) Assessing data based on dendrograms (fig. 3, 5) shows a clear the Fecal Microbiota: An Optimized Ion Torrent 16S separation of infected B10.O20 from infected O20 rRNA Gene-Based Analysis Protocol. PLoS ONE 8 (7): and control samples, especially in colon samples. e68739.
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[11] Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010 May;7(5):335-6. doi: 10.1038/nmeth.f.303. Epub 2010 Apr 11. PMID: 20383131; PMCID: PMC3156573.
35
Experimental study of model hepatocellular carcinoma cell lines
Bibiana Kvasnicová1*, Martin Otáhal1, Martin Gregor2
1Czech Technical University in Prague, Faculty of Biomedical Engineering, Kladno, Czech Republic 2Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic
*kvasnbib @fbmi.cvut.cz
Abstract: The measurement of cellular mechanical properties offers new perspective in biological sciences. It has a huge importance in cancer research, where cell mechanics are tool to study invasive potential of the cancerous cells or about stage of a tumour progression. The aim of the project is to elaborate a comparative study between two different hepatocellular carcinoma cell lines (Snu 475 and Huh 7), with artificially introduced mutations for cytoskeletal protein plectin and during epithelial-mesenchymal transition induced by TGF-β1, focusing on study of mechanical properties using atomic force microscopy (AFM). The AFM method called force spectroscopy is used for the mechanical analysis and the data is processed by JPK software, allowing to display and interpret measured force curves, considering stiffness the most important. So far, the cell preparation protocol has been elaborated and first experimental measurements by AFM have been performed on wild type cell lines. The stiffness was equal to 598±111 Pa for Huh 7 and 1109±188 Pa for Snu 475.
responsible for cellular mechanical properties. It is Introduction made of different types of filaments and Hepatocellular carcinoma (HCC) is the most cytoskeletal-associated proteins, such as above common type of liver cancer and one of the most mentioned plectin. Plectin is responsible for frequent complications for patients struggling with mechanical stabilization and filament’s chronical liver diseases such as cirrhosis or organization. Knocking out the gene expression of hepatitis type B or C [1]. Current research is plectin should have a severe impact on cellular focused on finding differences between healthy mechanical properties, especially lower stiffness and cancerous cells with a purpose of finding more [5][6]. convenient approaches of treatment, aiming Another influence on mechanical properties to cancerous cells with high specificity. Study of study is an epithelial-mesenchymal transition mechanical properties can be used to target specific (EMT) induced by transforming growth factor beta structures in cancerous cells, where specific types 1 (TGF-β1). EMT is a biologic process that allows of drugs or nanoparticles can be applied. an epithelial cell to assume a mesenchymal cell Mechanical properties can be measured by AFM phenotype, which includes enhanced migratory force spectroscopy evaluating Young’s modulus, capacity, invasiveness, elevated resistance to adhesion, indentation depth and other parameters apoptosis, and greatly increased production of [2]. extracellular matrix components. EMT is Our work is focused on two different HCC cell physiologically associated with embryonic lines – HuH 7 and Snu 475. HuH 7 is an immortal development (EMT I), wound healing (EMT II), HCC cell line composed of epithelial-like cells and yet also it is responsible for cancer progression its origin is from a liver tumour of a 57-year-old (EMT III) in carcinoma cells. In laboratory Japanese male. [3] Snu 475 is a cell line established conditions it is possible to induce the EMT by in 1990 from a primary HCC of a 43-year-old cytokine TGF-β1 that controls e.g. cell growth, Korean patient [4]. proliferation and differentiation [7]. A gene knockout (K.O.) is a molecular biological technique that is used to eliminate a function of certain gene. The purpose of this intervention is to observe, what influence the knocked-out gene has. Our K.O. introduces mutation for cytoskeletal protein plectin. The cytoskeleton is a dynamic structure mainly
36 Kvasnicová et al.:Experimental study of model hepatocellular carcinoma cell lines
Figure 1: The cell line HuH 7 during AFM measurement Figure 2: The cell line Snu 475 during AFM measurement
measurement, is based on sensing Coulomb Equipment and methods repulsive forces between the tip and the sample.
Biological samples Force spectroscopy and force mapping Two cell lines of hepatocellular carcinoma (HCC) The force spectroscopy is an AFM method based – HuH 7 and Snu 475 – were chosen for the on measurement of the cantilever deflection as a comparative study. Both cell lines are measured in function of vertical position (z-axis). Basically, it vitro and their preparation in tissue culture is a force depending on vertical coordinate, laboratory is also a part of the project. The cells are resulting in a desired output – force curve. AFM cultivated in DMEM (Dulbecco's Modified Eagle force spectroscopy is based on single point Medium) with 10% FBS (fetal bovine serum) and measurement, when the cantilever gets to contact 1% Pen/Strep (antibiotics). The cells are kept in a with surface of the specimen and then withdraws, standard tissue culture conditions of thermobox obtaining the extend and the retract curves. (37 °C and 5% CO2). The routine maintenance of Moreover, a suitable contact mechanics model splitting and medium change is needed to be done must be chosen for fitting the measured data, twice a week. considering tip-sample interaction specifics and Samples for the experiment are prepared in a type of the specimen. Widely used models are unified way and with the same time schedule to Hertz, JKR, Sneddon or DMT model [8]. ensure reproducibility of measured data. Proper calibration of the measuring system is also essential, laser focus on PSD, spring constant Atomic force microscopy (AFM) and deflection sensitivity are crucial for data AFM is a method from the family of scanning conversion and analysis, because the original probe microscopy. The scanning probe is a information obtained from AFM is in units of cantilever with a sharp tip mounted at the end. The voltage. Deflection sensitivity is set by calibration measuring system senses interaction forces on hard surface and is measured in nm/V, allowing between the tip and sample. These interactions lead conversion of volts from the PSD to nanometres. to cantilever deformation (deflection) that can be To obtain an information about the tip-sample measured by optical lever element and four- force, spring constant in N/m is needed. The spring quadrant position sensitive detector (PSD). constant is obtained via thermal tune method. The Information obtained from the PSD is processed force 퐹 can be expressed by Hooke’s law: and analysed by feedback electronics, which 퐹 = −푘 ∙ 훿(푧), (1) controls piezoelectric vertical positioning of the where 푘 is the spring constant and 훿(푧) the vertical AFM probe. displacement of the cantilever. See table 1 for There are two classical AFM modes – contact approximate values of the calibration. and tapping. The contact mode, used for this
37 Kvasnicová et al.:Experimental study of model hepatocellular carcinoma cell lines
Figure 3: AFM force mapping regime shown in JPK data Figure 4: Young’s modulus comparation for wild type HuH 7 processing software. Used grid: 16x16; scanning area and Snu 475 cell lines 10x10 μm. The scalebar represents setpoint height of the sensor’s head. Hertz/Sneddon model [8] As mentioned earlier, force spectroscopy When force curves are measured, it is necessary to means single point measurement, so if many use an appropriate contact-mechanic mathematical measurements are required, force mapping regime model to fit the data to be able to calculate Young’s can be used. It enables to set an area of a certain modulus (stiffness) of the sample in certain point. width and a grid (e.g. 8x8 or 16x16) where every The Hertz/Sneddon model was chosen for our data. square of the grid is filled with one force curve, in It makes series of assumptions [9]: other words, the tip is going to indent the sample in sample is isotropic and linear elastic solid which raster, so that we gain information about larger part occupies an infinite half space of the sample. indenter cannot be deformed there are no additional interactions between the Data setting indenter and the specimen
depth of indentation is negligible comparted to Table 1. Approximate calibration values sample thickness Gain laser Sensitivity Spring constant The model can be described by a mathematical [V] [nm/V] [mN/m] equation, as seen below, considering the shape of 3.5-4 45-55 70-90 the tip [9]. For soft biological samples, sphere shaped probes are recommended, and it is also used Table 2. Data setting for extend phase of force spectroscopy for our measurements. Setpoint z range Time Velocity [nN] [μm] [s] [μm/s] 4 퐸푠푎푚푝푙푒 퐹 = √푅 ∙ 푑3, (2) 4.186 5.000 5.000 1.000 3 1 − 휇2
The importance of calibration was mentioned where 퐹 is force, 퐸푠푎푚푝푙푒 is the Young’s modulus earlier in the “Force spectroscopy and force of the sample, 휇 is a Poisson ratio (equal to 0.5 for mapping” paragraph. In table 1, suitable values biological structures as cells), 푅 is the probe’s from our measurement are shown. radius and 푑 is depth of indentation. In the regime of force mapping we chose grid 16x16 and the scanning area was 10x10 μm. Results A sphere tip cantilever, radius 5.2 μm, was chosen. The setpoint value means the maximum force that As shown in figure 4, the cell line Huh 7 has lower cantilever can exert on the sample (recommended Young’s modulus or stiffness, respectively, than values are 0.1-30 nN [8]), z range is the maximum Snu 475. The stiffness of HuH 7 single-cell in position of the cantilever’s head and time setting average is equal to 598±111 Pa, in case of Snu 475 defines duration of the extend phase. is equal to 1109±188 Pa.
38 Kvasnicová et al.:Experimental study of model hepatocellular carcinoma cell lines
There were also differences observed during cell itself (it is not a homogenous flat solid), and splitting procedure of cells. Snu cell line is likely given thinner areas of the cell, where plastic to need more time for trypsinization and their shape substrate can interfere and increase the Young’s is more compact compared to HuH, as shown in modulus value. figures 1 and 2. Moreover, HuH cell line tends to form clusters and it has poor tolerance to low References seeding density after splitting procedure. [1] BALOGH, Julius, David VICTOR, Emad H ASHAM, et al. Hepatocellular carcinoma: a review. Journal of Discussion Hepatocellular Carcinoma. 2016, 3, 41-53. ISSN 2253- 5969. doi:10.2147/JHC.S61146 The value of Young’s modulus is influenced by many variables – every difference in measurement [2] MOEENDARBARY, Emad a Andrew R. HARRIS. Cell mechanics: principles, practices, and prospects. Wiley setting, change of cantilever, chosen part of the cell Interdisciplinary Reviews: Systems Biology and to be scanned, time of measurement when the cell Medicine. 2014, 6(5), 371-388. ISSN 19395094. is not in its comfort environment can affect results. doi:10.1002/wsbm.1275 Especially the temperature can cause cell [3] HuH-7 cell line origins and charateristics. 2020. contraction and therefore affect the stiffness [10]. https://huh7.com/ To stick to the chosen protocol deals with most [4] PARK, Jae-Gahb, Jae-Ho LEE, Myung-Soo KANG, et influences. The temperature influence and cell al. Characterization of cell lines established from human contraction during the measurement were not hepatocellular carcinoma. International Journal of observed, yet heated chamber would enable to Cancer. 1995, 62(3), 276-282. ISSN 00207136. measure more cells from one cell culture dish. doi:10.1002/ijc.2910620308 Moreover, the data processing is very [5] CASTAÑÓN, Maria J., Gernot WALKO, Lilli dependent on final fitting on the Hertz model, WINTER a Gerhard WICHE. Plectin–intermediate filament partnership in skin, skeletal muscle, and where a certain area of the force curve is needed to peripheral nerve: principles, practices, and be chosen. It is also important to recognize, when prospects. Histochemistry and Cell Biology. the force curve is not suitable to be included into 2013, 140(1), 33-53. ISSN 0948-6143. the final statistics – especially in thinner areas of doi:10.1007/s00418-013-1102-0 the cell, the glass or plastic substrate of the plate [6] BOUAMEUR, Jamal-Eddine, Bertrand FAVRE, Luca can interfere. BORRADORI a Gerhard WICHE. Plakins, a Versatile Family of Cytolinkers: Roles in Skin Integrity and in Human Diseases. Journal of Investigative Dermatology. Conclusion 2014, 134(4), 885-894. ISSN 0022202X. doi:10.1038/jid.2013.498 Used measurement protocol for AFM and data processing in JPK software seem to be suitable for [7] KALLURI, Raghu, Robert A. WEINBERG, Luca BORRADORI a Gerhard WICHE. The basics of obtaining valid results, as well as cell preparation epithelial-mesenchymal transition: Roles in Skin and maintenance routine are convenient. Integrity and in Human Diseases. Journal of Clinical Investigation. 2009, 119(6), 1420-1428. ISSN 0021- Further work 9738. doi:10.1172/JCI39104 [8] JPK Instruments NanoWizard® Handbook Version 6.0. For more gentle measurement and better conditions 07/2018. Berlin, Germany, 2018. for cells during AFM scanning, a use of a heated [9] RADMACHER, Manfred, C. MATHEW MATE, Kevin chamber would be appropriate. All experiments T. TURNER, et al. Studying the Mechanics of Cellular have been carried out in a room temperature, which Processes by Atomic Force Microscopy: a review. Cell is not ideal for the cells, given their comfort Mechanics. Hoboken, NJ: Elsevier, 2007, 2007, 3, 347- temperature is 37 °C. 372. Methods in Cell Biology. ISBN 9780123705006. Until now we’ve measured HCC wild types ISSN 2253-5969. doi:10.1016/S0091-679X(07)83015-9 without any biological treatment, which means two [10] LI, Mi, LianQing LIU, Ning XI, YueChao WANG, different gene knockouts on two different cell lines XiuBin XIAO a WeiJing ZHANG. Effects of temperature and cellular interactions on the mechanics will be studied. and morphology of human cancer cells investigated by Another thing is the evaluation of force curves, atomic force microscopy. Science China Life Sciences. which is very lengthy process. An automatized 2015, 58(9), 889-901. ISSN 1674-7305. programme with Hertz fitting could be designed, doi:10.1007/s11427-015-4914-4 but it is not a simple task given the nature of the
39
Improving properties of titanium alloy used for modern hip prosthesis
Lucie Košinová1,3*, Petr Písařík1, Jan Kaufman3, Zdeněk Tolde2, Jan Brajer3
1Czech Technical University in Prague, Faculty of Biomedical Engineering, nám. Sítná 3105, 272 01 Kladno, Czech Republic 2Czech Technical University in Prague, Faculty of Mechanical Engineering, Karlovo náměstí 13, Praha 2 – Nové Město, 121 35Czech Republic 3HiLASE Centre, Institute of Physics ASCR, Za Radnicí 828, 25241Dolní Břežany, Czech Republic
Abstract: Hip replacement is a surgical procedure in which a hip joint is replaced by a prosthetic implant, that is, a hip prosthesis. Hip replacement is currently one of the most common orthopaedic operations, although patient satisfaction short- and long-term varies widely. It is therefore important to investigate and improve hip replacement materials. One of the possible ways is through laser treatment of the material using the Laser Shock Peening (LSP) as promising method to increase/prolong life time of hip-joint implant beyond patient life expectancy technology. LSP hardens the material and thus its resistance in parts prone to cracking is improved.
Introduction Important factors for lifespan of hip prosthesis are improving its mechanical properties such Recently, there has been an increasing demand in as hardness, tensile strength modulus, elongation research and development of medical biomaterials and fatigue strength 4. with a focus on biomedical implants. In this article, we will present the potential of The increasing need lead to more focus on the Laser Shock Peening method as convenient developing more durable implants in the last years means for improving mechanical properties 1. of materials used for hip prosthesis. Past silver and gold were used which are considered bioinert materials, but they Methods are expensive and exhibit poor mechanical properties. Subsequently, metallic alloys have been Laser Shock Peening (LSP) or hardening developed to be used in medical implants because of the surface of a material by laser-induced shock of wider application potential due to their enhanced wave is an advanced and progressive technique mechanical properties, tribological properties and to modify surface and enhance mechanical better biocompatibility when compared to pure properties of metallic components. That allows metals 1, 2. a significant increase in fatigue life of cyclically Significant research has been focused stressed parts 5, 6. LSP induces high-amplitude ontitanium alloys (i.e. Ti6Al4V) and stainless steel. compressive residual stress on the metal surface, Specifically titanium and its alloys currently which can effectively suppress the fatigue crack constitute the most highly favoured implant generation and its subsequent propagation [7, 8]. materials for joint replacement because titanium The high-pressure shock wave is generated by alloy shows higher resistance to repeated stress the interaction between metallic samples loading and therefore is ideal for load-bearing and nanosecond pulsed laser beams (with a power orthopedic applications 2, 3. density in the range of GW/cm2). After this While joint replacement is a success interaction, plastic deformation and residual stress in orthopaedic surgery, maintaining an implant have been introduced in the surface layer, which for a long time is still a challenge. The average can effectively enhance mechanical lifespan of a hip replacement is about 15 years 4, properties of materials, such as microhardness, which is not satisfactory for patients under 60 years fatigue resistance and corrosion resistance [5, 9]. of age. Prolonging the life of the implant thus Compared to conventional methods of surface requires more attention. treatment and material reinforcement commonly
40 Košinová et al.: Improving properties of titanium alloy used for modern hip prosthesis used in mechanical engineering such as shot- Another suitable method for measuring peening and rolling, which affect the material up to the depth of the laser effect is to measure 0.3 mm, the LSP method can lead to a larger depth the hardness in the cross section of the sample. of effect approximately 1 mm due to higher The graph in Figure 5 shows the dependence compressive residual stress as well as generation of of hardness on depth below top surface after finer microstructures (even nanocrystallization) [5, different LSP treatments. The microhardness value 6, 9]. decreased continuously with depth and The resultant compressive stress slows down the maximum hardness value appeared at the top cracks propagation and prolongs lifetime of treated surface. The measurement of the hardness parts [10]. of the sample with 2 layers of LSP should harden Figure 1 shows the principle of LSP the material more and reach a greater depth, which technology, where the sample is coated with vinyl is not entirely clear from the graph in Figure 5. (black) tape at the processing site. When the laser These ambiguities were most probably created due beam hits the surface of the sample, a plasma to the small scope of the statistics. is generated, which creates a high-pressure shock wave. There is a 1 mm thick layer of water Conclusion on the surface of the sample in order to increase the pressure that the plasma exerts, thus introducing a As the average age of patients for joint replacement higher residual stress into the material. The high- surgery decreases and the average life expectancy pressure wave thus affects into larger depth and is of men and women increases worldwide, more efficient than conventional engineering the demands for the joint materials are growing. methods [5-8]. The required improvements in pivotal properties such as wear, corrosion and fatigue resistance Experiment are discussed in terms of the enhanced microstructures that can be achieved through Titanium alloys are used in a wide range advanced surface and metal treatments. of applications such as biomechanical where We succeeded in inducing compressive corrosion resistance plays an important role residual stress up to 0.7 mm by 1 layer LSP resp. (implants and prostheses). In the experiment, 1.1 mm by 2 layers LSP in Ti6Al4V material, but cylindrical coupons of Ti6Al4V were used with the it's important to connect the results with other diameter of 15 mm and thickness of 3 mm (Fig.2). method for increasing resistance to mechanical For the LSP process, Yb:YAG laser with 1030 nm wear like smart surfaces. The LSP method thus wavelength, 10 ns pulse duration and 10 Hz seems to be a suitable method for processing repetition frequency was used. The size of beam the material used for hip prosthesis. was 2×2 mm. Before LSP the sample was covered with black vinyl tape. Acknowledgements: The work was supported by grant Figure 3 shows the visual aspect of plastic Student grant competition CTU number SGS19/134/OHK4/2T/17. deformation created by laser-induced shock wave. For this particular case, the LSP layer is composed of 4 sequences of laser imprints on the surface. Each sequence consists of imprints that align one next to each other without overlapping. Additionally, each sequence is shifted from each other so that in the end combining four of them gives 50% overlap. X-Ray Diffraction (XRD) analysis was used to measure the residual stresses generated in the surface and subsurface layers. After electrochemical polishing and subsequent measurement by XRD, the relationship between residual stress and depth was found (Figure 4). Greater residual stress, both surface and depth, was measured in the case of LSP with 2 layers where the affected depth was approximately 1.1 mm.
41 Košinová et al.: Improving properties of titanium alloy used for modern hip prosthesis
Fig. 1: Principle of LSP technology (work piece before LSP, during LSP, with plastic deformation after LSP) 6
Fig.Fig.3: 3: SampleSample of material of Ti6Al4V: materialmaterial Ti6Al4V: a) unaffacted, b) 1 layer LSP, c) 2 layers LSP Fig. 2: LSP Setup scheme 11 a) unaffacted, b) 1 layer LSP, c) 2 layers LSP
Fig. 4: Dependence of residual stresses on depth Fig. 5: Dependence of the hardness of the material measured by XRD analysis using the method of HV 0.1 on the distance from the surface processed electrochemical polishing on a sample of by the LSP technology. Ti6Al4V affected by the LSP method.
42 Košinová et al.: Improving properties of titanium alloy used for modern hip prosthesis
[6] BRAJER, Jan, Vliv metody Laser Shock Processing References na integritu povrchu, Disertační práce, ČVUT v Praze, Fakulta strojní, 2018. Available from: 1 MAHMOUD Z. IBRAHIM et al., Biomedical materials https://dspace.cvut.cz/handle/10467/79047 and techniques to improve the tribological, mechanical and biomedical properties of orthopedic implants – [7] REN, XD et al., Microstructure evolution and grain A review article, Journal of Alloys and Compounds 714 refinementof Ti-6Al-4V alloy by laser shock processing. (2017) 636-667. DOI: 10.1016/j.jallcom.2017.04.231. Applied Surface Science363 (2016) 44-49. Available from: DOI: 10.1016/j.apsusc.2015.11.192. Available from: https://www.sciencedirect.com/science/article/abs/pii/S https://www.sciencedirect.com/science/article/abs/pii/S 092583881731441X 0169433215028998 2 RACHIT AGARWAL, ANDRÉS J. GARCÍA, [8] FENGZE, Dai et al., Effects of laser shock peening with Biomaterial strategies for engineering implants for contacting foil on micro laser texturing surface enhanced Osseointegration and bone repair, Advanced of Ti6Al4V, Optics and Lasers in Engineering 101 Drug Delivery Reviews 94 (2015) 53–62. (2018) 99-105. DOI: 10.1016/j.optlaseng.2017.09.024. DOI: 10.1016/j.addr.2015.03.013. Available from: Available from: https://www.sciencedirect.com/science https://www.sciencedirect.com/science/article/abs/pii/S /article/abs/pii/S014381661730619X 0169409X15000484 [9] WEI GUO et al., Effect of laser shock processing 3 KARL-HEINZ FROSCH, KLAUS MICHAEL on oxidation resistance of laser additive manufactured STÜRMER, Metallic Biomaterials in Skeletal Repair, Ti6Al4V titanium alloy, Corrosion Science 170 (2020) European Journal of Trauma 32 (2006) 149–59. 108655. DOI: 10.1016/j.corsci.2020.108655. Available DOI: 10.1007/s00068-006-6041-1. Available from: from: https://www.sciencedirect.com/science/article/ab https://link.springer.com/article/10.1007/s00068-006- s/pii/S0010938X19324217 6041-1 [10] GUJBA, A, K, .et al., Laser peening Process and Its 4 Zimmer Biomet, online, c2020 cit 2020-10-02, impact on Materials Properties in Comparison with Shot Available from: https://www.zimmerbiomet.com/patien Peening and Ultrasonic Impact Peening, Materials ts-caregivers/article/hip/hip-replacement- (Basel, Switzerland), 2014, Dec; 7(12): 7925-7974. longevity.html DOI: 10.3390/ma7127925. Available from: https://www.sciencedirect.com/science/article/abs/pii/S [5] WEI GUOA et al, .Laser shock peening of laser additive 0010938X19324217 manufactured Ti6Al4V titanium alloy, Surface & Coatings Technology 349 (2018) 503–510. 11 J.L. OCAÑAETAL, Improvement of fatigue life and DOI: 10.1016/j.surfcoat.2018.06.020. Available from: surface properties of metallic materials of biomedical https://www.sciencedirect.com/science/article/abs/pii/S interest by laser shock processing, Bramat 2017 -10th 0257897218305954 International Conference on Materials Science Engineering
43
Effects of a change in the direction of view to near uncorrected visual acuity following implantation of monofocal intraocular lens
Markéta Žáková1, Martin Fůs1,2, Ján Lešták1,2 and Šárka Pitrová1,2
1CTU in Prague, Faculty of Biomedical Engineering, 272 01 Kladno 2; 2Eye clinic JL FBMI CTU in Prague, 158 00 Prague 5, Czech Republic
Abstract. The objective of the present study was to evaluate the effect of the direction of view of the eye on the postoperative near visual acuity of patients with monofocal intraocular lens. A total of 121 eyes in which we performed conventional cataract surgery with implantation of a monofocal lens were included in the study group. The postoperative examination of near visual acuity was performed at two different positions of the eye at a constant distance from the reading table, with the assumption of improving visual acuity when looking perpendicularly to the plane of the floor. The mutual relation of the postoperative parameters central keratometry (Kc), keratometry in the visual axis (KVA) and anterior chamber depth (ACD) for the single axial length ranges was determined using the correlation coefficients. In the case of vertical position of the eye (visual axis of the eye perpendicular to the floor), the uncorrected visual acuity following implantation of the monofocal lens was higher or equal compared to the horizontal position of the eye (visual axis of the eye parallel to the floor). The mean visual acuity at the horizontal position of the eye was 0.508 according to Jaeger's tables (P<0.001); at the vertical position, the mean value was 0.555 (P<0.001). Within the entire group, a weak association at best was observed between the postoperative parameters (Kc, KVA and ACD) and subsequent near visual acuity. Different dependence was found after categorising the group according to the axial length of the eye. In conclusion, the near visual acuity in eyes with an implanted monofocal lens for emmetropy to distance reached higher values at the vertical vs. horizontal position of the eyes. However, neither of the observed parameters (KC, KVA or ACD) can be unambiguously determined as decisive for the assumption of the described feature.
even for a UNVA of 0.8, the AL was up to 22.5 Introduction mm. Theoretically, a significant role of the In the framework of cataract surgery, a comfortable postoperative pseudo-accommodation amplitude postoperative uncorrected distance visual acuity in such eyes may be expected, particularly the (UDVA) may be successfully achieved due to the effect of an axial shift of the IOL causing a development of modern biometric methods and reduction of the anterior chamber depth (ACD), calculation formulas. However, patients also which occurs even when the direction of view of require to be able to see a normal reading text the eye is changed. It was proven that a change in without further correction, which has led to the the position of the eye affects the ACD, albeit to a development of multifocal or accommodation lesser extent than predicted [2]. The hydrodynamic intraocular lenses (IOLs). The challenge of these status in the eye is practically indefinable with models is the number of contraindications, risk of regard to several influencing factors. However, the asthenopic problems caused by higher-order validity of Pascal’s law is hypothesised, and due to aberrations, as well as the additional required the hydrostatic pressure under the influence of a payment by the patient. small gravitational force, the shift of the IOL For pseudophakic eyes, in addition to the causes a slight myopisation that likely confers an optimal UDVA, the optimal uncorrected near increase in near visual acuity. visual acuity (UNVA) may also be seen, although The subject of the present study was a a monofocal IOL has been implanted. In a previous statistical evaluation of a change in the UNVA study [1] it was proven that optimal near visual depending on the position of the text or a change of acuity may be achieved even without targeted the direction of the eye, including an analysis of a postoperative myopisation in eyes with a short mutual correlation of the individual eye axial length (AL). For 30.33% of eyes with a parameters. UNVA of ≥0.6, the AL was ≤23.5 mm, whereas
44 Žáková et al.: Effects of a change in the direction of view to near uncorrected visual acuity following implantation of monofocal intraocular lens
Materials and methods Mean parameters of the input group following categorization Study parameters The mean age of the group was 71 years. The In total, 121 eyes were evaluated following surgery mean parameter values of the eyes included in the in 65 patients. Patients who underwent cataract study are presented in Table 2. surgery with implantation of a monofocal IOL were included in the study. The patient selection Mean values of UNVAH and UNVAV was performed randomly, depending on the time The paired t-test was used to compare visual acuity the patients came for the check-up examinations at the horizontal and vertical position of the eye for between January and March 2017. The surgery was the whole group of patients. The results revealed performed by a single surgeon using an identical that, in the case of UNVAH for the horizontal technique (phacoemulsification using the initial position of the eye, the values were lower incision of 2.2 mm), and the relation SRK/T was compared with UNVAV for the vertical position of used to calculate the optical power of the IOL for the eye (mean 0.51 vs. 0.56, respectively; emmetropy; the evaluation period was at least 1 P<0.001). A higher or identical UNVAV value month post-surgery. The following models of IOL compared with UNVAH was always achieved in all were implanted: MA50BM (58.68% of eyes), groups based on the AL. Visual improvement in SA60AT (23.14%), SN60WF (13.22%) and UNVAV was observed in 40.2% of the eyes. The SN6ATx (4.96%). Only the postoperatively lowest mean value for the UNVAH was recorded in emmetropic eyes among the eyes examined were eyes with an AL >23.5 mm. The highest mean included, with 97.52% of the eyes achieving a value for the UNVAV was achieved in short eyes vision of 1.0 or better (the remaining 2.48% of the (mean 0.58; P<0.001). The complete values are eyes achieved a vision of 0.8, whereas no summarised in Table 3. correction improved the vision). Input data for the study are summarised in Table 1. Correlation coefficients The evaluated postoperative parameters Evaluation of the association of the eye parameters included the typography values (Anterior Segment for UNVAH and UNVAH was performed using Analyser Orbscan II) for optical power in the correlation coefficients (Table IV). We did not central part of the cornea (KC), as well as in the identify a more significant than weak correlation visual axis (KVA), anterior chamber depth (ACD; value for the whole group. For the AL<22.5 group, OcuScan biometer) and eye axial length (AL; we observed a weak negative correlation of the OcuScan biometer). UNVAH with AL (-0.39), but a moderate negative To determine the near vision values, each eye correlation was observed for AL and UNVAV was examined separately using the Jäeger table (-0.45). In the AL22.5-23.5 group of eyes, the positive ZEISS at a distance of 40 cm and its perpendicular correlation of the UNVAH with KC (0.45) and KVA position relative to the eye viewing axis. First, the (0.34), and of UNVAV with KC (0.42) and KVA value of the least read text was recorded at the (0.33), was found to be more significant. For eyes horizontal position of the eye (UNVAH, viewing with an AL >23.5 mm there was only a weak axis of the eye parallel with the floor) and negative correlation of UNVAV with ACD (-0.28). subsequently at the vertical position (UNVAV, viewing axis of the eye perpendicular to the floor). Discussion Demonstration of the position of the mutual axes is shown on Fig. 1. The observed parameters were During the postoperative evaluation of patients evaluated for the whole group of patients, but also with implanted monofocal IOL following standard following categorisation of the group into three cataract surgery, an unexpectedly high cohorts according to the AL: The group of short postoperative near visual acuity was observed. To eyes (AL<22.5) included eyes up to 22.5 mm, AL predict this effect, scientific studies have gradually 22.5-23.5 mm was identified as the group of attempted to identify a correlation between eye normal eyes (AL22.5-23.5) and the cohort of long eyes parameters and this phenomenon. The pupil size had an AL >23.5 mm (AL>23.5). and AL were not conclusively found to be correlated with near vision in 84 patients. However, a pupil diameter <2.6 mm along with AL <23 mm demonstrated better near visual acuity (3). Our previous study partially supports these conclusions, as our data
45 Žáková et al.: Effects of a change in the direction of view to near uncorrected visual acuity following implantation of monofocal intraocular lens revealed a moderate negative correlation of the approved the final version of this manuscript for postoperative UNVA with a decreasing AL publication. (<22.5 mm) [1]. Association of age with UNVA was not Ethics approval and consent to participate proven in the present study, whereas Hayashi All patients included in the present study provided written informed to participate, and the study et al (4) confirmed that patient age is a negative protocol conformed to the principles outlined in the factor affecting the postoperative amplitude of Declaration of Helsinki under approval from the pseudo-accommodation(correlation coeffic. of ethic committee of JL Clinic FBME CTU in -0.49); however, that study also included Prague. patients aged <40 years, while only 3 patients were <60 years of age in the present study. A References relevant assessment of the dependence on age [1] Leštak J, Pitrová Š, Fůs M, Žáková M: Nekorigovaná would require a higher age range. According to zraková ostrost do blízka po implantaci monofokální Nanavaty et al [5], corneal astigmatism IOL. Cesk Slov Oftalmol. 2017;73(4):127-33. (against the rule) is a significant factor that [2] Lister LJ, Suheimat M, Verkicharla PK, Mallen EA, increases the possibility of pseudo- Atchison DA: Influence of Gravity on Ocular Lens accommodation up to 10-fold. Position. Invest Ophthalmol Vis Sci. 2016;57(4):1885-91. A more statistically significant dependence [3] Lim DH, Han JC, Kim MH, Chung ES, Chung TY: Factors affecting near vision after monofocal intraocular lens on preoperative ACD, Kc or Kva for the whole implantation. J Refract Surg. 2013;29(3):200-4. group of patients was not observed. When [4] Hayashi K, Haysashi H, Nakao F, Hayashi F: comparing different positions of the read text Aging changes in apparent accommodation in eyes with and the position of the eyes, there was a a monofocal intraocular lens. Am J probability of increasing the value of the near Ophtalmol 2003;135(4):432-6. vision for the vertical position (UNVAV). The [5] Nanavaty MA, Vasavada AR, Patel AS, Raj SM, Desai TH: mean values show an increase of near visual Analysis of patients with good uncorrected distance and near vision after monofocal intraocular lens implantation. acuity in all patients, particularly those with an J Cataract Refract Surg. 2006;32(7):1091-7. AL <22.5 mm. It is believed that, in short eyes with an implanted IOL of higher optical power, the same value of its displacement towards the cornea will cause a higher myopia compared with an IOL that of lower optical power.
Availability of data and materials The input data for the study are summarised in Table I and do not include any direct or indirect identifiers. The datasets generated and analysed in the present study are available from the corresponding author on reasonable request.
Authors' contributions Figure 1. Demonstration of the position of the mutual axes. (A) MZ conceived, designed and performed the data The viewing axis of the eye is parallel with the ground – analysis of this study. MF and JL coordinated UNVAH; (B) the viewing axis of the eye is pointing towards preoperative and postoperative examinations of the the ground – UNVAV. UNVAH, decimal value of uncorrected eyes and made general revisions. SP performed near visual acuity in the horizontal position; UNVAV, decimal cataract surgery in all the patients and made general value of uncorrected near visual acuity in the vertical position. revisions of study. All the authors have read and
46 Žáková et al.: Effects of a change in the direction of view to near uncorrected visual acuity following implantation of monofocal intraocular lens
Table 1. Input values and parameters of the study. A, AL < 22.5 mm
Eye no. AL (mm) IOL UDVA ACD (mm) KC (D) KVA (D) UNVAH UNVAV 1 21.16 27.5 SA 0.80 3.90 44.68 44.95 0.40 0.50 2 21.28 26.5 SA 1.00 3.90 43.77 42.65 0.80 0.80 3 21.28 27.0 SA 1.00 4.00 46.05 45.33 0.50 0.60 4 21.43 23.0 SA 1.20 4.10 46.50 46.71 0.60 0.60 5 21.46 30.5 SA 1.00 3.40 42.15 41.68 0.80 0.80 6 21.47 23.0 SA 1.20 4.10 46.50 46.74 0.60 0.60 7 21.54 25.5 SA 1.20 3.80 44.86 45.46 1.00 1.00 8 21.64 24.5 SA 1.20 3.80 46.10 45.77 0.80 0.80 9 21.68 24.0 SA 1.20 3.80 46.10 45.84 0.80 0.80 10 21.75 26.0 SA 1.00 3.80 43.12 44.14 0.40 0.60 11 21.75 25.5 SA 1.00 3.80 42.00 43.00 0.40 0.60 12 21.80 24.5 SA 1.00 4.19 45.57 45.45 0.80 0.80 13 21.80 25.0 SA 1.00 4.19 44.67 45.06 0.80 0.80 14 21.80 27.0 SA 1.20 3.80 47.16 45.55 1.00 1.00 15 21.87 24.0 SA 1.20 3.50 45.20 45.67 0.50 0.50 16 21.87 24.5 SA 1.00 3.70 44.78 44.45 0.50 0.60 17 21.94 24.5 MA 1.00 3.30 45.91 44.76 0.30 0.40 18 21.95 24.0 SA 1.50 3.80 45.81 45.19 0.20 0.30 19 21.95 24.5 SA 1.00 3.50 45.00 44.81 0.40 0.40 20 21.96 24.0 SA 1.20 3.50 45.58 45.54 0.50 0.50 21 21.96 23.5 SA 1.50 3.90 45.64 45.93 0.20 0.30 22 21.99 25.0 SA 1.00 3.50 45.07 44.36 0.40 0.40 23 21.99 24.5 MA 1.00 3.50 45.70 44.47 0.30 0.40 24 22.12 26.5 SA 1.00 4.00 46.06 45.54 0.50 0.60 25 22.13 24.5 SA 1.00 3.50 44.84 44.53 0.50 0.60 26 22.14 22.0 SA 1.20 3.60 46.35 45.79 0.40 0.40 27 22.16 24.0 IQ 1.00 2.00 45.64 45.66 0.60 0.60 28 22.20 22.5 SA 1.20 3.80 45.93 46.25 0.50 0.60 29 22.22 25.0 IQ 1.20 4.20 44.00 44.01 0.30 0.30 30 22.28 22.0 SA 1.20 3.80 46.26 46.48 0.50 0.60 31 22.32 23.5 IQ 1.00 2.00 45.61 44.80 0.50 0.60 32 22.35 22.0 SA 1.00 3.60 45.94 46.52 0.60 0.60 33 22.38 24.5 SA 1.00 3.70 43.94 43.30 0.50 0.50 34 22.40 22.0 SA 1.00 3.70 45.28 45.11 0.60 0.60 35 22.42 24.5 IQ 1.20 3.60 44.27 44.38 0.30 0.30 36 22.46 24.0 SA 1.00 3.70 42.72 42.65 0.50 0.50 37 22.47 23.0 SA 1.20 4.20 44.45 44.80 0.30 0.40 AL, axial length of eye; IOL, intraocular lens - power and model (SA = SA60AT, MA = MA50BM, IQ = SN60WF IQ); UDVA, decimal value of uncorrected distance visual acuity; ACD, anterior chamber depth; KC postoperative keratometry value - centre of the cornea; KVA, postoperative keratometry value - visual axis of the cornea; UNVAH, decimal value of uncorrected near visual acuity in the horizontal position; UNVAV, decimal value of uncorrected near visual acuity in the vertical position.
47 Žáková et al.: Effects of a change in the direction of view to near uncorrected visual acuity following implantation of monofocal intraocular lens
B, AL 22.5 – 23.5 mm
Eye no. AL (mm) IOL UDVA ACD (mm) KC (D) KVA (D) UNVAH UNVAV 38 22.51 23.0 SA 1.20 4.30 44.46 44.42 0.30 0.40 39 22.56 23.0 SN 1.00 3.27 42.50 45.34 0.50 0.50 40 22.58 23.0 SA 1.20 3.70 46.47 44.55 0.40 0.40 41 22.58 23.0 SA 1.00 3.40 47.90 47.48 0.80 0.80 42 22.64 23.0 SA 1.00 3.97 43.62 43.96 0.40 0.50 43 22.66 23.0 SA 1.00 3.80 46.48 44.41 0.80 0.80 44 22.70 22.0 SA 1.20 3.70 46.50 45.44 0.80 0.80 45 22.73 21.0 SA 1.00 4.20 46.56 46.78 0.60 0.80 46 22.74 26.5 IQ 1.20 3.80 41.24 40.46 0.40 0.50 47 22.80 22.0 SN 1.20 3.28 44.50 44.24 0.50 0.50 48 22.80 21.5 SA 1.20 3.80 45.14 46.19 0.80 0.80 49 22.81 22.5 SA 1.50 3.96 45.15 45.16 0.60 0.60 50 22.81 22.0 SA 1.50 3.80 44.36 44.44 0.60 0.60 51 22.81 21.5 SA 1.50 3.90 43.93 43.74 0.50 0.60 52 22.85 22.0 SA 1.00 3.90 42.15 41.15 0.40 0.50 53 22.87 20.0 SA 1.00 4.20 47.18 46.32 0.60 0.80 54 22.87 22.0 SA 1.20 4.30 42.81 42.44 0.50 0.60 55 22.92 21.5 IQ 1.50 4.00 45.12 44.66 0.60 0.60 56 22.94 22.0 MA 0.80 3.60 44.10 45.59 0.40 0.50 57 22.95 22.0 MA 0.80 3.70 45.60 45.69 0.40 0.50 58 22.96 22.0 SA 1.00 2.20 43.73 43.50 0.50 0.50 59 22.96 22.5 SA 1.00 3.80 45.19 43.50 0.50 0.60 60 22.98 22.0 SA 1.00 3.80 42.37 43.06 0.50 0.60 61 22.99 22.0 SA 1.00 4.00 45.96 45.71 0.50 0.50 62 23.00 21.5 SA 1.00 3.60 43.93 44.73 0.60 0.60 63 23.10 20.5 SA 1.00 3.80 44.65 44.57 0.50 0.50 64 23.10 22.5 MA 1.50 4.10 42.48 43.13 0.40 0.50 65 23.12 24.0 SA 1.50 4.10 40.82 40.93 0.60 0.60 66 23.17 21.5 SA 1.00 4.20 42.78 41.76 0.50 0.50 67 23.24 24.5 IQ 1.20 3.80 40.20 41.07 0.40 0.40 68 23.26 21.0 MA 1.00 4.20 45.66 44.75 0.60 0.60 69 23.29 21.0 MA 1.00 4.20 44.32 45.19 0.60 0.60 70 23.30 23.0 IQ 1.20 3.60 42.09 42.12 0.50 0.60 71 23.31 21.0 SA 1.50 3.80 43.90 43.45 0.50 0.60 72 23.32 23.0 IQ 1.00 3.60 43.09 43.29 0.50 0.60 73 23.35 20.0 MA 1.20 3.60 45.36 45.26 0.60 0.60 74 23.37 20.5 SA 1.20 3.70 43.54 44.35 0.50 0.50 75 23.40 21.5 SA 1.00 3.60 44.26 44.94 0.60 0.60 76 23.40 24.5 SA 1.50 4.10 41.62 42.64 0.60 0.60 77 23.40 23.0 SA 1.20 4.20 41.88 42.71 0.40 0.50 78 23.41 20.0 MA 1.20 3.90 44.45 45.69 0.50 0.60 79 23.41 20.5 MA 1.20 3.80 44.79 45.02 0.50 0.50 80 23.41 21.5 MA 1.20 3.90 43.94 41.59 0.50 0.50 81 23.42 21.5 MA 1.20 3.80 42.43 43.26 0.60 0.80
48 Žáková et al.: Effects of a change in the direction of view to near uncorrected visual acuity following implantation of monofocal intraocular lens
82 23.42 22.0 MA 1.20 3.90 42.29 43.29 0.40 0.40 83 23.44 20.5 SA 1.20 3.90 44.15 44.36 0.50 0.50 84 23.44 21.0 MA 1.20 3.80 43.44 43.37 0.50 0.50 85 23.48 20.5 SN 1.20 4.30 43.22 44.76 0.50 0.50 86 23.50 20.0 SN 1.20 4.40 44.90 45.30 0.50 0.50 87 23.50 21.5 MA 1.20 3.60 45.65 44.57 0.60 0.60 88 23.50 20.0 SA 1.20 4.00 43.62 43.51 0.30 0.30 89 23.50 20.0 SA 1.50 3.70 43.83 45.61 0.20 0.30
C, AL >23.5 mm
Eye no. AL (mm) IOL UDVA ACD (mm) KC (D) KVA (D) UNVAH UNVAV 90 23.51 21.5 MA 1.20 3.80 41.93 42.99 0.40 0.40 91 23.53 19.5 IQ 1.00 3.70 44.56 44.47 0.50 0.50 92 23.54 23.5 SA 1.20 3.70 40.92 41.41 0.60 0.80 93 23.55 21.5 SA 1.00 4.20 43.05 43.81 0.60 0.60 94 23.57 20.0 MA 1.20 3.90 44.22 44.57 0.50 0.50 95 23.58 20.5 SN 1.00 3.96 46.21 44.91 0.50 0.50 96 23.60 21.5 IQ 1.50 4.10 45.90 45.86 0.60 0.60 97 23.61 19.5 MA 1.00 4.10 45.13 45.66 0.20 0.30 98 23.63 18.5 IQ 1.00 3.80 45.43 45.99 0.50 0.50 99 23.66 20.0 MA 1.00 4.20 45.81 46.16 0.20 0.30 100 23.67 19.5 SN 1.00 4.00 43.54 44.85 0.50 0.50 101 23.69 18.5 MA 1.20 4.20 45.96 44.33 0.50 0.60 102 23.73 22.5 SA 1.20 3.80 42.53 42.05 0.60 0.80 103 23.74 21.0 MA 1.20 4.10 43.57 42.96 0.30 0.30 104 23.78 21.0 SA 1.00 4.20 41.24 44.98 0.50 0.60 105 23.81 20.5 MA 1.20 4.10 42.83 44.14 0.30 0.30 106 23.85 20.5 MA 1.20 3.80 43.19 43.56 0.60 0.80 107 23.88 22.5 SA 1.20 4.20 41.28 41.42 0.50 0.50 108 23.90 20.0 SA 1.00 3.90 46.56 45.50 0.50 0.60 109 23.90 20.0 SA 1.00 3.90 45.81 43.06 0.50 0.60 110 23.92 22.5 SA 1.20 4.10 41.04 41.07 0.50 0.50 111 23.97 22.0 SA 1.00 4.00 40.43 40.80 0.50 0.50 112 24.12 21.5 IQ 1.20 3.90 41.40 41.60 0.20 0.30 113 24.13 21.5 IQ 1.20 3.80 41.50 41.97 0.20 0.30 114 24.27 21.5 SA 1.00 3.90 40.68 41.15 0.50 0.50 115 24.35 17.5 MA 1.00 3.90 44.72 44.94 0.50 0.60 116 24.46 17.0 MA 1.50 3.90 44.84 44.91 0.60 0.80 117 24.65 22.0 MA 1.00 4.10 41.66 41.68 0.50 0.50 118 24.75 18.5 IQ 1.50 4.20 42.74 42.50 0.50 0.50 119 24.76 18.5 IQ 1.50 4.40 42.85 42.80 0.50 0.50 120 24.81 15.5 MA 1.20 4.00 44.71 45.81 0.60 0.60 121 24.83 22.0 MA 1.00 4.10 41.26 41.15 0.50 0.50
49 Žáková et al.: Effects of a change in the direction of view to near uncorrected visual acuity following implantation of monofocal intraocular lens
Table 2. Mean parameters of the input group following categorization into the groups.
Values based on the AL groups Values of the whole group Parameters <22.5 ≤22.5-23.5≥ >23.5
Count (eyes) 121 37 52 32
AL (mm) 22.96±0.85 21.93±0.36 23.09±0.31 23.96±0.43
ACD (mm) 3.83±0.37 3.68±0.47 3.84±0.35 3.68±0.47
KC (D) 44.19±1.73 45.11±1.21 44.04±1.65 43.36±1.91
KVA (D) 44.21±1.58 44.96±1.17 44.10±1.55 43.53±1.73
Values are presented as mean ± standard deviation. AL, axial length of eye; ACD, anterior chamber depth; KC postoperative keratometry value - centre of the cornea; KVA, postoperative keratometry value - visual axis of the cornea.
Table 3. Mean values of UNVAH and UNVAV. Category Mean (mm) SD t P-value
UNVAH 0.51 0.15 All AL -8.18 <0.001 UNVAV 0.56 0.15
UNVAH 0.53 0.20 AL<22.5 -4.62 <0.001 UNVAV 0.58 0.18
UNVAH 0.52 0.12 AL22.5-23.5 -5.25 <0.001 UNVAV 0.56 0.12
UNVAH 0.47 0.13 AL>23.5 -4.19 <0.001 UNVAV 0.52 0.16
AL, axial length of eye (different groups); UNVAH, decimal value of uncorrected near visual acuity in the horizontal position; UNVAV, decimal value of uncorrected near visual acuity in the vertical position; SD, standard deviation.
Table 4. Correlation coefficients for the whole group and different ALs. AL (mm) Variables
Whole group ACD KC KVA AL
UNVAH -0.06 0.24 0.18 -0.21
UNVAV -0.04 0.22 0.17 -0.22 <22.5
UNVAH 0.06 0.11 0.06 -0.39
UNVAV 0.07 0.06 0.04 -0.45 22.5-23.5
UNVAH -0.09 0.45 0.34 -0.21
UNVAV 0.01 0.42 0.33 -0.29 >23.5
UNVAH -0.11 0.03 -0.01 0.12
UNVAV -0.28 0.07 0.02 0.05
50 Žáková et al.: Effects of a change in the direction of view to near uncorrected visual acuity following implantation of monofocal intraocular lens
AL, axial length of eye; ACD, anterior chamber depth; KC postoperative keratometry value - centre of the cornea; KVA, postoperative keratometry value - visual axis of the cornea; UNVAH, decimal value of uncorrected near visual acuity in the horizontal position; UNVAV, decimal value of uncorrected near visual acuity in the vertical position.
51
Comparison of multipotent mesenchymal stromal cells from human adipose tissue and Wharton's jelly for soft tissue reconstruction
Simona Stuchlíková1,2*, Eliška Vavřinová1,2, Taťána Jarošíková1, Yuriy Petrenko2
1Czech Technical University in Prague, Faculty of Nuclear Sciences and Physical Engineering, Prague, Czech Republic 2Institute of Experimental Medicine AS CR, Prague, Czech Republic
Abstract: Adipose tissue engineering, based on the application of multipotent mesenchymal stromal cells (MSCs) represents a promising strategy to restore soft tissue defects. The source of MSCs for the application in adipose tissue engineering may determine the efficiency of reconstruction strategy. The capacity to differentiate towards adipogenic lineage is one of the criteria defining fibroblast-like cells to MSC. However, the efficiency of the differentiation depending on the source of cell isolation is not determined as a standard and comparative studies are needed to find the optimal source of MSC for adipose / soft tissue reconstruction. The aim of the study was to evaluate the potential of MSCs derived from adipose tissue (AT) and Wharton’s jelly (WJ) for adipose tissue engineering. We assessed the effect of the presence of serum in the adipogenic differentiation medium. Finally, we assessed the possibility of using established culture and differentiation conditions for the adipose tissue engineering in 3D collagen scaffolds.
possible to use various dermal fillers, which can Introduction significantly improve or even remove scarring. [5] Soft tissue reconstruction is performed mainly in There are natural and synthetic fillers. plastic surgery, where a number of procedures are Soft tissue reconstruction deals with adipose performed due to adipose tissue transfer, such as: tissue engineering, where it is necessary to breast augmentation, facial rejuvenation, skin comprehensively understand the physiology of treatments after deformation caused by injuries tissue in vivo. This knowledge will allow the (burns, frostbite, electric shock, burns, etc.) [1]. designed components to be integrated so that the Adipose tissue is a sophisticated and dynamic set tissue that is regenerated is mechanically and of heterogeneous populations that are capable of functionally suitable for the implantation site. [3] producing and responding to hormones, Tissue engineering is a field in which various areas vasculature formation, energy storage, and the are connected - medicine, cell and molecular transformation of inactive precursor cells into biology, technical fields, especially materials mature cells and stimuli [2]. There are two main science and biotechnology. This engineering uses types of adipose tissue (brown (BAT - brown both cells and biomaterials as well as engineering adipose tissue) and white (WAT - white adipose approaches. [6] [7] tissue)) in the human body [3]. Cells can be considered a major component of Lipotransfer is a plastic operation in which tissue engineering. Choosing the right type of cells two procedures are performed simultaneously and with high adipogenic differentiation capacity is a the patient's own fat is used [4]. In this way, not key factor for adipose tissue reconstruction. [8] only rejuvenation, filling wrinkles, enlarging the Mesenchymal stem cells (MSCs) are lips, where the fat itself is already missing, but also multipotent cells with unique therapeutic potential. replenishing the fat in places where it is already, These cells are expected to have a highly effective such as breasts, buttocks, etc. [3] [4]. Tissue treatment mainly in the overall response of the collection is performed with a special liposuction organism to infection, transplant medicine and cannula, as with conventional liposuction, but autoimmune diseases. They have the capacity of should be fine [1]. immune modulation and can differentiate into tree Undesirable phenomena occurring in the face lineages: adipogenic, osteogenic, and are post-traumatic scars, acne scars, wrinkles as chondrogenic [9]. MSCs produce cytokines and a sign of aging and the like. Today, it is already
52 Stuchlíková et al.: Comparison of multipotent mesenchymal stromal cells from human adipose tissue and Wharton's jelly for soft tissue reconstruction growth factors, which affect the host cells and shaking After centrifugation at 200 x g for 10 tissues. minutes, the stromal vascular fraction was washed MSCs can be derived from the variety of twice PBS and cultured. Discarded human tissues such as bone marrow, adipose tissue, umbilical cords (UC) were obtained from healthy peripheral blood, or internal organ harvesting, if neonates (N = 3) after spontaneous delivery. About present for life [10]. In newborns, MSCs can be 10 cm UC was aseptically stored in sterile PBS obtained from umbilical cord, amniotic fluid, (IKEM) with antibiotic-antifungal solution (AA) at placenta or amniotic membrane. 4 ° C and transported to the laboratory within 24 In the application of tissue engineering, there hours. After washing several times in PBS-AA and is a need to create so-called scaffolds that provide brief washing in 10% betadine (EGIS a cell-like environment to promote cell Pharmaceuticals PLC, Budapest, Hungary), blood proliferation, differentiation and tissue vessels were removed and the remaining Wharton's regeneration. Numerous techniques and jelly tissue (WJ) was cut into small fragments (1-2 biomaterials are used to mimic native tissues. The mm3) and weighed. WJ-MSCs were isolated from most interesting are hydrogels formed from fragments (1 g) by digestion in PBS-AA solution biopolymer networks. Protein-based hydrogels containing 0.26 U / ml Liberase® (Roche Custom with important components such as silk, keratin, Biotech, Mannheim, Germany) 62 and 1 mg / ml elastin, or resilin proteins are used. [14] Scaffold - hyaluronidase at 37 ° C with constant shaking after the so-called carrier is actually a supporting for 2 hours. After removing undigested fragments structure in which cells grow and multiply, or using 40 μm cell filters, the cells were centrifuged tissues develop. It is made of synthetic or (450 x g, 10 minutes), diluted in complete culture biological materials, it must decompose well medium, and cultured. according to how the tissue is formed (these are Cells were cultured in complete culture months), it must be porous so that the cells can medium (KKM) containing α-MEM (LONZA, settle, nourish them and drain waste fluids. [16] Basel, Switzerland), 10% fetal bovine serum (FBS) These scaffolds should be non-immunogenic, or 5% platelet lysate (PL) (Bioinova, Ltd.) and 10 promoting cell adhesion and growth during culture μg / ml gentamicin (Sandoz, Holzkirchen, [3], [16]. The materials used Germany) at 37 ° C, in a humidified atmosphere decompose over time into non-toxic products containing 5% CO 2 with regular changes of and are excreted naturally from the human body. medium twice a week. Cells derived in passage The disadvantages are low mechanical strength and 3 were used for the following studies. insufficient reproducibility. [16] To determine the colony formation potential, The aim of the study was to evaluate the MSCs were seeded at a concentration of 1,000 cells potential of multipotent mesenchymal stromal cells per 100 mm cell culture dish (TPP, Switzerland) derived from adipose tissue and Wharton's jelly for and cultured for 14 days in α-MEM supplemented adipose tissue engineering and soft tissue with 20% FBS. The medium was changed once reconstruction. after 7 days of cultivation. After 14 days of culture, cells were fixed with 70% ethanol and stained with Material and methods Giemsa using standard procedures. The number and size of colonies were evaluated visually. In our study, we worked with cells isolated Colony formation efficiency (CFU-F efficiency) from adipose tissue and umbilical cord (Wharton's was determined as the ratio between the number of jelly), which were already taken from human colonies generated to the total number of tissues at the Institute of Experimental Medicine of inoculated cells. the Academy of Sciences of the Czech Republic The metabolic activity of MSCs was and cryopreserved. The adipose tissue (AT) determined using Alamar Blue (AB). The samples used were obtained from healthy alamarBlue® test is designed to quantitatively volunteers (N = 3) who underwent liposuction measure the proliferation of various human and procedures for aesthetic reasons and were animal cell lines, bacteria and fungi. AB is an processed (lipoaspirate was repeatedly washed in integral indicator of redox reactions that occur in phosphate buffered saline (PBS; IKEM, Prague, living cells. Alamar Blue (AB) was used for this Czech Republic) and enzymatically digested with experiment to reach a concentration of 10%. After collagenase (0.3 PzU / ml, collagenase NB 6 GMP; 3 hours of further incubation, the level of Serva Electrophoresis GmbH, Heidelberg, fluorescence of reduced AB was determined using Germany) at 37 ° C for 2 hours with constant
53 Stuchlíková et al.: Comparison of multipotent mesenchymal stromal cells from human adipose tissue and Wharton's jelly for soft tissue reconstruction
TECAN GENios microplate reader (Tecan) with an excitation wavelength of 550 nm and an emission wavelength of 590 nm [19]. The ratio between the fluorescence of the experimental and blank samples (same medium without cells) was used as the AB value. Adipogenic differentiation medium consisted of glucose-free DMEM (Gibco) supplemented with ITS (insulin, transferrin and selenium mixture) (Gibco), glucose (5.5 mM), 10% fetal bovine serum (FBS) and subsequent adipogenic A. stimulators 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone and 100 μM indomethacin (all from Sigma-Aldrich, USA). Complete media changes were performed every 3-4 days. After 14 days of culture with adipogenic supplements, MSCs were fixed in 4% buffered formalin for 30 minutes at 4 ° C and stained with Nile red solution (1 μg / ml in PBS), (Sigma- Aldrich, USA) according to the manufacturer's instructions. Cells were evaluated using a fluorescence microscope (Nikon, Japan). Quantitative evaluation of Nile red fluorescence intensity in the examined groups was prepared in a B. microtiter plate reader (Tecan Genios; Tecan, Austria) at wavelengths of 488 nm / 530 nm, using a multipoint reading mode (12 reading points per well). Data were presented as the mean Nile red fluorescence intensity for each condition, after subtracting negative control values (obtained from uninduced cells). Positive staining of Nile red was confirmed by fluorescence microscopy (Nikon, Japan) or laser confocal microscopy (Zeiss, LSM 710, Germany). Sowing of MSCs into collagen scaffolds was performed by static method [20]. The method C. consisted of applying a minimum volume (20 μl) of concentrated cell suspension (3 x 105 cells / ml) to the surface of the 3D scaffold using an automated pipette. The cell carrier was incubated for 3 hours at 37 ° C and then transferred to a 50 ml tube (with filter) containing 10 ml of medium A media change was prepared every 4 days. Cell morphology was assessed after 24 hours of 3D culture using double fluorescence staining using the LIVE / DEAD® Vability / Cytotoxicity Kit (2 μM Calcein AM and 5 μM Ethidium homodimer-1; Invitrogen). D. Results Figure 1: Adipogenic differentiation of AT-MSCs (A, B) and WJ-MSCs (C,D) in the presence (A, C) or absence (B,D) of In AT-MSC cultures, lipid accumulation was FBS. clearly visible, with droplets distributed throughout the cell surface (Figure 1A). In contrast, there was
54 Stuchlíková et al.: Comparison of multipotent mesenchymal stromal cells from human adipose tissue and Wharton's jelly for soft tissue reconstruction no significant accumulation of lipids in the WJ- MSC (Figure 1C).
Conclusion In this work, we studied the potential of Figure 2: Colony formation efficiency after 14 days (* - values in AT-MSC are significant (p <0.05) higher compared to WJ- multipotent mesenchymal stromal cells derived MSC) Figure 3: Morphology and viability of AT-MSC in 3D collagen carriers (Live / Dead test, fluorescence microscopy).
We also monitored the effect of the from adipose tissue and Wharton's jelly for adipose composition of the culture medium on the tissue engineering. adipogenic differentiation of AT-MSC and WJ- The efficiency of adipogenic differentiation in MSC. In the absence of FBS, the efficiency of vitro was found to be higher in MSCs derived from adipogenic differentiation was significantly lower human adipose tissue, compared to Wharton's jelly. (Figure 1B). The absence of serum in the differentiation The ability of MSCs to form colonies of medium almost completely prevented the fibroblast-like cells in vitro is one of the adipogenesis of MSCs. The efficiency of colony characteristics of early progenitor cells, within formation was higher in AT-MSCs compared to a functionally heterogeneous population of MSCs. WJ-MSCs, but the proliferative activity of colony- In our study, to evaluate colony formation activity, forming cells was higher in WJ-MSC cultures. We cells were seeded at a density of 40 cells per cm 2 assessed the possibility of using 3D collagen to ensure the distribution of individual cells on the carriers for adipose tissue engineering and surface of the tissue culture. After 14 days of confirmed their suitability. culture, the cells were fixed, stained with Giemsa, and the number and size of colonies formed were Acknowledgements: The work was supported by the determined visually using a ruler without Department of Biomaterials and Biophysical Methods at the instrumentation. After 14 days of culture, the Institute of Experimental Medicine of the Academy of manifestation of CFU-f activity on AT-MSC was Sciences of the Czech Republic (BIOCEV). significantly higher than on WJ-MSC. (Figure 2) To confirm the capacity of AT-MSCs for References adipogenic differentiation in 3D environment, [1] MĚŠŤÁK, J., M. MOLITOR, O. MĚŠŤÁK a L. 5 approximately 5 x 10 AT-MSCs were inoculated KALINOVÁ. Základy plastické chirurgie. Vydání into commercially available porous lyophilized druhé. V Praze. ISBN 978-80-246-2839-4. collagen foam (VUP Medical, Czech Republic) by [2] BRETT, E., N. CHUNG, W. T. LEAVITT, A. slow pipetting and saturation. After one hour of MOMENI, M. T. LONGAKER a D. C. WAN. A Review incubation in a minimum volume of culture of Cell-Based Strategies for Soft Tissue Reconstruction. medium (required for cell adhesion), the cells were Tissue Engineering Part B: Reviews [online]. 2017, 23(4), 336-346 [cit. 2020-04-30]. DOI: cultured for one week in medium supplemented 10.1089/ten.teb.2016.0455. ISSN 1937-3368. Dostupné with 5% PL. Cell morphology and viability 24 z:https://www.liebertpub.com/doi/10.1089/ten.teb.2016 hours after inoculation were determined by Live / .0455 Dead fluorescence microscopy (Figure 3). [3] CHOI, J. H., J. M. GIMBLE, K. LEE, K. G. MARRA, After the successful Live / Dead test, we J. P. RUBIN, J. J. YOO, G. VUNJAK-NOVAKOVIC a induced the adipogenic differentiation of MSCs. D. L. KAPLAN. Adipose Tissue Engineering for Soft We detected the accumulation of intracellular lipid Tissue Regeneration. Tissue Engineering Part B: Reviews [online]. 2010, 16(4), 413-426 [cit. 2020-04- droplets that were positively stained with Nile red. 30]. DOI: 10.1089/ten.teb.2009.0544. ISSN 1937-3368. Dostupné z:
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56
The application of contact lenses as carriers of multipotent mesenchymal stromal cells for regenerative medicine applications
Eliška Vavřinová1,2, Simona Stuchlíková1,2, Taťána Jarošíková2, Yuriy Petrenko1
1 Institute of Experimental Medicine AS CR, Prague, Czech Republic 2Czech Technical University in Prague, Faculty of Biomedical Engineering, Kladno, Czech Republic . *[email protected]
Abstract: Cell transplantation at the site of injury is one of the main strategies of regenerative medicine. This modern approach could reduce the need of corneal transplantation and prevent possible adverse effect during surgery, overcome low availability of donor tissue or graft rejection. The aim of the work was to find a suitable contact lens, which could provide attachment and growth of multipotent mesenchymal stromal cells (MSCs). We selected 8 different types of contact lenses with different technical, optical and material parameters. Only one type of contact lens ensured adhesion and proliferation of MSCs on the surface of the contact lens. The obtained data allows to provide more information and opens new strategies for the application of multipotent mesenchymal stromal cells in ophthalmological regenerative medicine.
MSCs can differ depending on the Introduction isolation and expansion methods or tissue of origin. The cornea is transparent front part of eye which For this reason, the Committee on Mesenchymal protects the internal structures [1,2]. On the surface and Tissue Stem Cells of the International Society of the cornea is a tear film, which hydrates the for Cell Therapy and Gene Therapy (ISCT) has optical surface and at the same time is a source of proposed a set of standards for defining human oxygen, immunoglobulins, lysozymes and MSCs to unify biological properties. The reference lactoferrins [3]. Very common cause of impaired criteria are: (1) plastic adhesion; (2) expression vision or blindness is corneal damage, which can of specific surface antigens CD105, CD73 and become in the event of mechanical damage, CD90; (3) absence of hematopoietic markers infection or pathology of the eye [1]. Today, CD45, CD34, CD11b, CD14, CD19, CD79a, corneal transplantation is the most commonly used HLA-DR; and (4) the ability to differentiate into treatment for opaque corneas. Complications after chondrocytes, osteoblasts or adipocytes in vitro, surgery and lack of donor tissues have aroused if appropriate stimuli are provided [8,11]. efforts to find an alternative to this treatment [2,4]. MSCs cells have been already used for The regenerative medicine could be corneal epithelial regeneration, which achieved an alternative to the transplantation, which encouraging results and for this reason study of combines the application of stem or progenitor corneal epithelial regeneration and reparation is cells [5,6]. The most important factors influencing currently underway [12]. However, regenerative the continuance of corneal regeneration are medicine is still not widely used in clinical practice sufficient number of cells, the presence of growth due to regulations, logistics and economic factors and proper biocompatible carrier [2]. complexity [6]. MSCs are present in various organs and Scaffolds are important for the cell therapy tissues. The most commonly used source is bone they may ensure local application of MSCs [2]. marrow [1,7]. A rich source of proliferative MSCs The advantage of using contact lenses as carriers comes from Wharton's jelly tissue and blood from for MSCs are simplicity, sterility of the material, umbilical cord [8]. It has been found to MSCs can shape, availability, permeability to oxygen activate several repair paths in response to a local and water, they are not toxic to the cornea stimulus from damaged tissue by releasing growth or to stem cells. For these reasons, the aim was find factors, chemokines, enzyme and other substances a proper contact lens as carriers of a stem cells. [8,9,10]. After application the MSCs can promote The contact lens material is most often based the development of new blood vessels, prevent on a polymer of silicone or hydrogel [13]. apoptosis, stimulate mitotic division or protect against various damaging factors [8].
57 Vavřinová et al.: The application of contact lenses as carriers of multipotent mesenchymal stromal cells for regenerative medicine applications
Proliferation was assessed by alamarBlue on day 1 and 5. To confirm the possibility of using Alcon contact lenses as carriers for MSCs, we studied WJ-MSCs proliferation.
Results
After seeding of MSCs onto the surface of the majority of contact lenses from different companies, the formation of spheroidal aggregates was observed, indicating low adhesion properties of the surface of contact lens surface (Fig. 2A). Figure 1: Contact lens from company Alcon. Materials and methods In our study, we worked with cells which have been already obtained, expanded and cryopreserved at the Institute of Experimental Medicine the Czech Academy of Sciences. Mononuclear cells were cultured in complete culture medium containing α-MEM (LONZA, Figure 2: Figure A shows formation of sphere. Figure B shows the correct cell morphology, where cells covering the entire Basel, Switzerland), 5% platelet lysate (Bioinova, surface of a suitable contact lens. Ltd.) and 10 μg/ml gentamicin (Sandoz, Holzkirchen, Germany) at 37 °C, in humidified This may be caused by the ratio of water and environment containing 5% CO2. The media was material in the contact lenses. changed regularly twice a week [14]. Contact lenses from company Alcon Cells were washed with PBS and harvested (containing 67 % Lotrafilcon B and 33 % H2O) with 0.05% Trypsin/EDTA solution (Life provided good initial cells attachment (Fig. 2B). To Technologies, Carlsbad, CA, USA). Subsequently, determine the viability and morphology of cells trypsin was neutralized and centrifuged (450 G, 10 adhered to the surface of contact lens, we used the minutes). Cell density and viability were counted LIVE / DEAD ® Viability / Cytotoxicity Kit using a Bürker chamber and MSCs were reseeded (Thermo Fisher Scientific, Roskilde, Denmark). at a density of 5·103 cells/cm2 [15]. After culturing the MSCs for 24 hours on a suitable Number of living cells was determined using contact lenses, we added Calcein AM and after the vital dye Trypan Blue. Viability of cells were 10-20 minutes we observed the cells using a expressed as the percentage of viable unstained fluorescence microscopy (Fig. 3). cells in suspension relative to the total number of cells counted in the Bürker chamber. Metabolic and proliferative activity was measured using the alamarBlue assay (Thermo Fisher Scientific, Roskilde, Denmark). We applied dye and after 2 hours we determined the level of fluorescence of reduced AB using a TECAN GENios microplate reader (Tecan) with excitation wavelength 550 nm and emission wavelength 590 nm. The AB values was presented as ratio between the fluorescence of the experimental and blank samples (same medium without cells) [15]. We used 8 different types of contact lenses with variant material properties and producer, for find Figure 3: The viability and morphology of cells adhered to the the most suitable contact lens which may ensure surface of contact lens. MSCs adherence. MSCs were cultured on a contact lens for 24 hours and then metabolic activity were determined by the alamarBlue assay.
58 Vavřinová et al.: The application of contact lenses as carriers of multipotent mesenchymal stromal cells for regenerative medicine applications
The proliferation on the Alcon contact lens is Concepts and Future Directions. Frontiers in similar to a monolayer culture of cells on the Bioengineering and Biotechnology [online]. 2019, 7 [cit. 2020-03-04]. DOI: 10.3389 / fbioe.2019.00135. ISSN surface of a tissue culture plastic (Fig. 4). 2296-4185. Available from: https://www.frontiersin.org/article/10.3389/fbioe.2019. 00135/full [3] SRIDHAR, Mittanamalli S. Anatomy of cornea and Proliferation WJ-MSC ocular surface. Indian J Ophthalmol [online]. 2018, 190– 194. [feeling. 2020-03-03]. DOI: 10.4103 / 5 ijo.IJO_646_17. Available from: 4 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC58190 93/ 3 [4] SACCHETTI, Marta, Paolo RAMA, Alice 2 BRUSCOLINI and Alessandro LAMBIASE. Limbal Stem Cell Transplantation: Clinical Results, Limits, and 1 Perspectives. Stem Cells International [online]. 2018, 0 2018, 1-12 [cit. 2020-04-20]. DOI: 10.1155 / Alamar fluorescence (RFU)Alamar Blue TCP Alcon Contact lens 2018/8086269. ISSN 1687-966X. Available from: https://www.hindawi.com/journals/sci/2018/8086269/ 1. day 3. day [5] JESSOP, Zita M., Ayesha AL-SABAH, Wendy R. Figure 4: Comparison of WJ-MSC proliferation on a tissue FRANCIS and Iain S. WHITAKER. Transforming culture plastic (TCP) and contact lens from Alcon. healthcare through regenerative medicine. BMC Medicine [online]. 2016, 14 (1) [cit. 2020-03-03]. DOI: Conclusion 10.1186 / s12916-016-0669-4. ISSN 1741-7015. Available from: The aim of the work was to determine the https://www.ncbi.nlm.nih.gov/pmc/articles/PMC49808 possibility of using polymeric contact lenses as 02/ carriers for multipotent mesenchymal stromal [6] ROMALDINI, Alessio, Maddalena cells. MASTROGIACOMO, Ranieri CANCEDDA and We selected 8 different types of contact lenses Fiorella DESCALZI. Platelet Lysate Activates Human Subcutaneous Adipose Tissue Cells by Promoting Cell with different technical, optical and material Proliferation and Their Paracrine Activity Toward parameters. Only one type of contact lens from Epidermal Keratinocytes. Frontiers in Bioengineering company Alcon ensured adhesion and proliferation and Biotechnology [online]. 2018, 6 [cit. 2020-03-30]. of MSCs on the surface of the contact lens. The DOI: 10.3389 / fbioe.2018.00203. ISSN 2296-4185. proliferation of MSCs on the Alcon contact lens Available from: https://www.frontiersin.org/article/10.3389/fbioe.2018. and TCP was almost identical, confirming the 00203/full suitability of using such contact lens as a carrier for [7] SHIVAKUMAR, Sharath Belame, Dinesh BHARTI, MSCs. The obtained data allows to provide more Raghavendra Baregundi SUBBARAO, et al. DMSO- information and opens new strategies for the and Serum-Free Cryopreservation of Wharton's Jelly application of multipotent mesenchymal stromal Tissue Isolated from Human Umbilical Cord. Journal of cells in ophthalmological regenerative medicine. Cellular Biochemistry [online]. 2016, 117 (10), 2397- 2412 [cit. 2020-03-28]. DOI: 10.1002 / jcb.25563. ISSN 07302312. Available from: Acknowledgements: The work was done in the department of http://doi.wiley.com/10.1002/jcb.25563 Biomaterials and Biophysical Methods in Institute of Experimental Medicine AS CR (BIOCEV facilities). [8] MARQUEZ-CURTIS, Leah A., Anna JANOWSKA- The work was supported by the Ministry of Education, Youth WIECZOREK, Locksley E. MCGANN and Janet A.W. and Sports of the Czech Republic: BIOCEV (CZ.1.05 / 1.1.00 ELLIOTT. Mesenchymal stromal cells derived from / 02.0109) and the National Sustainability Program II various tissues: Biological, clinical and cryopreservation (LQ1604) (BIOCEV-FAR project). aspects. Cryobiology [online]. 2015, 71 (2), 181-197 [cit. 2020-03-13]. DOI: 10.1016 / j.cryobiol.2015.07.003. ISSN 00112240. Available References from: [1] JAVORKOVA, Eliska and Vladimir HOLAN. https://linkinghub.elsevier.com/retrieve/pii/S00112240 Prospects for cell therapy in ophthalmology: 1. Use of 15002047 stem cells in regeneration of the ocular surface damage. [9] OSTRO, Alexander and Frantisek LESNIK. Biological Czech and Slovak ophthalmology [online]. 2016, (1), aspects of regenerative medicine. Olomouc: Publishing 268–271 [cited. 2020-02-15]. house Olomouc, 2008. ISBN 978-80-7182-250-9. [2] MOBARAKI, Mohammadmahdi, Reza ABBASI, [10] MURPHY, Matthew B, Kathryn MONCIVAIS and Sajjad OMIDIAN VANDCHALI, Maryam Arnold I CAPLAN. Mesenchymal stem cells: GHAFFARI, Fathollah MOZTARZADEH and Masoud environmentally responsive therapeutics for MOZAFARI. Corneal Repair and Regeneration: Current
59 Vavřinová et al.: The application of contact lenses as carriers of multipotent mesenchymal stromal cells for regenerative medicine applications
regenerative medicine [online]. 2013, 45 (11), e54-e54 [13] MUSGRAVE, Christopher Stephen Andrew and [cit. 2020-03-04]. DOI: 10.1038 / emm.2013.94. ISSN Fengzhou FANG. Contact Lens Materials: A Materials 2092-6413. Available from: Science Perspective. Materials [online]. 2019, 12 (2) http://www.nature.com/articles/emm201394 [cit. 2020-03-03]. DOI: 10.3390 / ma12020261. ISSN 1996-1944. Available from: [11] DOMINICI, M., K. LE BLANC, I. MUELLER, et al. http://www.mdpi.com/1996-1944/12/2/261 Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular [14] SEBEK, Jaroslav. Cell culture in medicine. Prague: Therapy position statement. Cytotherapy [online]. 2006, Galen, [2018], 148 pp. ISBN 978-80-7492-380-7. 8 (4), 315-317 [cit. 2020-03-04]. DOI: 10.1080 / 14653240600855905. ISSN 14653249. Available from: [15] PETRENKO, Yuriy, Irena VACKOVA, Kristyna https://linkinghub.elsevier.com/retrieve/pii/S14653249 KEKULOVA, et al. A Comparative Analysis of 06708817 Multipotent Mesenchymal Stromal Cells derived from Different Sources, with a Focus on Neuroregenerative [12] MA, Yanling, Yongsheng XU, Zhifeng XIAO, Wei Potential. Scientific Reports [online]. 2020, 10 (1) [cit. YANG, Chun ZHANG, E. SONG, Yiqin DU and 2020-03-28]. DOI: 10.1038 / s41598-020-61167-z. Lingsong LI. Reconstruction of Chemically Burned Rat ISSN 2045-2322. Available from: Corneal Surface by Bone Marrow-Derived Human http://www.nature.com/articles/s41598-020-61167-z Mesenchymal Stem Cells. Stem Cells [online]. 2006, 24 (2), 315-321 [cit. 2020-02-15]. DOI: 10.1634 / stemcells.2005-0046. ISSN 10665099. Available from: http://doi.wiley.com/10.1634/stemcells.2005-0046
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Tribological properties of contact lenses
Alena Škubníková1*, Petr Písařík1,
Czech Technical University in Prague, Faculty of Biomedical Engineering CTU in Prague, Kladno, Czech Republic
Abstract: This article deals with the measurement of tribological properties of contact lenses, especially a friction coefficient and a contact angle. The measurements are performed on various types of contact lens – hydrogel and silicone-hydrogel. It provides information about the methodology of work with contact lenses, tribometer and experimental part of the measurement of the friction coefficient and the contact angle. It was found that the friction coefficient decreased with increasing normal load and the increase in the friction coefficient with the sliding speed.
Friction is a force that acts at the interface Introduction between two solid surfaces and prevents them from Tribology is the science of the interrelationships moving relative to each other. The level of between surfaces in motion. He deals consistently resistance is a property of the surface, which we processes we experience every day: friction, call Coefficient of Friction (CoF). Coefficient of lubrication and wear. Friction consists two separate components: the In recent years, tribology has also been used in forces, which is needed to move objects and the the study of influences that affect the comfortable forces holding the surfaces together. Both forces wearing of contact lenses. Subconsciously, we are directly measurable and the Coefficient of sense the interconnectedness of the various factors Friction can be expressed according to the such as lower friction between the eyelid and following formula [1]: contact lens, comfort level and the number of 퐹푟푖푐푡푖표푛 푓표푟푐푒 symptomatic carriers. 퐶표퐹 = An increasing number of people are stopping 푁표푟푚푎푙 푓표푟푐푒 wearing contact lenses due to discomfort, contactologists and manufacturers of contact lenses If the coefficient of friction is higher, than has have to focus on the factors involved - in particular to be the greater resistance due to friction, and the friction and lubrication. [1,2] greater frictional energy is required for each other displacement of surfaces to a given distance. If we The friction energy know the friction force and the distance, where the shift is, than we can figure the friction energy Our eyes work during blinking, because the according to the following formula [1]: energy is needed to overcome frictionalforces acting against to the movement of the lid. The friction energy (Ef) = Fortunately the tear film lubricates our eyelid and Friction force ∙ Distance eye surface. But what happens, if the friction between the lid and surface of the eye gradually The measurements of the friction increases during the day? It is bad, when this situation happens with contact lenses and then the energy surface of contact lens is less slippery and less The work, which the lid performs, when it is wettable. During the day, dirt settles also on the blinking, it is straight friction energy needed to surface of lenses. In this case, it creates a thousand move the eyelid along the cornea (or contact times a day repeated movement of the eyelid, lenses). The friction energy expended is which one keep changing a rougher surface of proportional to the Coefficient of Friction, because contact lens and then it is needed a larger amount both equations describe the friction force. The of excess work to move them.[1,2] relationship between the Coefficient of Friction and comfort in contact lens has been determined by measurement, that the loss of friction energy
61 Škubníková et al.: Tribological properties of contact lenses during a simulated blinking can give us an insight, their intended application. In the case of hydrogel how different types of contact lens behave on the materials for contact lens, it is in particular eye. [2] transparency, good oxygen permeability for proper Although the measurement is a little bit corneal metabolism and more. That is features that difficult and it is not possible to measure this ensure clear and stable vision and the necessary directly, there are many measuring instruments, comfort during wearing contact lens. which one can already measure frictional forces The basic material for the production of soft between the lid and the contact lens. This contact lens has become PHEMA, which is formed measurement can be in artificial conditions (in- by copolymerizing vitro) or in physiological conditions in accordance 2-hydroxyethylmethacrylate monomer with the with the eye, it is important step in the assessment crosslinking agent ethylene dimethacrylate of the comfort of contact lens. [2] (EDMA). Contact lens prepared from this material are among the “standard hydrogels”. In this time Materials of contact lenses there are many hydrogel materials with a specific composition on the market. [3,4,5] Contact lenses can be divided into many different categories. The basic division is the SILICON-HYDROGELS division of contact lenses by material. The Silicon hydrogel contact lens are among the soft, individual materials significantly affect the hydrophilic materials, precisely because of their resulting properties of contact lens, such as the more or less hydrogel character. Silicon hydrogels interaction between the contact lens and the are a combination of hydrogels and silicone gas anterior segment of the eye. Contact lens can be permeable materials. divided into [3,4,5]: Their typical property, why they were Hard contact lenses developed is greater oxygen permeability o Hard impermeable (glass, compared to hydrogels. Therefore, wearing of PMMA) contact lens, which one are made from this o Hard permeable (Rigid Gas material, is healthier and gentler for eyes. Thanks Permeable) to the high permeability, it is possible to sleep in Soft contact lenses contact lens or to wear them without removal for o Hydrogels up to thirty days and twenty-nine night o Silicon-hydrogels occasionally. Silicone hydrogel lens are slightly stiffer than hydrogels lens and due to the content of Materials of contact lenses are sparsely water, they are roughly at the level of standard crosslinked polymers. These are solid materials, hydrogels and it is recommended to use additional formed by high molecular weight chains, which wetting agents. Silicon hydrogel contact lenses consist of small, constantly repeating units, have reached three generation since their launch monomers. These chains are linked in places by a until today. [3,4,5] suitable crosslinking agent to a three-dimensional polymer net - a gel. Selection of monomer units and Experiment their arrangement, length of their chains, density of net, these are parameters, which one can influence The aim of this work was to make out the the resulting properties of the material or contact influence of load and sliding speed on the lens. [3,4,5] coefficient of friction. The measurement was provided using a Tribometer, which you can see at Soft contact lenses the picture number one. There had to be made a HYDROGELS special cell for attaching lens. During measurement From a chemical point of view, the hydrogel is a contact lens were in a lubricated state. Three three-dimensional sparsely crosslinked polymer different contact lenses were selected for the structure, that swells on contact with water until tribology experiment. Unfortunately our equilibrium is established. Tribometer was not able to achieve low forces. The As a biomaterial, the hydrogel has to meet contact lenses were mounted into a sample holder certain general requirements. These requirements specially designed to match the internal curvature include good biocompatibility, almost zero toxicity of the lens during measurement. The lens was then and it cannot damage tissues, etc. The specific clamped with the upper part to be held in place. requirement and behavior of materials are based on
62 Škubníková et al.: Tribological properties of contact lenses
Results The following figure1 and figure 2 show the evolution of the coefficient of friction with normal applied load – 0,25 N and sliding speed.
Figure 2: Evolution of the CoF with 0,25N of load
Figure 1: Tribometer used to during the measurement
The presence of liquid on the surface of the hydrogel is known to affect dramatically its friction properties. Therefore, in order to avoid the dehydration, the contact lenses were tested Figure 3: Evolution of the CoF at the same type of the completely immersed in saline solution. contanct lens with different loads
Three types of contact lenses were applied. Used contact lenses you can see in the table 1 and table.
Table 1: Used contact lenses – Silicon hydrogels
Type of Content Oxygen Modulus of contact lenses of water permeability elasticity Siliconhydrogel 56% 86 Dk/t One Day 0,5 MPa
Air Optix Aqua 33% 138 Dk/t 1,0 MPa Figure 4: Evolution of the CoF at different lenses with the same sliding speed, and the load Table 2: Hydrogels contact lenses Type of Conclusion Content of Oxygen contact Modulus of water permeability lenses elasticity This article presents the results of tribological Dailies experiment on soft contact lenses. The
Aqua 69% 26 Dk/t 0,89MPa measurement for such soft materials was Comfort successfully applied using the Tribometer and dedicated contact lens holder. During the experimental conditions the The measurement for the tribological test normal load was 0,25N and 0,5N and the linear consisted either in the increase of normal load sliding speed was changing from 0,2 cm/s under constant speed or in the increase of the to 0,4 cm/s. sliding speed under constant load.
63 Škubníková et al.: Tribological properties of contact lenses
It was found that the coefficient of friction Executive Summary. Invest Ophthalmol Vis Sci. decreased with the increase of normal load and also 2013;54:TFOS7- TFOS13. [cit. 2020-10-04] with the increase of the sliding speed. [3] Charakterizace povrchu. Anton Paar [online]. [cit. 2020- 10-04]. Dostupné z: https://www.anton-paar.com/cz- References cs/charakterizace-povrchu/. [4] NATHAN EFRON. Contact lens practice. 2nd ed. St. [1] SUBBARAMAN, LN a LW JONES. Measuring Louis, Mo.: Butterworth Heineman, 2010, 510 s., ISBN Friction and Lubricity of Soft Contact Lenses: A 9780702047633. Review. Contact Lens Spectrum [online]. June 2013 [cit. 2020-10-04]. Dostupné z: http:// [5] PETROVÁ, Sylvie, Zdeňka MAŠKOVÁ a Tomáš www.clspectrum.com/articleviewer.aspx?articleID=10 JUREČKA. Základy aplikace kontaktních čoček. Vyd.2. 8560 přeprac. a dopl. Brno: Národní centrum ošetřovatelství a nelékařských zdravotnických oborů v Brně, 2008, 219 s. [2] Nichols JJ, Willcox MDP, Bron AJ, et al. The TFOS ISBN 978-80-7013-470-2. International Workshop on Contact Lens Discomfort:
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Data Analysing and Processing
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Functional differences of the brain in patients in the presymtomatic and manifestic phases of Parkinson´s disease
Jana Kukačková1*, Radim Krupička2
1Czech Technical University in Prague, Faculty of Biomedical Engineering, Kladno, Czech Republic 2 Clinic of Neurology, General University Hospital in Prague, Czech Republic
Abstract: Parkinson's disease (PD) is a degenerative disorder of the central nervous system and PD causes movement disorders. REM Sleep Behavior Disorder (RBD) is a disease manifested by patients acting out their dreams that can lead to injury. RBD is classified as a premotor manifestation of Parkinson's disease. The work aim is to analyze the differences in functional magnetic resonance imaging (fMRI) of the brain in patients with RBD, patients with PD, and healthy controls. Processing, segmentation, and quantification will be performed by implemented scripts in SPM. The results for each group are compared and statistically processed. The results of this work can be used in the future to detect PD in early disease stages and enable more effective treatment.
Introduction Methods and tools
Parkinson's disease (PD) Functional magnetic resonance imaging (fMRI) PD is a neurodegenerative disease of the It is a non-invasive imaging method that central nervous system that is closely related to the allows mapping of the human brain. loss of nerve cells (neurons) in a part of the brain This method uses the principles of classical known as Substantia nigra (part of basal ganglia). magnetic resonance imaging to indirectly evaluate First motor signs in patients are visible after the changes in regional neuronal activity. The local loss of 31 % of dopaminergic cells in this brain increase in neuronal activity is accompanied by an region. The most significant manifestation of the increase in the supply of oxygenated blood to this disease is the gradual loss of movement control. area and a given location begins to outweigh the [1,2,6] amount of oxygenated blood over the oxygenated. There is an increase in the ratio of oxyhemoglobin Rem Sleep Disorder (RBD) (diamagnetic) and deoxyhemoglobin RBD is characterized by abnormal behavior (paramagnetic properties) in the blood, which is during REM sleep, which is associated with manifested by a local increase in the intensity of pronounced dream production. Dreams are usually the MRI signal. The method using the detection of unpleasant, and therefore there is a frequent different magnetic properties of blood, depending interruption of sleep. A symptom of RBD is on the level of oxygenation, is called BOLD (Blood insufficient muscle atony. Oxygen Level Dependent). [1,3] The disorder may be primary (idiopathic) or secondary. Secondary forms are related to the use SPM or removal of pharmaceuticals or other co-ongoing SPM stands for Statistical Parametric neurological diseases – most often narcolepsy or Mapping. This is an additional library that runs Parkinson's disease. through MathWorks (MATLAB) mathematical There is a connection between RBD and PD. software. The SPM library can process images Numerous studies indicate that RBD may be an from EEG, PETu, and mainly fMRI. It can be important risk factor and predictor of Parkinson´s downloaded as Freeware from disease. Some studies predict a worse prognosis of https://www.fil.ion.ucl.ac.uk/spm/software/downl PD in the presence of RBD. [2,7,8] oad/ . The program allows the processing of a large number of imagesequences from different patients..
67 Kukačková et al.: Functional differences of the brain in patients in the presymtomatic and manifestic phases of Parkinson´s disease
Figure 3: SPM 12 program main menu
Subjects The research will be performed on sets of images provided by the Clinic of Neurology, General University Hospital in Prague. These patients underwent an fMRI examination with a well-defined protocol. The Clinic will provide T1 Figure 4: Three dialog windows of SPM weighted images for anatomical orientation and 1. Step: Realign & Unwarp EPI images (echo-planar imaging), which inform us about the function - perfusion. It is a function that makes an estimate that The study involved 51 patients with PD, 50 aligns a time series of images taken from the same patients with RBD a 46 controls. patient. It uses the method of least squares and spatial transformation of 6 parameters. The first 3 parameters describe the displacements in the x, y, Results z axes in mm and the second 3 are described as rotations in the space pitch, roll, yaw in degrees. Data Pre-processing The first image taken of a given patient is used as a "reference image" for all subsequent images that We will perform preprocessing in the version of the are aligned accordingly. The main goal is to program for the fMRI device. The program works remove the movement artifact caused by in three dialog windows - the first Menu window, involuntary small movements of the patient. where we select the function, the second Batch Functional images (EPI) inserted. editor window, where we set the parameters, and the third window, where we see the course and 2. Step: Slice Timing speed of processing. First, we will convert the images from the When we use Slice Timing, it is necessary to DICOM format with the *.IMA extension to the adjust the input parameters. We set the Number of Nifty format with the *.nii extension. Slices to 30 and TR (time to repeat) to 2. We set In SPM, select the Batch button, a dialog box TA () as the difference TA = TR - (TR / Number of will pop up, where you can select File in the upper Slices), in our case equal to 1.933. We then set the toolbar. Then we chose Load Batch. We found the Slices Order [1, 2… 29, 30] and the Reference slice SPM library folder on our computer, selected for the 15th slide. batches in it, and loaded the preproc_fmri.m 3. Step: Segmentation protocol in batches. We also made a selection of This step modifies the T1 structural images. Slice time correction (STC), where we chose STC 4. Step: Get Pathnames after realignment. 5. Step: Image Calculator In the new Batch Editor work window, are These steps can be skipped. shown the steps required to pre-process the images 6. Step: Coregister: Estimate in the following order: [5] The step in which the structural and functional image is compared. 7. Step: Normalise: Write and 9. Step: Normalise: Write
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In step no. 7 the normalization of the functional image is performed and in step no. 9 the normalization of the structural image. A comparison of the scanned brain with a standard brain is performed as part of normalization. The result is a "deformation" of the scanned brain into a standard brain. Each patient's images should be distorted differently. When performing normalization, it is necessary to select the image to which it will normalized (Images to Align) for each patient - usually it is the first image, and the images that we want to normalize - ie. all images of the patient. As part of the normalization, the estimated parameters are deformed relative to the first image. You can change various parameters, but usually we can do with the default. One of these parameters is the size of the voxels given in coordinates (x, y, z) in mm. It is necessary to change the size of the voxels from [2 2 2] to [3 3 3].
8. Step: Smooth Smoothing suppresses data noise in individual voxels. We choose the functional images we want to smooth. Figure 5: Example result of preprocessing. An example of a result of preprocessing can be seen in figure 3. Each software package has a different sequence of preprocessing steps - this is called Conclusion pipeline or workflow. So far, there is no clear In conclusion, it can be said that the process of consensus on a specific pipeline, so pre-processing preprocessing had to be described, because in this results from other software may disagree. It is way we prepared the data (images of patients) for necessary to choose the most suitable pipeline for statistical processing and evaluation. We will now your study (according to the required preferences). statistically process the prepared data in DPABI. Acknowledgements: I would like to thank my supervisor In addition to SPM, preprocessing can also be Radim Krupička for all the time he has given me. I would also performed in AFNI (Analysis of Functional like to thank Clinic of Neurology, from whom we have the NeuroImages), ANTs (Advanced Normalization patient´s data. Tools), FreeSurfer or FSL (FMRIB Software Library) software. The disadvantage of these Further work software is that they work in programming languages other than SPM. Other lesser-known The master's project aims to analyze software working with the MATLAB differences in functional magnetic resonance of the programming language are for example DPABI (a brain in patients suffering from REM sleep toolbox for Data Processing and Analysis for Brain disorder and Parkinson's disease. Quantification of Imaging), DPARSF (Data Processing Assistant for differences between the control group, Parkinson's Resting-State fMRI), SnPM (Statistical patients and patients with RBD in each part of the NonParametric Mapping), BCT (Brain brain will be quantified. The differences found Connectivity Toolbox) or CONN (Functional could serve in the future to timely PD, allowing for Connectivity Toolbox). [4] more effective treatment.
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References
[1] Pyatigorskaya N.; Gallea C.; Garcia-Lorenzo D.; Vidailhet M.; Lehericy S. A review of the use of magnetic resonance imaging in Parkinsosn´s disease, Therapeutic Advances in Neurological Disorders. 201, 7 (4). DOI: 10.1177/1756285613511507 [2] ŠONKA, Karel. Porucha chování v REM spánku. Neurologie pro praxi. 2008, 9(5), 297-299.
[3] CHLEBUS, Pavel, Michal MIKL, Milan BRÁZDIL a Petr KRUPA. Funkční magnetická rezonance – Úvod od problematiky. Neurologie pro praxi. 2005, 6(3), 133- 139.
[4] PHINYOMARK, Angkoon, Esther IBANEZ- MARCELO a Giovanni PETRI. Resting-State fMRI Functional Connectivity: Big Data Preprocessing
Pipelines and Topological Data Analysis. IEEE Transactions on Big Data [online]. 2017, 3(4), 415-428 [cit. 2020-09-30]. ISSN 2332-7790. DOI:10.1109/TBDATA.2017.2734883 [5] ASHBURNER, John, et al. SPM12 Manual. The FIL Methods Group (and honorary members). UCL Queen Square Institute of Neurology. London WC1N 3BG, UK January 13, 2020. Dostupné z: https://www.fil.ion.ucl.ac.uk/spm/ [6] CHENG, Hsiao-Chun, Christina M. ULANE a Robert E. BURKE. Clinical progression in Parkinson disease and the neurobiology of axons. Annals of Neurology [online]. 2010, 67(6), 715-725 [cit. 2020-10-
28]. ISSN 03645134. DOI: 10.1002/ana.21995
[7] KIM, Ryul, Dallah YOO, Jin Hee IM, Han-Joon KIM a Beomseok JEON. REM sleep behavior disorder predicts functional dependency in early Parkinson's disease. Parkinsonism & Related Disorders [online]. 2019, 66, 138-142 [cit. 2020-10-28]. ISSN 13538020. DOI: 10.1016/j.parkreldis.2019.07.025
[8] LIN, Yi-Qi a Sheng-Di CHEN. RBD: a red flag for cognitive impairment in Parkinson's disease? Sleep Medicine [online]. 2018, 44, 38-44 [cit. 2020-10-28].
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Comparison of brain iron levels in basal ganglia and thalamus in patients with REM sleep disorder and Parkinson's disease
Pavla Marousková1*, Radim Krupička1, Petr Dušek2
1Czech Technical University in Prague, Faculty of Nuclear Sciences and Physical Engineering, Prague, Czech Republic 2Clinic of Neurology, General University Hospital in Prague, Czech Republic
Abstract: Increased levels of iron in the brain results in degeneration of dopaminergic neurons. The death of dopaminergic neurons leads to the development of neurodegenerative diseases such as Parkinson’s disease (PD). Due to the loss of the dopaminergic neurons prior to appearance of the motoric symptoms of the PD there is an effort to find diagnostic methods to discover the symptoms in advance. One of the indicators could be the changed levels of iron in certain areas of the basal ganglia and thalamus. We focused on methodology of processing raw data from T1 images and QSM maps and implemented automated segmentation scripts of the basal ganglia and thalamus in the FSL software. The results of quantification of iron levels of Parkinson's disease, REM sleep behavior disorder patients and control group of healthy patients in five different regions of interest are compared and statistically processed.
Introduction serious complications can occur due to either iron deficiency or excess. [3] [4] Parkinson's disease (PD) is a neurodegenerative Iron accumulates with age in the basal ganglia, disease that mainly affects people over 60 years of which are the main sites where excessive iron age. However, the disease begins long before the accumulation may also occur in neurodegenerative manifestations of motor dysfunction occur, when disorders. The main region in PD is substantia the disease is diagnosed. Motor manifestations of nigra (SN), where iron is stored in myelized the disease are preceded by non-motor symptoms, dopaminger neurons. [3] [5] As research which may occur 10 years earlier. Previous studies progresses, excessive iron accumulation appears to have found that 15-60% of patients with be the onset of neuronal degeneration. The Parkinson's disease have a REM sleep behavior accumulation of iron in SN is the result of a disorder (RBD). This is the main non-motor combination of increased imports and reduced symptom. Due to the non-existent treatment, leaching and redistribution of intracellular iron. [6] emphasis is also placed on the earliest possible Quantitative susceptibility mapping (QSM) is diagnosis of this disease. [1][2] used to quantify the spatial distribution of magnetic The amount of iron in the adult human body is susceptibility of biological tissues. Magnetic around 3.5-4 g [3]. It is the most abundant trace sensitivity is a physical quantity that describes the metal in the healthy human brain and its level change in magnetization of a material in response increases linearly with age up to 20 years, then to an applied external magnetic field. The magnetic remains constant and starts to increase again from susceptibility of a material depends on its the age of 60. Iron is essential for many processes composition and comes from the rotation and such as oxygen transport and storage in the brain. motion of the nuclei and electrons of the molecules Also, in the synthesis of the metabolism of that make up the material both positive and neurotransmitters including dopamine and negative, depending on whether the magnetization noradrenaline. Under normal conditions, the brain of the material equalizes with or against the does not respond at all to changes in peripheral external field. The values can be magnetization of iron, due to the existence of a hemoencephalic the material equalizes with or against the external barrier. Accurate iron homeostasis in the brain is field. Phase images of the GRE sequence or maintained by regulatory proteins, iron asymmetric spin echo sequence (SE) are used to transporters, and transport receptors. This function create QSM maps. The GRE sequence is used more is important for maintaining healthy brain function. often because it is faster than the SE sequence. For If the fragile iron homeostasis is disrupted, then
71 Marousková et al.: Comparison of brain iron levels in basal ganglia and thalamus in patients with REM sleep disorder and Parkinson's disease
Figure 1: QSM map before and after repair in MATLAB. Reconstructed QSM map from GRE in wrong anatomical shape at the top (shown in blue) and corrected QSM map to correct anatomical shape of the brain at the bottom. Sagittal plane shown on the left, coronary plane shown on the right. GRE sequences, it is exploited that phase shifts are the primary result of field inhomogeneity. [7] The QSM map is a useful tool in the diagnosis Figure 2: Segmented areas of the basal ganglia and thalamus using the first tool displayed on the T1 image, which was used and treatment of PD due to its sensitivity to as a reference image for segmentation above. The eroded changes in iron levels. (In vivo studies have shown segmented areas are displayed below using a script in that iron values detected by the QSM map are MATLAB. Thalamus is green, globus pallidus is blue, higher in patients with PD than those measured in putamen is yellow and nucleus caudatus is red. The coronary plane shown on the left, the transverse plane shown on the healthy controls.) Studies show that the QSM map right. is more sensitive than R2* in distinguishing between patients and healthy controls. It can images were reconstructed offline using a virtual quantify abnormal iron deposition in PD much reference coil approach [9]. QSM reconstruction better [8]. consisted of (i) continuous Laplacian unwrapping; In this thesis, we focused on the quantification (ii) variable, spherical mean value property-based of iron levels in the basal ganglia and thalamus in background field removal (starting radius, 25 mm; patients diagnosed with REM sleep behavior 1-mm radius at the brain boundary; without disorder and on the comparison of iron levels deconvolution) and (iii) non-linear MEDIN between patients with RBD, patients with PD and inversion with λ=500 [10]. healthy controls (CON). Quantification of iron in MRI images and QSM were converted form appropriate areas of the brain would reveal the DICOM format to Nifti format (the analysed early onset of Parkinson's disease in patients with format), which works in MATLAB and FSL RBD. sowfware. Firstly, we repaired QSM images and Methods coregistration on T1 images in MATLAB. The wrong QSM and repaired QSM can be seen on Patients for this project were selected from Figure 1. MRI studies in General University Hospital in Next, we used the model-based segmentation Prague. The research sample consisted of three tool called first in FSL. The script first_run_all can groups. The first group included 30 patients with segment all the subcortical structures. We choose PD, of which 5 were women and 25 were men aged putamen, globus palladius, nucleus caudatus and 55.7 ± 12.4 years. The second group consisted of thalamus for calculating susceptibility value of 24 patients with RBD, of which 1 female and 23 iron. [11] men aged 65.7 ± 9.5 years. The last group consisted Secondly, we must create a mask from images of 32 healthy control patients, of which 2 were of the segmented structures. It’s necessary because women and 30 were men aged 64.9 ± 8 years. The this image contains the value between 0 – 1 research sample used to quantify iron in the (represent probability each voxel is part of segment substantia nigra consisted of a smaller selection of structures). We turned this image into a binary patients. image with a threshold value of 0.5 (50 % QSM maps were reconstructed from 3D multi- probability). In binary image each voxel has a gradient echo (GRE) acquisitions with TR/TEs = value of 0 or 1. When inspecting the segments of X/X ms, voxel resolution = 0.5x0.5x2 mm3. Phase the basal ganglia and thalamus, we found that the
72 Marousková et al.: Comparison of brain iron levels in basal ganglia and thalamus in patients with REM sleep disorder and Parkinson's disease
* * * * * * * *
Figure 3: Box-plot graph comparing the measured median values of iron of individual groups in the areas of interest. The cross shows the average value, the horizontal line the median. Black lines with symbol * show statistically significant difference (p <0.05) of magnetic susceptibility values between the given groups. It was used Mann-Withney U-test for statistic comparsion. boundaries of the areas were larger than we between two groups. The symbol * indicates a expected and were near other areas of similar statistically significant difference (p <0.05) of the intensity. Therefore, we performed erosion to magnetic susceptibility median values between two eliminate possible bias in iron quantification groups, which lay under the black line in the graph results. We have already taken over the finished in the Figure 3. masks from the thesis “Quantification of The graph in Figure 3 shows the trend of neuromelanin in the substantia nigra in measured values in the substantia nigra, where the neuromelanin-sensitive MRI images” [12]. group of patients with PD has the highest values Segmentation of the substantia nigra here was (0.0285), the group of healthy control patients has performed using manual anatomical localization the lowest values (0.0198) and the group of patients and using the signal intensity threshold to correct with RBD is among them (0.0237). Statistic values not belonging to the substantia nigra area comparison of the median values in the substantia after manual localization [12]. These masks were nigra shows that a significant difference arises only segmented from neuromelanin-sensitive images between the group PD and CON (p = 0.03). This aligned with T1-weighted images. These were the measured trend in the substantia nigra is consistent same T1-weighted images, from which I with the results of studies [14] [15] [16] [17] [18] segmented the remaining areas of interest and co- [19]. registered QSM maps according to T2 * echo. In other areas, either the values of the Finally, we applied masks on QSM maps and individual groups do not differ much, or the calculate the magnetic susceptibility value of iron measured values of magnetic susceptibility of iron in regions of interest. We did this with the help of in patients with PD is lower than in patients with fslstats tool. Fslstats is a general tool for RBD and CON. Statistical processing and calculating various values/statistics from the image comparison of measured values, always between intensities [13]. the two groups, showed that a significant difference was found between the PD and CON Results groups (p = 0.02) in the GP region and between the PD and RBD groups (p = 0.046) in the PN region. The uptake of iron was measured in thalamus The result for the GP area is the opposite of the (TH), nucleus caudatus (NC), putamen (PN), study [15], but for the PN area the results are the globus palladius (GP) and substantia nigra (SN). same. In studies [16] and [17], lower iron levels Each step is described above, and we used FSL were also measured in GP and PN areas in PD software and MATLAB. patients than in CON. This is consistent with a The results of measurement can be seen in the study [20] that reported that patients with PD have graph in the Figure 3. We compared the measured increased cellular metabolic activity in these nuclei values using the Mann-Whitney U-test always and increased activity of the GABAergic pathway
73 Marousková et al.: Comparison of brain iron levels in basal ganglia and thalamus in patients with REM sleep disorder and Parkinson's disease from PN to GP. Disruption of GABA metabolism [2] KIM, Yoon, Young Eun KIM, Eun Ok PARK, et al., then results in a reduction of iron in these areas 2018. REM sleep behavior disorder portends poor prognosis in Parkinson’s disease: A systematic [17]. The same results are obtained in the study review. Journal of Clinical Neuroscience. 47(11), 6-13. [21], but the opposite in the study [18]. Both DOI: 10.1016/j.jocn.2017.09.019. studies used QSM maps to quantify iron. [3] ZUCCA, Fabio A., Juan SEGURA-AGUILAR, In the Table 1 there can be seen the result of Emanuele FERRARI, et al., 2017. Interactions of iron, Spearman correlation test. Spearman test was used dopamine and neuromelanin pathways in brain aging to compare the measured values with the age of the and Parkinson's disease. Progress in patients. Neurobiology. 155(11), 96-119. DOI: 10.1016/j.pneurobio.2015.09.012.
Table 1: Results of spearman correlation test between [4] STÜBER, Carsten, David PITT, Yi WANG, et al., 2016. measurement value and age of patient. The R value takes Iron in Multiple Sclerosis and Its Noninvasive Imaging values from -1 to 1. The closer the R value is to zero, the less with Quantitative Susceptibility Mapping. International the measured values correlate with age. Conversely, the closer Journal of Molecular Sciences. 17(1), 96-119. DOI: R is to the value 1, it is a positive strong correlation of 10.3390/ijms17010100. quantities, and the closer R is to the value -1, it is a negative [5] WEINREB, Orly, Silvia MANDEL, Moussa B.H. strong correlation of quantities. YOUDIM a Tamar AMIT, 2013. Targeting R-value dysregulation of brain iron homeostasis in Parkinson's ROI PD RBD CON disease by iron chelators. Free Radical Biology and Medicine. 62(9996), 52-64. DOI: TH 0.2 -0.06 0.3 10.1016/j.freeradbiomed.2013.01.017. ISSN 08915849. CN 0.2 0.2 0.4 [6] JIANG, Hong, Jun WANG, Jack ROGERS a Junxia GP 0.05 0.3 0.2 XIE, 2017. Brain Iron Metabolism Dysfunction in NP 0.1·10-3 0.3 0.4 Parkinson’s Disease. Molecular Neurobiology. 54(4), SN 0.9·10-3 -0.12 -0.3 3078-3101. DOI: 10.1007/s12035-016-9879-1. [7] WANG, Yi a Tian LIU, 2015. Quantitative susceptibility The correlation coefficient R did not turn out mapping (QSM): Decoding MRI data for a tissue magnetic biomarker. Magnetic Resonance in to be greater than 0.4 in any regions of interest, and Medicine. 73(1), 82-101. DOI: 10.1002/mrm.25358. this indicates a weak correlation, and in some areas [8] ST LOUIS, Erik K., Bradley F. BOEVE, C. B. SAPER, only a very weak correlation between age and the et al., 2017. REM Sleep Behavior Disorder: Diagnosis, magnetic susceptibility of iron. This finding shows Clinical Implications, and Future Directions. Mayo that there is no correlation between the amount of Clinic Proceedings. 92(11), 1723-1736. DOI: iron and age of patients. 10.1016/j.mayocp.2017.09.007. [9] D.L. Parker et al, MRM 72:563-9, 2014 Conclusion [10] J. Acosta-Cabronero et al, Neuroimage, 183:7-24, 2018 This thesis was focused on quantification of [11] Patenaude, B., Smith, S.M., Kennedy, D., and Jenkinson iron in the brain in patients with REM sleep M. A Bayesian Model of Shape and Appearance for behavior disorder and Parkinson’s disease. Subcortical Brain NeuroImage, 56(3):907-922, 2011 First, we determined the procedure for [12] LANG, Martin, 2019. Kvantifikace neuromelaninu v processing and editing QSM maps and T1 substantia nigra v neuromelanin-sensitivní MRI weighted images. Then, we made measurement in snímcích. Kladno. Diplomová práce. ČVUT, Fakulta biomedicínského inženýrství specific regions of brain: thalamus, nucleus caudatus, globus pallidus, putamen and substantia [13] JENKINSON, Mark, Christian F. BECKMANN, Timothy E.J. BEHRENS, Mark W. WOOLRICH a nigra. Stephen M. SMITH. FSL. NeuroImage [online]. The results of quantification correspond to the 2012, 62(2), 782-790. DOI: trends presented in the monitored studies, except 10.1016/j.neuroimage.2011.09.015. for the area of globus pallidus, where we found [14] BARBOSA, Jeam Haroldo Oliveira, Antonio Carlos studies confirming the trend in our measured SANTOS, Vitor TUMAS, Manju LIU, Weili ZHENG, values of iron and studies that refute this trend. E. Mark HAACKE a Carlos Ernesto Garrido SALMON, 2015. Quantifying brain iron deposition in patients with Parkinson's disease using quantitative susceptibility References mapping, R2 and R2*. Magnetic Resonance Imaging. 33(5), 559-565. DOI: [1] KALIA, Lorraine V a Anthony E LANG, 2015. 10.1016/j.mri.2015.02.021. Parkinson's disease. The Lancet. 386(9996), 896-912. DOI: 10.1016/S0140-6736(14)61393-3. ISSN [15] SHAHMAEI, Vahid, Fariborz FAEGHI, Ahmad 01406736. MOHAMMADBEIGI, Hasan HASHEMI a Farzad ASHRAFI, 2019. Evaluation of iron deposition in brain
74 Marousková et al.: Comparison of brain iron levels in basal ganglia and thalamus in patients with REM sleep disorder and Parkinson's disease
basal ganglia of patients with Parkinson's disease using Disorder. Movement Disorders [online]. 35(3), 478-485 quantitative susceptibility mapping. European Journal DOI: 10.1002/mds.27929. ISSN 0885-3185. of Radiology Open [online]. 6, 169-174. DOI: 10.1016/j.ejro.2019.04.005. [19] AN, Hedi, Xiaoyan ZENG, Tengfei NIU, et al., 2018. Quantifying iron deposition within the substantia nigra [16] AZUMA, M., T. HIRAI, K. YAMADA, et al., 2016. of Parkinson's disease by quantitative susceptibility Lateral Asymmetry and Spatial Difference of Iron mapping. Journal of the Neurological Deposition in the Substantia Nigra of Patients with Sciences [online]. 386, 46-52. DOI: Parkinson Disease Measured with Quantitative 10.1016/j.jns.2018.01.008. Susceptibility Mapping. American Journal of Neuroradiology [online]. 37(5), 782-788. DOI: [20] LEVY, R., L.-N. HAZRATI, M.-T. HERRERO, et al., 10.3174/ajnr.A4645. 1997. Re-evaluation of the functional anatomy of the basal ganglia in normal and Parkinsonian [17] KOSTA, P., M. I. ARGYROPOULOU, S. states. Neuroscience [online]. 76(2), 335-343 [cit. 2020- MARKOULA a S. KONITSIOTIS, 2006. MRI 05-13]. DOI: 10.1016/S0306-4522(96)00409-5. evaluation of the basal ganglia size and iron content in patients with Parkinson's disease. Journal of [21] MURAKAMI, Y., S. KAKEDA, K. WATANABE, et Neurology [online]. 253(1), 26-32. DOI: al., 2015. Usefulness of Quantitative Susceptibility 10.1007/s00415-005-0914-9. Mapping for the Diagnosis of Parkinson Disease. American Journal of [18] SUN, Junyan, Zhaoyu LAI, Jinghong MA, et al., 2020. Neuroradiology [online]. 36(6), 1102-1108. DOI: Quantitative Evaluation of Iron Content in Idiopathic 10.3174/ajnr.A4260. Rapid Eye Movement Sleep Behavior
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Summary of the substantia nigra atlases
Vendula Lhotáková1*, Radim Krupička1
1Czech Technical University in Prague, Faculty of Biomedical Engineering, Kladno, Czech Republic 2Clinic of Neurology, General University Hospital in Prague, Czech Republic
Abstract: Parkinson's disease is an incurable neurodegenerative disease that causes loss of nerve cells. This causes a lack of dopamine, which regulates the function of the basal ganglia, and thus affects the mobility of the individual affected by the disease. Parkinson's disease can be detected by magnetic resonance imaging, which allows the detection of neuromelanin content in the substantia nigra. The aim of the work was to verify the accuracy of segmentation of the substantia nigra, which is part of the basal ganglia. Segmentation of individual parts of the brain using atlases performed semi-automatically or automatically allows faster and objective evaluation of MRI images. In this work, we focused on segmentation method using TPM also tissue probability maps. We used MATLAB and its extended software SPM12 to process MRI images.
Introduction suitable for diagnostics in neurology, oncology and orthopedics.. MRI images of the brain are used for Parkinson's disease (PD) is the most common our work, where we focus directly on the structure neurodegenerative diseases, affecting up to 3% of of the substantia nigra. It is a small paired structure the population, and most frequently occurs after the in the midbrain. It is part of the basal ganglia and 65th year of life. The disease is caused by is involved in controlling movement. It consists of progressive loss of dopamine from the first two parts - pars compacta and pars reticularis, symptoms, according to studies (see: Tolosa E Parkinson's disease affects the above part of pars Katzenschlager R. Pharmacological Management compacta [2]. of Parkinson disease in: Jankovic, Tolosa: SPM (Statistical parametric mapping) is a Parkinson Disease and Movement Disorders, freely available software package that Lippincott Williams and Wilkins, 2007) to complements the MATLAB program from decrease amount of dopamine below 60% of the MathWorks. It is used to analyze data sequences original amount. Parkinson's disease initially that represent the brain. Currently available for manifests as tremor with rest, rigidity and data from the following modalities: fMRI, PET, bradykinesia. SPECT, EEG and MEG. Another toolkit is the For the diagnosis of PD is preferably used LEAD-toolbox, which works with MRI or CT MRI imaging or SPECT. In the late stage, the images within MATLAB and there is an effort to symptoms are diverse, including motor disorders create the best neuroimaging tool. Provides (dysnesia), mental disorders, postural instability, automatic image normalization to standard MNI autonomic dysfunction, eating disorders sensitive space. Another is FSL (The FMRIB Software symptoms and pain. PD causes atrophy of Library). It is a software package freely available pigmented neurons in the substantia nigra which for academic purposes. It can be run from the causes a decrease in dopamine in the basal ganglia. command line or via a graphical interface. FSL In PD, it is not possible to accurately determine the contains a number of imaging tools for analysis and cause and it can not be cured. Sofaronly the quality statistics [3]. of life can be improved by supplementing the precursor of dopamine [1]. Methods MRI and image segmentation using atlases The anatomy of the human brain is very variable MRI is a 3D imaging method for imaging soft among individuals, so there is an effort to facilitate tissues and organs. Unlike X-rays, CT and SPECT, diagnosis. To solve this problem, digital which use ionizing radiation for imaging, MRI uses probabilistic atlases can help, which serve as an a strong magnetic field of the size 1 to 7 Tesla. It is anatomical template. They contain statistically preferably used for soft tissue imaging, so it is generated information about the variable position
76 Lhotáková et al.: Summary of the substantia nigra atlases of individual parts of the brain. Each of the atlases was created in a certain standardized space. Here is a simplified summary of the spaces used: MNI Colin 27 DARTEL - A Fast Diffeomorphic Registration Algorithm, a field calculation that allows forward and backward deformation. Creating the average of all images, which serves as a template - the deformations are calculated from the template to individual images - the template is then regenerated using inverse deformation. 452 ICBM - called. Average brain. MNI-ICBM 152 - used in SPM12 and was created by nonlinear registration of 152 T1 Figure 7: Atlas of the Basal Ganglia and Thalamus (left) and weighted images, it is suitable for group CIT168 Reinf Leartn (right). In the pictures you can see the white area of the substantia nigra. In the picture on the right, studies. you can see only one part of substantia nigra, the pars compacta. We used Mango software developed by the University of Texas to display atlases. Mango program is freeware and may be used only for scientific purposes[4].
Figure 8: Human Motor Thalamus (left) and MNI PD25 subcortical (right). In the pictures you can see the white areas of the substantia nigra of both hemispheres.
Figure 6: Mango software, where we displayed atlases Atlas of the Basal Ganglia and Thalamus defining the substantia nigra (SN) area. The main author of this work states Xiaosong He. In this program, we used a simple procedure There were 96 patients with epilepsy, they were (Figure 1) to display individual subcortical atlases, divided into 3 groups of 32 subjects. The groups which contain the SN region monitored by us. The were divided according to condition, all of whom following figures (Figures 2 and 3) show the underwent Focal Impaired Awareness Seizures selected atlases shown in this program. The figures (FIAS). The first group of patients included those show the areas of the substantia nigra. Mango who had never had FBTCS (focal to bilateral tonic- offers the ability to unify planes for multiple clonic seizures). The second group were patients images using options - Link Slice Navigation and who had experienced FBTCS in their lifetime but we used this function. had not developed within 1 year prior to scanning. And the last group included patients who had FBTCS in the last year before the scan [5].
77 Lhotáková et al.: Summary of the substantia nigra atlases
CIT168 Reinf Learn Table 1: An overview of subcortical atlases that define the The main author of this work states Wolfgang M. substantia nigra. The authors of the atlas, the number of subjects that were used to create the atlas. Pauli. 168 adults aged 22-35 were used for the MNI creation. Scans of 84 women and 84 men were used Atlases (Author): Description: for balancing. A template was created from 3D T1 space: 30 young healthy and T2 weighted scans using diffeomorphic ATAG_Linear subjects (14 women, MNI04 normalization [6]. (Kauken 2014) [9] mean age 24.2) Human Motor Thalamus 31subjects; 13 (24.38, 6 The main author of this work states Igor Ilinsky. ATAG_Nonlinear women) MNI Four postmortem human brain samples were used (Keuken 2014) [10] + 9 (50.67, 5 women) to generate this atlas. Two women and two men, + 9 (72.33, 4 women) these samples were stored in 4% paraformaldehyde. These samples were provided Atlas of the Basal 96 patients with by The University of Iowa Department of Ganglia and epilepsy - divided into 3 MNI Anatomy Deeded Body Program [7]. Thalamus (He 2020) groups (32 patients [5] each group)
MNI PD25 subcortical 168 adults (22-35 The main author of this work states Yiming Xiao. CIT168_Reinf_Learn years) 84 women and Twenty-five patients with Parkinson's disease were (Pauli 2017) - MNI 84 men, mean age ± in used for this atlas. There were a total of 13 men in imported from ICBM152 both groups = 28.9 ± CIT168 [6] the group, the mean age in these patients was 58.77 3.6 years) years. The possibility of fusing T1 and T2 weighted images was used to create the atlas [8]. Human Motor 4 samples after death – MNI Thalamus (Ilinsky 2 women and 2 men ICBM152 Results 2017) [7]
We observed how atlases were created. The names MNI PD25 25 PD patients (58 ± MNI of the atlases, their authors, and information on the subcortical (Xiao years, 13 men) ICBM152 number of patients from whose MRI scans were 2017) [8] atlases are listed in the table below (Table 1). These Nigral organization 485 healthy subjects are probabilistic atlases containing the anatomical atlas (Zhang 2017) FNIRT (29.1 ± 3.5 years, 202 location of the substantia nigra (SN). The table also [11] shows whether the individuals were healthy. In females) Ultra-high field atlas several cases, scans from patients with PD or MNI for DBS planning 12 healthy subjects ICBM152 epilepsy are used. (Wang 2016) [12]
Conclusion aim to determine the most appropriate atlas that The aim of the work was to study individual best displays this structure in practice. As a next subcortical atlases that depict SN, ind out how they step we try to average probability atlases that were created, what was the proportion of men and contain SN representations, the combination of women in each atlas. We also focused on the which could contribute to accurate segmentation. possibility of displaying these atlases using Mango Later, it would be compared with Czech patients software and filtering the ROI of the SN area. With who have an SN region defined by neuromelanin this display, we are convinced that the atlases detection. We will then use the atlas created by us provide a good basis for identifying the SN region. to evaluate the stage of Parkinson's disease. But we must not forget the fact that the individual atlases differ from each other. We will address this References fact in our next work. [1] POEWE, Werner, Klaus SEPPI a Caroline M. The continuation of the work is therefore TANNER, 2017. Parkinson disease. NATURE the above-mentioned comparison of individual REVIEWS. 21. atlases and proving their differences. As well we [2] MORRIS, Shaine A. a Timothz C. SLESNICK, 2018. Magnetic Resonance Imaging. Visual Guide to Neonatal Cardiology. 5.
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[3] SPM – Statistical Parametric Mapping [online]. [cit. [8] XAIO, Yiming, Vladimir FONOV a M. Mallar 2020-09-15]. Available from: CHAKRAVARTY, 2017. A dataset of multi- https://www.fil.ion.ucl.ac.uk/spm/ contrast population-averaged brain MRI atlases of a Parkinson's disease cohort. Data in Brief. 10. [4] LORIO, s., S. FRESARD a S. ADASZEWSKI, 2016. New tissue priors for improved automated [9] KEUKEN, M.C., P.-L. BAZIL a L. CROWN, 2014. classification of subcortical brain structures on MRI. Quantifying inter-individual anatomical variability NeuroImage. 10. in the subcortex using 7 T structural MRI. NeuroImage. 7. [5] HE, Xiaosong, Ganne CHAITANYA a Burcu ASMA, 2019. Disrupted basal ganglia– [10] KEUKEN, Max C., Pierre-Leuis BAZIN a Andreas thalamocortical loops in focal to bilateral tonic- SCHÄFER, 2013. Ultra-High 7T MRI of Structural clonic seizures. BRAIN. 16. Age-Related Changes of the Subthalamic Nucleus. doi:10.1093/brain/awz361 The Journal of Neuroscience. 5. [6] PAULI, Wolfgang M., Amanda N. NILI a J. [11] ZHANG, Yu, Kevin M.-H. LARCHER a Bratislav Michael TYSZKA, 2018. Data Descriptor: A high- MISIC, 2017. Anatomical and functional resolution probabilistic in vivo atlas of human organization of the human substantia nigra and its subcortical brain nuclei. DATA. 13. connections. ELife. 23. doi: doi:10.1038/sdata.2018.63 https://doi.org/10.7554/eLife.26653 [7] ILINSKY, Igor, Andreas HORN, Perrine PAUL- [12] WANG, Brian T., Stefan POIRIER a Ting GUO, GILLOTEAUX a Pierre GRESSENS, 2018. Human 2016. Generation and evaluation of an ultra-high- Motor Thalamus Reconstructed in 3D from field atlas with applications in DBS planning. 10. Continuous Sagittal Sections with Identified doi:10.1117/12.2217126 Subcortical Afferent Territories. ENeuro. 17.
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Reconstruction and properties of T2* maps based on the number of iterations and their correlation with QSM in Parkinson's disease patients
Jozefína Vaľková1*, Radim Krupička2, Petr Dušek3
1Czech Technical University in Prague, Faculty of Biomedical Engineering, Kladno, Czech Republic 2Clinic of Neurology, General University Hospital in Prague, Czech Republic
Abstract: Parkinson's disease (PD) is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. Nowadays, magnetic resonance imaging has significant role at enhanced understanding of the pathological progression and clinical diagnosis of PD. The aim of the project is to choose from the available tools for the reconstruction of the T2* maps from the multi-echo GRE and to develop a methodology for the reconstruction of the T2* maps in healthy people, Parkinson's patients and patients with sleeping disorder. The project is also focused on evaluating the impact of the number of iterations on the T2* map quality. Sum of squared differences, comparison between the values of pixels in the image matrix and cross-correlation as quality indicators between images with different number of iterations have been done. According these calculations the T2* parametric maps made with 130 iterations calculated by open source image processing program ImageJ with right settings are considered as mapss with appropriate quality for further research.
Introduction The sequence of a multiecho gradient recalled echo (GRE) T2*-weighted imaging is a relatively Parkinson's disease (PD) is the second most new magnetic resonance imaging (MRI) technique. common neurodegenerative disorder of the elderly In contrast to T2 relaxation, which acquires a spin and is clinically characterized by the motor echo signal, T2* relaxation acquires a gradient symptoms of tremor, bradykinesia, and rigidity. echo signal. The sequence of a GRE T2*WI Non-motor features including cognitive and requires high uniformity of the magnetic field. neuropsychiatric disturbances are also commonly GRE T2*WI can detect the smallest changes in manifest, dispelling previous beliefs that PD is uniformity in the magnetic field and can improve solely a disorder of movement. The main the rate of small lesion detection. In addition, the pathological characteristic of PD is the progressive T2* value can indirectly reflect changes in tissue loss of dopamine neurons in the substantia nigra biochemical components. The measurement of the pars compacta [1]. T2* value of tissues on the GRE T2*WI can Iron overload has been implicated in the indirectly reflect the iron content of the tissues, pathology and pathogenesis of PD. The substantia which demonstrates two advantages: non- nigra, where the selective loss of dopaminergic invasiveness and rapidness. The increase in iron neurons occurs, is the primary region in the brain content of tissues causes a shortening in the T2* known to deposit iron. Additionally, aberrant iron relaxation time. This is conducive to quantitatively concentrations have been observed in other brain study iron deposition [4]. regions such as red nuclei, globus pallidus and T2* value is widely used in clinical cortex of PD patients, despite of unknown applications. T2* mapping can measure the amount pathology [2]. Many studies have shown that some of iron deposition in the brain as a biomarker for MRI sequences such as Quantitative Susceptibility prognostic the PD [4]. Therefore, the main aim of Mapping (QSM), T2-weighted imaging, T2*, and the project is to choose from the available tools for R2* mapping can measure the amount of iron the reconstruction of the T2 * maps from the multi- deposition in the brain as a biomarker for echo GRE and to develop a methodology for the prognostic the disease and help diagnosis of the reconstruction of the T2 * maps. disease more accurately [3].
80 Val’ková et al.: Reconstruction and properties of T2* maps based on the number of iterations and their correlation with QSM in Parkinson's disease patients
Figure 2. T2* parametric map calculated with 30 iterations (left) and T2* parametric map calculated with 200 iteration (right).
15 parametric maps with different number of Figure 1. ImageJ program with appropriate parameters for iterations (from 50 to 190 iteration). calculation T2* parametric maps for the research. Quality indicators Subject and Methods After that we compared quality of these maps in MATLAB 2018a. We wrote the matlab script for Subject consisted of 8 parkinsonian patients. calculation of three parameters of quality: cross- Patients were selected from Clinic of Neurology, correlation, pixels and sum of squared differences. General University Hospital in Prague. All subjects We decided to choose 3 parameters to get the most were scanned on a 3T MRI system. accurate results. Like the pattern matrix we used the T2* parametric map (8-bit image) with 200 Primary data adjustment iteration. We elected the image with 200 iterations First of all, we had to convert available DICOM because there were no differences or they were (Digital Imaging and Communications in negligibly small between neighbouring images Medicine) format images to NIfTI (Neuroimaging (image made with 190 iterations and image made Informatics Technology Initiative) format images with 200 iterations). by using free available programme Mricron. The NIFTI file is generally made up of a header Results followed by volumetric data. The format supports up to 5 dimensions. One of the key capabilities of Three parameters (cross-correlation, sum of the format (stored in the header) is an affine squared differences and pixels) of each map have mapping between voxel indices (i,j,k) and real been calculated in MATLAB. world spatial location (x,y,z) [5]. The cross-correlation was the least precise parameter. It was insensitive to changes in images Post-processing of images from 80 iteration. For images with higher number The next step was the calculation parametric maps of iteration than 70 was cross-correlation 1 (table from T2* images. From the available tools for the 1). reconstruction of the T2* maps from the multi- echo GRE we selected program ImageJ. ImageJ is Table 1. Values of cross-correlation parameter in T2* parametric maps calculated with 50-90 iterations for an open source image processing program 8 patients. designed for scientific multidimensional images with thousands of plugins and scripts. For our Iter 50 60 70 80 90 research we used the plugin Mri processor which 1 0.9870 0.9984 0.9996 1 1 calculates parametric maps in MRI images. We 2 0.9824 0.9974 0.9994 1 1 selected an appropriate parametres for calculation 3 0.9853 0.9980 0.9995 1 1 such as Levenberg–Marquardt fit algorithm, cap-T 4 0.9853 0.9981 0.9996 1 1 map at 100 and echo times of images (figure 1). 5 0.9834 0.9980 0.9996 1 1 We used free available program MicroDicom 6 0.9863 0.9984 0.9996 1 1 viewer to find out the echo times of our images 7 0.9808 0.9976 0.9995 1 1 from available raw data. For each patient we made 8 0.9866 0.9984 0.9997 1 1
81 Val’ková et al.: Reconstruction and properties of T2* maps based on the number of iterations and their correlation with QSM in Parkinson's disease patients
Figure 3. Non-log scale graph of pixels. Figure 4. Log scale graph of pixels.
After that we calculated sum of squared Conclusion differences parameter. The resulting values were considered to be a little more accurate than in the The first part of project was focused on the previous case. There were negligible changes in detecting the program for postprocessing of T2* images from 90 iteration (table 2). maps. We chose the program called ImageJ and then we defined and validated parameters for Table 2. Values of sum of squared differences parameter in creation T2 * parametric maps. T2* parametric maps calculated with 50-90 iterations for 8 In the second part we compared quality of patients. individual maps for all 8 patients. We wrote the Iter 50 60 70 80 90 script in MATLAB for calculation three image 1 e+06 e+05 e+04 e+02 e-05 quality indicators (cross-correlation, sum of 2 e+06 e+05 e+05 e+03 e-05 squared differences and pixels). Following, the 3 e+06 e+05 e+05 e+03 e-04 different values between images were evaluated. 4 e+06 e+05 e+04 e+02 e-02 The quality of T2* parametric maps created with 5 e+06 e+05 e+04 e+03 e-03 130 iterations is considered as adequate. These 6 e+06 e+05 e+04 e+02 e-04 images will be used in further research. 7 e+06 e+05 e+04 e+02 e-06 8 e+06 e+05 e+04 e+02 e-04 Further work The pixels (comparison between the values of Our following goal is to create T2* parametric pixels in the image matrix) were the best quality maps using 130 iterations for images in healthy image indicator. The resulting values are shown in people, patients with Parkinson's disease and figure 3. The figure 4 shows the log-scale graph of patients with sleep disorder (at least for 20 patients these values. As we can see the values in pixels for in each group). Then we correlate these T2* maps images are really similar without significant with same QSM images and compare the amount differences in images from 130 iterations. For of iron in the substantia nigra pars compacta. images with 140 iterations and higher values differed just in 1 to 3 pixels per image. References [1] POLITIS, Marios a Flavia NICCOLINI. Serotonin Discussion in Parkinson's disease. Behavioural Brain Research [online]. 2015, 277, 136-145 [cit. 2019-06-20]. DOI: Sum up, according to cross-correlation and 10.1016/j.bbr.2014.07.037. ISSN 01664328. sum of squared differences parameters there are no Available from: significant changes in quality between images with https://linkinghub.elsevier.com/retrieve/pii/ 90 iterations and higher. Although, the pixels S0166432814004860 parameter pointed out the differences between [2] WANG, Jian-Yong, Qing-Qing ZHUANG, Lan- individual images until 130 iterations. From Bing ZHU, et al. Meta-analysis of brain iron levels images with 140 iterations and higher the of Parkinson’s disease patients determined by individual values were negligibly small. For postmortem and MRI measurements. Scientific Reports [online]. 2016, 6(1) [cit. 2019-06-20]. DOI: obtaining the most relevant results in further work 10.1038/srep36669. ISSN 2045-2322. Available we decided to work with maps of 130 iterations. from:: http://www.nature.com/articles/srep36669 [3] SHAHMAEI, Vahid, Fariborz FAEGHI, Ahmad MOHAMMDBEIGI, Hasan HASHEMI a Farzad ASHRAFI. Evaluation of iron deposition in brain basal ganglia of patients with Parkinson's disease using quantitative susceptibility mapping. European Journal of Radiology Open [online]. 2019, 6, 169-
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174 [cit. 2019-06-18]. DOI: https://www.hindawi.com/ 10.1016/j.ejro.2019.04.005. ISSN 23520477. journals/bmri/2014/312142/ Available from:: https://linkinghub. elsevier.com/retrieve/pii/S2352047719300206 [5] LAROBINA, Michele a Loredana MURINO. Medical Image File Formats. Journal of Digital [4] TANG, Meng Yue, Tian Wu CHEN, Xiao Ming Imaging [online]. 2014, 27(2), 200-206 [cit. 2019- ZHANG a Xiao Hua HUANG. GRE T2 * -Weighted 06-22]. DOI: 10.1007/s10278-013-9657-9. ISSN MRI: Principles and Clinical Applications. BioMed 0897-1889. Available from:: Research International [online]. 2014, 2014, 1-12 http://link.springer.com/10.1007 [cit. 2019-06-20]. DOI: 10.1155/2014/312142. /s10278-013-9657-9 ISSN 2314-6133. Available from::
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