4th International Conference for Innovation in Biomedical Engineering and Life Sciences: Proceedings of ICIBEL 2022, December 10–13, 2022, Kuala Lumpur, Malaysia (IFMBE Proceedings, 107) 3031564375, 9783031564376

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IFMBE Proceedings 107

Fatimah Ibrahim Juliana Usman Mohd Yazed Ahmad Norhamizan Hamzah Editors

4th International Conference for Innovation in Biomedical Engineering and Life Sciences Proceedings of ICIBEL 2022, December 10–13, 2022, Kuala Lumpur, Malaysia

IFMBE Proceedings

107

Series Editor Ratko Magjarevi´c, Faculty of Electrical Engineering and Computing, ZESOI, University of Zagreb, Zagreb, Croatia

Associate Editors Piotr Łady˙zy´nski, Warsaw, Poland Fatimah Ibrahim, Department of Biomedical Engineering, Faculty of Engineering, Universiti Malaya, Kuala Lumpur, Malaysia Igor Lackovic, Faculty of Electrical Engineering and Computing, University of Zagreb, Zagreb, Croatia Emilio Sacristan Rock, Mexico DF, Mexico

The IFMBE Proceedings Book Series is an official publication of the International Federation for Medical and Biological Engineering (IFMBE). The series gathers the proceedings of various international conferences, which are either organized or endorsed by the Federation. Books published in this series report on cutting-edge findings and provide an informative survey on the most challenging topics and advances in the fields of medicine, biology, clinical engineering, and biophysics. The series aims at disseminating high quality scientific information, encouraging both basic and applied research, and promoting world-wide collaboration between researchers and practitioners in the field of Medical and Biological Engineering. Topics include, but are not limited to: • • • • • • • • • •

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Fatimah Ibrahim · Juliana Usman · Mohd Yazed Ahmad · Norhamizan Hamzah Editors

4th International Conference for Innovation in Biomedical Engineering and Life Sciences Proceedings of ICIBEL 2022, December 10–13, 2022, Kuala Lumpur, Malaysia

Editors Fatimah Ibrahim Center for Innovation in Medical Engineering (CIME), Department of Biomedical Engineering, Faculty of Engineering Universiti Malaya Kuala Lumpur, Malaysia Mohd Yazed Ahmad Center for Innovation in Medical Engineering (CIME), Department of Biomedical Engineering, Faculty of Engineering Universiti Malaya Kuala Lumpur, Malaysia

Juliana Usman Department of Biomedical Engineering Universiti Malaya Kuala Lumpur, Malaysia Norhamizan Hamzah Department of Rehabilitation Medicine, Faculty of Medicine Universiti Malaya Kuala Lumpur, Malaysia

ISSN 1680-0737 ISSN 1433-9277 (electronic) IFMBE Proceedings ISBN 978-3-031-56437-6 ISBN 978-3-031-56438-3 (eBook) https://doi.org/10.1007/978-3-031-56438-3 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Paper in this product is recyclable.

Preface

The 4th International Conference for Innovation in Biomedical Engineering and Life Sciences (ICIBEL 2022) convened in Kuala Lumpur from December 10 to 13, 2022. The conference served as a dynamic platform for researchers and industry leaders to share cutting-edge breakthroughs, engage in discussions, and exchange perspectives on biomedical engineering and sciences. Under the thematic umbrella of “Fusion of Healthcare & Technology in the New Era,” the event featured distinguished keynote and invited speakers specializing in advanced biomedical, green, and medical device technologies. Additionally, ICIBEL 2022 incorporated workshops on current issues, on medical device registration by the government’s Medical Device Authority, as well as on commercialization. Within these pages, readers will discover a collection of 25 peer-reviewed papers addressing timely issues in biomechanics, ergonomics, rehabilitation, biosensing, and life sciences. The volume also explores solutions for technology transfer, telemedicine, and point-of-care healthcare. Rigorous evaluation criteria, including relevance to the conference, contribution to academic discourse, appropriateness of research methods, and clarity of presented results, guided the selection process, resulting in an acceptance rate of 85%. This biannual conference owes its success to the unwavering dedication of numerous contributors, including the conference committee, the Center of Innovation in Medical Engineering (CIME) at Universiti Malaya, the Faculty of Engineering at Universiti Malaya, Malaysia’s Society of Medical and Biological Engineering (MSMBE), the International Federation for Medical and Biological Engineering (IFMBE), sponsors, reviewers, speakers, presenters, and delegates. Special acknowledgment goes to Dr. Noraisyah Mohamed Shah for her invaluable assistance in the editing process. Without the tireless efforts and support of these individuals and organizations, ICIBEL 2022 would not have been possible. We extend our heartfelt gratitude to each of them, and we trust that this volume will stand as a faithful record of the diverse topics discussed at the event while serving as a source of inspiration for new ideas and collaborations. Fatimah Ibrahim Juliana Usman Mohd Yazed Ahmad Norhamizan Hamzah

Conference Details

Name 4th International Conference for Innovation in Biomedical Engineering and Life Sciences

Short Name ICIBEL 2022

Venue December 10–13, 2022 Pullman KLCC, Kuala Lumpur, Malaysia

Proceedings Editors Fatimah Ibrahim Juliana Usman Mohd Yazed Bin Ahmad Norhamizan Hamzah

Organized by Center for Innovation in Medical Engineering (CIME) Universiti Malaya, Malaysia

Co-organized by Malaysia’s Society of Medical and Biological Engineering (MSMBE)

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Conference Details

Endorsed by International Federation for Medical and Biological Engineering (IFMBE)

Supported by IFMBE Asia Pacific Working Group IEEE Universiti Malaya Student Branch Universiti Malaya Centre of Innovation & Commercialization (UMCIC) Medical Device Authority (MDA), Ministry of Health Malaysia STEM Center, Universiti Malaya

Organization

Organizing Committee Chairperson Fatimah Ibrahim

Faculty of Engineering, Universiti Malaya, Malaysia

Co-chair Tan Maw Pin

Faculty of Medicine, Universiti Malaya, Malaysia

Publication Fatimah Ibrahim Juliana Usman

Faculty of Engineering, Universiti Malaya, Malaysia Faculty of Engineering, Universiti Malaya, Malaysia

Secretary Noraisyah Mohamed Shah

Faculty of Engineering, Universiti Malaya, Malaysia

Treasurer Mas Sahidayana Mohktar

Faculty of Engineering, Universiti Malaya, Malaysia

Publicity/Logistic Norhayati Soin

Faculty of Engineering, Universiti Malaya, Malaysia

x

Organization

Technical Mohd Yazed Ahmad Wan Safwani Wan Kamarul Zaman Norhamizan Hamzah

Faculty of Engineering, Universiti Malaya, Malaysia Faculty of Engineering, Universiti Malaya, Malaysia Faculty of Medicine, Universiti Malaya, Malaysia

Sponsorship/Exhibition Wan Safwani Wan Kamarul Zaman

Faculty of Engineering, Universiti Malaya, Malaysia

Secretariat Committee Wan Safwani Wan Kamarul Zaman Nurul Fauzani Jamaluddin Mohd Faiz Zulkeflee Yuslialif Mohd Yusup

Faculty of Engineering, Universiti Malaya, Malaysia Faculty of Engineering, Universiti Malaya, Malaysia Faculty of Engineering, Universiti Malaya, Malaysia Faculty of Engineering, Universiti Malaya, Malaysia

International Advisory Board Alessandro Luzio Bojan Petrovic Ratko Magjarevic Goran Stojanovic Marc J. Madou Elman El Bakri Azman Hamid Anis Nurashikin Nordin Ng Kwan Hoong

Istituto Italiano di Tecnologia—IIT, Italy University of Navi Sad, Serbia International Federation for Medical and Biological Engineering, (IFMBE), USA University of Novi Sad, Serbia Autonomous Medical Devices Inc., USA Malaysian Society of Medical and Biological Engineering Malaysia (MSMBE), Malaysia Malaysian Society of Medical and Biological Engineering Malaysia (MSMBE), Malaysia International Islamic University Malaysia, Malaysia Universiti Malaya, Malaysia

Contents

Aerospace and UAV in Medical Application Healthcare Delivery in the Era of IR4.0: The Rise of the Drone . . . . . . . . . . . . . . Zaleha Abdullah Mahdy, Rahana Abd Rahman, Mohamad Afiq Hidayat Zailani, Raja Zahratul Azma Raja Sabudin, Aniza Ismail, Shamsuriani Md Jamal, and Ismail Mohd Saiboon Path Optimization Algorithms for Unmanned Aerial Vehicles (UAVS) Collision Avoidance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Muhammad Isyraf Ismail, Anees Ul Husnain, Norrima Mokhtar, and Noraisyah Mohamed Shah

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Biotechnology, Life Sciences and Biosensing Effect of Fluid Properties on Bone Scaffold Permeability . . . . . . . . . . . . . . . . . . . . Sook Wei Chan, Norhana Jusoh, and Adlisa Abdul Samad Antimicrobial Properties of Nanoporous Hydroxyapatite Doped with Polyphenols Extracted from Euphorbia tirucalli L. (Pokok Tetulang) . . . . . Alwani Ibrahim, Tun Iqmal Haziq Tun Rashdan Arief, Nur Farahiyah Mohammad, Nashrul Fazli Mohd Nasir, Khairul Farihan Kasim, Siti Shuhadah Md Saleh, and Farah Diana Mohd Daud Fundamental Research for Leg Band Type Wearable Electrocardiogram Monitor-Examination of Electrode Position and Construction of Basic System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reina Kobayashi and Akinori Ueno Glutathione Precursors Supplementation Effects on Renal Function, Lipid Profile and Body Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nur Rasyidah Hasan Basri, Mas Sahidayana Mohktar, Wan Safwani Wan Kamarul Zaman, and Selvam Rengasamy Fabrication and Characterization of Inkjet Printed Flexible Fractal-Type Temperature Sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saima Qureshi, Lazar Mili´c, Varun Jeoti, and Goran M. Stojanovi´c

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Development of Kirigami-Based Strain Sensors Printed on Medical Dressing for On-Skin Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sajjad Hossen, Anis Nurashikin Nordin, Muhammad Irsyad Suhaimi, Lim Lai Ming, Norsinnira Zainul Azlan, Rosminazuin Ab Rahim, Mohd Saiful Riza, and Zambri Samsudin Multidisciplinary Approach in Primary Teeth Bite Marks Analysis on Prehistoric Pottery Sherds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nikola Jovanovi´c, Bojan Petrovi´c, Miroslav Ðo´coš, Jovana Kalamkovi´c, Lazar Mili´c, Marija Vejin, Sanja Koji´c, Sofija Stefanovi´c, and Goran Stojanovi´c Partially Automatic Detection of Mental and Panoramic Mandibular Indexes for Diagnosis of Osteoporosis in Clinical and Medieval Samples . . . . . . Lazar Mili´c, Sanja Koji´c, Bojan Petrovi´c, Miroslav Ðo´coš, Marija Vejin, Sofija Stefanovi´c, and Goran Stojanovi´c Development of a Mechanomyography (MMG)-Based Muscle Strength Monitoring Tool for Long Covid Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Harinivas Rao Suba Rao, Nur Azah Hamzaid, Norhamizan Hamzah, Mohd Yazed Ahmad, and Jannatul Naeem

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The Effects of Bowling Ball Drilling Angle in Tenpin Bowling; A Preliminary Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Azrena Zaireen Ahmad Zahudi, Juliana Usman, and Noor Azuan Abu Osman Design and Fabrication of Surface Acoustic Wave (SAW) Device for Cell Migration Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Mazlee Mazalan, Anas Mohd Noor, Nor Farhani Zakaria, Mohd Rosydi Zakaria, Arif Mawardi Ismail, Wan Safwani Wan Kamarul Zaman, Mohammad Shahrazel Razalli, Tien-Dung Do, and Yufridin Wahab E-Stethoscope: Preliminary Classification of Chest Sound for Proper Intubation in Paediatrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 Siti Hajar Juhari and Mohd Yazed Ahmad Embedded System for Medical Devices Smart Wheelchair Navigation Using ROS with Collision Avoidance and 2D Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Nawaf Mohammed, Sulaiman Azzan, Vedanta Jaitoo, Vickneswari Durairajah, and Suresh Gobee

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An Intelligent Autonomous Wheelchair for Hospital . . . . . . . . . . . . . . . . . . . . . . . . 144 Meng Kiat Chua, Boon Jian Chun, Kai Sheng Lee, Yi Chen Wong, Vickneswari Durairajah, and Suresh Gobee Metamaterial-Based Textile Antenna for Wearable Medical Applications . . . . . . 158 Hussein Yahya Alkhalaf, Mohd Yazed Ahmad, Harikrishnan Ramiah, and Fatimah Fawzi Hashim A Compact Flexible Wideband Antenna with Low SAR for Biomedical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Fatimah Fawzi Hashim, Wan Nor Liza Binti Mahadi, Tariq Bin Abdul Latef, Mohamadariff Bin Othman, and Hussein Yahya Alkhalaf Medical and Engineering Future Education One Hand Braille Assistive for Computer Application . . . . . . . . . . . . . . . . . . . . . . 181 Alif Aiman Bin Rosdi, Farah Amira Binti Fazlur, Kum Shou Shun, Ting Kwong Jian, Vickneswari Durairajah, and Suresh Gobee A Communication Assistive Glasses for Hearing Impaired Users . . . . . . . . . . . . . 191 Schubert Tan Su Min, Lim Wei Qi, Cheong Soon Hou, Vickneswari Durairajah, and Suresh Gobee Assistive Vest for Visually Impaired Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Chun You Low, Min Kai Dan, Chern Wei Ship, Yik Seng Tee, Vickneswari Durairajah, and Suresh Gobee Advanced Ergonomics Technology and Robotics Rehabilitation Design and Structural Analysis of Patient-Specific Knee Guide Using Automated FEA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Christopher James Aeria, Suresh Gobee, and Vickneswari Durairajah Physiological Changes in Male Pelvic Floor Muscles During Salat Movement: A Preliminary Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 Fatimah Ibrahim, Woo Kai Wen, Nurul Fauzani Jamaluddin, and Azad Hassan Abdul Razack Functional Electrical Stimulation (FES) Study for Foot Drop Rehabilitation Using Arduino Nano Atmega328p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 Kuryati Kipli, Muhd Nazreen Ulia, Lidyana Roslan, Annisa Jamali, and Norhayati Soin

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Special Track on Industry: Medical Device and Commercialization The Effect of Inverter and Non-inverter Air-Conditioning, Air Movement, CO2 Concentration and Aircon Set Temperature on Sleep Wellness . . . . . . . . . . . 253 Kenny James Ling Neng Hui, Fatimah Ibrahim, Mas Sahidayana Mokhtar, and Nurul Fauzani Jamaluddin Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

Aerospace and UAV in Medical Application

Healthcare Delivery in the Era of IR4.0: The Rise of the Drone Zaleha Abdullah Mahdy1(B) , Rahana Abd Rahman1 , Mohamad Afiq Hidayat Zailani2 , Raja Zahratul Azma Raja Sabudin2 , Aniza Ismail3 , Shamsuriani Md Jamal4 , and Ismail Mohd Saiboon4 1 Department of Obstetrics and Gynaecology, Faculty of Medicine, Universiti Kebangsaan

Malaysia Medical Centre, Jalan Yaacob Latif, 56000 Cheras, Kuala Lumpur, Malaysia [email protected] 2 Department of Pathology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, 56000 Cheras, Kuala Lumpur, Malaysia 3 Department of Community Health, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, 56000 Cheras, Kuala Lumpur, Malaysia 4 Department of Emergency Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, 56000 Cheras, Kuala Lumpur, Malaysia

Abstract. The advent of the drones began with unmanned balloons in the eighteenth century, but the uptake into healthcare delivery was as recent as 2014. A systematic review in 2020 produced only three publications involving drones in blood samples delivery. Since then, there has been an exponential increase in research and publications on the medical drone, particularly for healthcare service delivery. Two economic analyses comparing medical drone transportation to motorcycles and to ambulances respectively, revealed rather contrasting outcomes. Local legislation varies among countries, posing a formidable obstacle to conducting on site research into drone flights for healthcare delivery. Differences in climate and topography make it impossible to extrapolate the drone’s capability from one geographical location to another, hence the importance of local experience and studies. From the technical point of view, several outstanding issues need to be solved before the drone can take to the skies much more effectively in its role in healthcare service delivery. Topping the list are the cost of drone acquisition, the capacity to monitor autonomous drone flights beyond visual line of sight versus the local network coverage, the drone’s power source versus its capacity to fly long distances, its navigation accuracy, and climate challenges. Keywords: healthcare delivery · drone · review

1 Introduction The autonomous aerial vehicle (UAV) or drone as it is better known, has made its debut in the crude form of a balloon about two centuries ago, but its potential in medical transportation was only realized recently. Its transport potential in healthcare delivery was demonstrated for the first time in 2014 by Médecins Sans Frontières, by sending © The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 F. Ibrahim et al. (Eds.): ICIBEL 2022, IFMBE Proceedings 107, pp. 3–7, 2024. https://doi.org/10.1007/978-3-031-56438-3_1

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sputum samples for a diagnosis of tuberculosis in Papua New Guinea using a drone [1]. Despite its significant potential, uptake has been slow, compared to its exploitation for military functions, or even ordinary goods delivery. Reports of drone transfer of clinical items have largely been anecdotal, with the only large-scale usage being Zipline activities in Rwanda, where the focus has been in the field of obstetrics [2, 3]. This mini review shall focus on the controversies surrounding usage of the drone as a mode of medical transportation in the era of the fourth industrial revolution (IR4.0), its benefits, obstacles, and future potential.

2 Benefits In a systematic review of publications until July 2019, there were only three publications on the use of drones for transportation of blood samples or product [4]. A PubMed search today using the key words “drone”, “healthcare” and “transport” retrieved 44 publications (https://pubmed.ncbi.nlm.nih.gov/?term=drone+healthcare+transport), whereas narrowing it down to blood transport retrieved 32 publications (https://pubmed.ncbi.nlm. nih.gov/?term=drone+healthcare+blood+transport), out of which 12 were original articles. Obviously there has been increased interest in the subject, accompanied by much research and reports, implying rising confidence in the feasibility of the system. Outstanding among the benefits of managing blood transportation using drones is the timeliness of delivery and drastic reduction in wastage of blood products [2]. The time saved was almost two thirds on average, with a range of 3 to 211 min depending on the actual distance from the location of need to the source of blood products. Concurrently, drone transportation of blood products resulted in a reduction of 7.1 units of blood products wastage per month, which translated into two thirds reduction in blood products wastage over 12 months. Without the use of drones, peripheral blood bank units have to be set up in order to beat the delay in transportation when blood products are urgently needed. Because such peripheral units serve smaller populations and the demand for blood products is unpredictable, some of the blood packs may not be used up on time, resulting in expiry of these packs [2]. The savings in terms of time and blood supply with the use of the drones are indeed significant achievements in terms of savings of invaluable resources that are potentially lifesaving. There are three fundamental arms of medical drone function for healthcare application: search and rescue, medical care, and transport or delivery systems [5]. Search and rescue is most useful in disaster management. Medical care involves combination with remote telemedicine, and transport or delivery systems such as the delivery of blood products, vaccines, medicines, anti-venom, laboratory samples, organs for transplant, and the Automated External Defibrillator (AED) device.

3 Obstacles 3.1 Cost of Drones Economic evaluations of drone usage in this regard have not been uniformly impressive. Ochieng et al. in 2020 [6] reported the cost per sample of a routine non-emergency transportation as USD0.65 by motorcycle, compared to USD0.82 by drone. In an emergency,

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the cost was USD24.06 for motorcycles, USD27.42 by drone for an unadjusted model without adequate geographical coverage, and USD34.09 for an adjusted drone model with complementary motorcycles. Motorcycles were more cost-effective compared to short-range drones, but increasing the range and operational lifespans of the drone made it more cost-effective. However, the study did not discuss the human risks of motorcycle transport, such as the possibility of loss of life and limb due to road traffic accidents, which may be significantly common in certain countries. When compared to transport by ambulance, which is the norm in some parts of the world, the drone costs less due to the shorter travel time, despite a higher cost per minute [7]. The shortening of the journey by almost half resulted in an Incremental Cost Effectiveness Ratio (ICER) of –RM2.95, i.e., a cost saving of RM2.95 per minute using the drone rather than the ambulance. The detailed calculation can be found in the article by Zailani et al., which was published in 2021 [7]. Cost may also be influenced by locality – urban versus rural – as time savings differ between the two settings [8]. The drone may save delivery time by about 20–30% in urban areas, whereas in rural areas the time savings may rise up to 65–74%. The main component that contributes to the high cost of drones is the acquisition cost [7]. It is possible that, as with many other technological innovations, the drone may become more affordable as time passes and its manufacture becomes more widespread. Manufacturer competition may lead to this. 3.2 Legislation and Societal Acceptance The law pertaining to drones vary from one locality to another, even between one township and another, depending on the local authority regulations. The lag time between innovation and legislation may hamper integration of useful innovation to benefit society, and this is certainly true to some extent in the case of the drone [9]. Societal acceptance may play a significant role in influencing the legislative climate. Recent surveys in Malaysia and abroad have shown good acceptance of unmanned aerial vehicles for usage in healthcare [10], especially for the purpose of rescue and research [11]. 3.3 Technological Limitations Currently, several technological issues limit the progress of the drone. In the background, the wireless mobile network system that is available in the locality is important [9]. Without a good system in place, it is impossible to deploy the drone and pilot it remotely, especially beyond visual line of sight (BVLOS). The capability to operate the drone BVLOS is essential for its function as an effective vehicle for healthcare delivery. The flight capacity of the drone in terms of power to cover long distances and carry a reasonable payload is vital for its delivery efficiency. Our study limited the payload to 2.5kg [7], but significant progress has been made in this field, with payloads up to 16kg reported recently [12]. In this regard, agricultural drones have progressed to usage of hybrid engines that are more powerful in terms of both payload and flight distance [13]. Whether this can be adopted by the medical drone remains to be seen.

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Aerial navigation and geofencing is another aspect of the drone that requires much research at present, to improve its competence in healthcare delivery. The accuracy of drone landing is important to ensure correct delivery of medical items. Geographical terrain and climate challenges may be issues in tropical countries, with jungle-clad hills and mountains, and strong winds, heavy rains and thunderstorm to surmount. Whether the drone can eventually be flown amidst these harsh conditions, or its function has to be limited to flights within favorable weather conditions only, remains to be seen. Specifically, for blood transportation by drone, temperature maintenance matters, in order to retain the quality of the blood samples or blood products. In this regard, the material and design of the drone carriage play a significant role [14]. The required temperature and its maintenance need to be investigated for delivery of other healthcarerelated materials such as vaccines, medicines, and organs for transplantation.

4 The Future The role of the drone in healthcare transportation is still in its infancy and has a long way to go before becoming fully developed as an effective means of transportation. Much research is warranted not only pertaining to the technological aspect but also the social, safety and legislative aspects of drone usage. Impact studies looking at effect of drones on clinical outcomes is an important perspective to explore. Also useful will be innovations to create drones that are more resilient in harsh weather such as tropical thunderstorms. Empowering drones to travel further with larger payloads will widen its potential and improve the feasibility of drone usage. Unmanned Transport Management (UTM) is a whole new area to look into. Nonetheless, once fully functional, the medical drone is an invaluable asset in delivering healthcare services to otherwise inaccessible or poorly accessible locations.

References 1. Knoblauch, A.M., et al.: Bi-directional drones to strengthen healthcare provision: experiences and lessons from Madagascar, Malawi and Senegal. BMJ Glob. Health 4, e001541 (2019). https://doi.org/10.1136/bmjgh-2019-001541 2. Nisingizwe, M.P., et al.: Effect of unmanned aerial vehicle (drone) delivery on blood product delivery time and wastage in Rwanda: a retrospective, cross-sectional study and time series analysis. Lancet Glob. Health 10(4), e564–e569 (2022). https://doi.org/10.1016/S2214-109 X(22)00048-1 3. Amukele, T.: Using drones to delivery blood products in Rwanda. Lancet Glob. Health 10, e463 (2022) 4. Zailani, M.A., Sabudin, R.Z., Rahman, R.A., Saiboon, I.M., Ismail, A., Mahdy, Z.A.: Drone for medical products transportation in maternal health care: a systematic review and framework for future research. Medicine 99(36), e21967 (2020). https://doi.org/10.1097/MD.000 0000000021967 5. Zailani, M.A., Azma, R.Z., Aniza, I., Rahana, A.R., MohdSaiboon, I., Mahdy, Z.A.: Drone technology in maternal healthcare in Malaysia: a narrative review. Malays. J. Pathol.Pathol. 43(2), 251–259 (2021)

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6. Ochieng, W.O., et al.: Uncrewed aircraft systems versus motorcycles to deliver laboratory samples in west Africa: a comparative economic study. Lancet Glob. Health 8(1), e143–e151 (2020). https://doi.org/10.1016/S2214-109X(19)30464-4 7. Zailani, M.A., et al.: Drone versus ambulance for blood products transportation: an economic evaluation study. BMC Health Serv. Res. 21(1), 1308 (2021). https://doi.org/10.1186/s12913021-07321-3 8. Johannessen, K.A.: A conceptual approach to time savings and cost competitiveness assessments for drone transport of biologic samples with unmanned aerial systems (drones). Drones. 6, 62 (2022). https://doi.org/10.3390/drones6030062 9. Comtet, H.E., Johannessen, K.-A.: A socio-analytical approach to the integration of drones into health care systems. Information 13, 62 (2022). https://doi.org/10.3390/info13020062 10. Sham, R., et al.: Drone usage for medicine and vaccine delivery during the COVID-19 pandemic: attitude of health care workers in rural medical centres. Drones. 6, 109 (2022). https:// doi.org/10.3390/drones6050109 11. Eibfeldt, H., et al.: The acceptance of civil drones in Germany CEAS. Aeronaut. J. 11, 665–676 (2020).https://doi.org/10.1007/s13272-020-00447-w 12. Avidrone Homepage. https://www.prnewswire.com/news-releases/avidrone-aerospace-exh ibits-flagship-210tl-tandem-drone-integrated-with-iris-automations-casia-onboard-detectand-avoid-technology-at-idex-2021-301230814.html. Accessed 18 Dec 2022 13. Skyfront Homepage, https://skyfront.com/. Accessed 31 May 2022 14. Zailani, M.A.H., et al.: Influence of drone carriage material on maintenance of storage temperature and quality of blood samples during transportation in an equatorial climate. PLoS ONE 17(9), e0269866 (2022). https://doi.org/10.1371/journal.pone.0269866

Path Optimization Algorithms for Unmanned Aerial Vehicles (UAVS) Collision Avoidance Muhammad Isyraf Ismail1 , Anees Ul Husnain1 , Norrima Mokhtar1,2(B) , and Noraisyah Mohamed Shah1,2 1 Department of Electrical Engineering, Faculty of Engineering, Universiti Malaya, 50603

Kuala Lumpur, Malaysia [email protected] 2 Malaysia Japan Research Institute, Universiti Malaya, 50603 Kuala Lumpur, Malaysia

Abstract. Unmanned aerial vehicles (UAVs) are frequently being applied at numerous applications and implementations in a tricky task. UAVs cannot operate effectively without path planning. Path planning is a key component of the overall performance of automation systems, particularly when it comes to industrial robots or drones. The mobility of an industrial drone is predetermined by a variety of algorithms that make up the industrial drone path planning system. The purpose of route planning algorithms is to identify safer, more efficient, collision-free, and least-cost travel paths for mobile robots and unmanned aerial vehicles. The path planning for Unmanned Aerial Vehicles (UAVs) can be categorized which is Conventional, Intelligent and Fusion algorithms. In this work, three algorithms were chosen which are A*, Hybrid A* and Dynamic Window Approach (DWA). All the algorithms were being tested in the multiple obstacle setup that had been created. Different obstacle setup was created to portray different complexity of the real situation in software simulation. All the data results for each algorithm were recorded and compared with each other. Based on the simulation generated, all path planning algorithms show the ability to reach the targeted point and avoid the obstacle in all maps created with different time computational and smoothness of the path. The results in terms of computational time and performance based on different complexity of the environment have shown a great potential for automatic UAVs path planning with collision avoidance in a known environment. Keywords: Unmanned aerial vehicles · path search algorithm · collision avoidance

1 Introduction Path planning is a computer issue that involves determining the sequence of viable configurations that will transport an item from the node source to the targeted destination [1]. It is also known as motion planning in a field such as robotics, computational geometry, computer animation and computer games. The objective of the path planning algorithm is to generate a computation of continuous path that connects a start configuration point and a goal/targeted configuration © The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 F. Ibrahim et al. (Eds.): ICIBEL 2022, IFMBE Proceedings 107, pp. 8–18, 2024. https://doi.org/10.1007/978-3-031-56438-3_2

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point, while avoiding the collision with known obstacles [2]. The obstacle geometry and the robot are described in 2D or 3D environments workspace while the motion is represented as a path in configuration space [3]. According to [2], algorithms for UAV route planning study have been offered by a variety of academics, and they may be loosely split into three categories: conventional algorithms, intelligent algorithms, and hybrid or fusion algorithms, which mix traditional algorithms with intelligent algorithms. The conventional path planning method (CPPM) is the classic approach utilized by researchers for mobile robot route planning throughout the years. Intelligent approaches are methods and tactics that have anything to do with the mimicking of natural events. [4]. The Dijkstra and A-Star (A*) algorithms are wellknown examples of traditional route planning methods [5]. Other classic route planning approaches based on division maps, such as the Rapidly exploring random tree (RRT) algorithm and artificial potential field algorithms, are available. Intelligent algorithms, such the Particle Swarm Optimization Algorithm (PSO), the Ant Colony Algorithm (ACA), and the Genetic Algorithm (GA), among others, combine environmental element knowledge with their own position to design real-time pathways. Each algorithm has their own advantages and disadvantages that differ from other algorithms. To achieve a superior route planning fusion algorithm, the benefits of several methods are combined in a third category called fusion algorithm. Table 1. Comparison of path planning for UAV collision avoidance Path planning Algorithm

Principle

Advantage

Disadvantage

Dijkstra Algorithm [6]

State space search

Strong searchability

Low computational efficiency

A* Algorithm [7]

Heuristic function Introduce cost function

Simple, easy to implement and fast calculation algorithm

Inefficient when dealing with multi-target points and 3D path planning

RRT Algorithm [8]

Stochastic tree expansion

Strong searchability

The randomness of the algorithm leads to only probabilistic completeness

Dynamic Window Approach (DWA) algorithm [9]

Local path planning algorithm

Good obstacle avoidance

Only detect when the obstacle is close

Based on the result of the literature review summarized in Table 1, the algorithm identified for this experiment is the A*, Hybrid A* and DWA.

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2 Methodology The methodology in this section describes the five distinct obstacles setup for static environment simulation, and the path optimization algorithm used. Comparative analyses are then conducted from the result obtained using a set of performance metrics. All simulation was conducted in Spyder IDE (Python 3.8). The computer configuration is AMD Ryzen 5 3550H with Radeon Vega Mobile Gfx 2.10 GHz, 12 GB. 2.1 Obstacles Setup for Static Environments Five distinct environments were selected, each of which featured a unique set of spatial characteristics. An assumption is made that one grid cell distance is equal to 1 m. The grid size for the environment has been set with the grid size of 27 × 18 grid cell. The initial point was set at coordinate (5,5) and the targeted point at coordinate (23,14). Firstly, the environment was initialized to consist entirely of open ground, creating a road across it is the simplest undertaking possible. This environment is illustrated in Fig. 3(a) where the red mark is the initial point, and the blue mark is the target point. Its total surface area is 27 × 18 = 486 m2 . The optimum pathways for this environment are straight lines from the start node to the finish node. The algorithm will be tested by setting a few patterns of obstacles near to the beginning point but far from the target point. The dots in the figures below indicate the obstacles that need to avoid by the drone. Next, the complexity of the obstacle is improved by set a few shapes of obstacle patterns in the grid map. The graphical representation of each map is given in Fig. 1 and their respective description is as follows. Map 1: Wall Environment. The proposed map was to set a barrier like a wall near the starting point and the end node. The distance between each obstacle is set 1 m apart. The width and the length of the drone are both changed to 1.2 m. This size is to ensure that the drone will not pass through a gap of less than 1 m. Map 2: “U” Shape Environment. The proposed map is using a combination of “U” shape and a mirror “U”. The initial point is set in the center of the small “U” while the goal point is set outside the obstacle at coordinate (23,14). The distance between each obstacle is set 1 m apart. The width and the length of the drone are both changed to 1.2 m. This size is to ensure that the drone will not pass through a gap of less than 1 m. Map 3: Comb Shape Environment. The initial point is positioned in between the “E” configuration and is identified by a red mark, while a blue mark identifies the destination target. The width and the length of the drone are both changed to 1.2 m. This size is to ensure that the drone will not pass through a gap of less than 1 m. Map 4: Dense Convex Environment. For Map 4, the obstacles are orderly arranged at a distance of 2 m apart from other obstacles. This is to ensure that the size of the drone is fixed before can pass through it. This convex environment was set up in between the initial point which is above the axis of 5 (is it x-axis?) and the target point below the axis of 14 in the y-axis. The boundary for this map is 27 × 18. This scenario is identified by growing difficulty as the heavy barriers create tiny routes.

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(b) Map 1: Wall environment (a) Collision free environment setup

(c) “U” shape environment

(d) Comb shape environment

(e) Dense convex environment

(f) Spiral maze environment

Fig. 1. Different obstacle setup for the experiment

Map 5: Spiral Maze Environment. The fifth map is the spiral maze shape with the initial point being set in the center of the spiral. The distance between each obstacle is set 1 m apart. The width and the length of the drone are both changed to 1.2 m. This size is to ensure that the drone will not pass through a gap of less than 1 m. The success is determined when the path generated follows the maze shape to reach the target point that is set outside the spiral maze shape.

2.2 Path Planning Algorithm From the environment that has been created, the path planning for the algorithm is determined by the red line created in the grid map. The performance of the algorithm is

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considered a success when the red line can reach the target point and manage to avoid the obstacle that has been set. A* and Hybrid A* algorithm A* algorithm was proposed as a heuristic algorithm (Weiguang & Xia, 2015), and as a solution to the Dijkstra algorithm’s problem of having a large amount of computation and low efficiency. Using a heuristic function, the A* algorithm calculates the ideal route by comparing the value of each generation with the search operation time and distance cost of each path point (Guruji et al., 2016). A* select the path that minimizes the following equation: f (n) = g(n) + h(n)

(1)

where n is the next node on the path, g(n) represents the cost of the path from the initial node to n, and h(n) is a heuristic function that calculates the cheapest path from n to the target. Originally, the algorithm computes the cost to all its immediate adjacent nodes, n, and selects the one with the lowest cost. This method is repeated until no new nodes can be selected and all paths have been explored. Then, choose the best path among them. If f(n) represents the total cost, then it may be written as Eq. (1). Figure 2 illustrate the workflow for both A* and the hybrid A*. Hybrid A* algorithm incorporates the vehicle dynamics, thus the path generated is smoother. Figure 3 shows the comparison of A* algorithm and Hybrid A* where the dot is symbolic of obstacles. As can be seen, the path generated by Hybrid A* is smoother. Dynamic Window Approach (DWA) Algorithm. Dynamic Window Approach introduced by [10]. Its workflow can be summarized into three steps [11]: 1. Based on how the robot is moving right now, several different paths can be made and simulated. Such paths are ruled out if they lead to obstacles or are too fast for the configuration. This dynamic window shows all possible routes. 2. Cost functions based on the distance between the path and the goal and the direction towards the goal are used to evaluate the paths that are made. 3. Execution of the best course of action, which is the one that costs the least overall. Because of this, the robot moves in this direction. The DWA was chosen because it was easy to add more cost functions and make it bigger. Any projected trajectories can be tested as hypotheses because they are made in different ways. Because the algorithm already includes the basic ideas of avoiding obstacles and following a path, these things do not need to be considered further. 2.3 Performance Metrics To evaluate the performance ability of each algorithm, the following parameter were considered. Number of Steps Count Towards Goal. The distance of the path measured to reach the target point. Number of steps counts =

Pathsize−1  i=1

No of iteration to reach target point

(2)

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Fig. 2. Flowchart for A* and Hybrid A*

Fig. 3. (a)Path generated by A* (b) Path generated by Hybrid A*. The x and y-axis are coordinates of location

Number of Corners. To calculate the number of corners, the vertices made were counted to calculate the number of corners. Number of corners = No of vertices − 1

(3)

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The ratio of smoothness for the path is determined by calculating using the formula: Ratio Smoothness =

(number of corners) (path length)

(4)

where the path length is the number of steps towards the goal. Success rate. The success rate of each algorithm in that environment can be calculated as follows: Success rate =

(number of successful path generate) ×100 (numbers of successsive goals the algorithm must produce) (5)

3 Result and Discussion Table 2 below gives a visualization example for the path generated by all algorithms for Map 4 and 5. Map 4 was found to have the shortest execution time for all algorithms, while Map 5 was the longest. Figure 4 gives the result for all algorithms in terms of time, length and smoothness of path generated towards the goal for all maps. In general, the computational time for A* algorithms is the fastest with an average time taken is 0.2383 s, followed by Hybrid A* with 3.6061 s and DWA with the longest which is 194.7447 s. A* and hybrid A* use global planner for path finding search while DWA uses local planner search. Global planner assumes that a holistic view of the robot’s environment is accessible. The advantage of global approaches comes in the fact that a complete trajectory from the beginning point to the target location can be computed offline. While local planner only uses a small fraction of the whole environment model to generate the path. To recalculate the path at a given rate, the map is reduced to the vehicle’s surroundings and is updated as the vehicle moves. The entire map cannot be used since the sensors are unable to update the map in all places, and a high number of cells would increase the processing cost. Map 4 or Dense Convex environment has the fastest computational time in all three graphs that have been compared. This is due to the simplicity of the map itself. The obstacle is set to narrow the path generated to the goal node. However, it does not affect the path generated since the size of the drone is set at 1.2-m width and length which is smaller than the gap between the obstacle that has been set 2 m apart. From all three graphs, the longest computational time taken is map 5 or spiral maze shape environment. Since the size of the drone is 1.2-m × 1.2-m is bigger than the gap between the obstacle, which is set to 1 m, the only option for a path to be generated is to follow the spiral maze shape to reach the goal point. The next parameter is the number of steps taken to reach the target point, which is given in Fig. 4(b). In general, the lowest number of steps to target represents the optimal path possible. All algorithms have the highest steps in map 5 compared to other maps since they must follow the spiral maze shape to reach the goal. When the number of steps taken towards the goal point increase, the time execution to generate path will become longer.

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Table 2. A sample of the path generated for Map 4 and Map 5 obstacles. The x and y-axis are coordinate of location.

Path generated for Map 5

DWA Algorithm

Hybrid A* Algorithm

A* Algorithm

Path generated for Map 4

To compare the smoothness of the path generated, critical edges and vertices taken were used to determine the number of corners of each algorithm. Theoretically when the number of corner decrease, the smoothness of the path will increase. And the longer the length of the path, the smoother the path will be. The number of corners produced in the path generated for Map 5 is compared between A*, hybrid A* and DWA algorithm. DWA algorithm has the lowest numbers of corners produced which is 20 corners followed by A* which is 23 corners made and hybrid A* has the highest value which is 44 corners produced.

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(a) Time taken to generate the path to target

(b) Number of steps taken towards goal Fig. 4. Comparative analysis between A*, Hybrid A* and DWA algorithms.

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(c) Smoothness of the path generated

(d) Number of vertices taken towards goal Fig. 4. (continued)

4 Conclusion Five different environments were created to simulate the path planning for A*, Hybrid A* and Dynamic Window Approach (DWA). Based on the simulation generated, all path planning algorithms show the ability to reach the targeted point. Execution time varies for each algorithm, where A* has the shortest time with an average 0.2383 s and the

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DWA has the highest computational time with an average of 194.7447 s. The average computational time for hybrid A* is 3.6061 s. Time taken to generate the path is directly proportional to the number of time steps to reach the target point. The smoothness of the path was compared in the map to determine the complexity of the map. Map with the “U” shape obstacle setup has the highest complexity of the map based on the ratio of smoothness. DWA algorithm performs the best overall with 80% of the total maps but it took longer to compute the generated path. However, A* & Hybrid A* has the shortest computational time and perform best in 60% of the total environment.

References 1. Azadi, S., Kazemi, R., Nedamani, H.R.: Trajectory planning of tractor semitrailers. In: Vehicle Dynamics and Control, pp. 429–478. Elsevier (2021). https://doi.org/10.1016/B978-0-32385659-1.00010-0 2. Wang, H., Pan, W.: Research on UAV path planning algorithms. In: IOP Conference Series: Earth and Environmental Science (2021) 3. Guo, J., Liang, C., Wang, K., Sang, B., Wu, Y.: Three-dimensional autonomous obstacle avoidance algorithm for UAV based on circular arc trajectory. Int. J. Aerosp. Eng. 2021, 1–13 (2021). https://doi.org/10.1155/2021/8819618 4. Ayawli, B.B.K., Chellali, R., Appiah, A.Y., Kyeremeh, F.: An overview of nature-inspired, conventional, and hybrid methods of autonomous vehicle path planning. J. Adv. Transp. 2018, 1–27 (2018). https://doi.org/10.1155/2018/8269698 5. Guruji, A.K., Agarwal, H., Parsediya, D.K.: Time-efficient A* algorithm for robot path planning. Proc. Technol. 23, 144–149 (2016). https://doi.org/10.1016/j.protcy.2016.03.010 6. Dijkstra, E.W.: A note on two problems in connexion with graphs. Numer. Math. 1(1), 269–271 (1959) 7. Song, R., Liu, Y., Bucknall, R.: Smoothed A* algorithm for practical unmanned surface vehicle path planning. Appl. Ocean Res. 83, 9–20 (2019). https://doi.org/10.1016/j.apor.2018. 12.001 8. Guitton, J., Farges, J.-L., Chatila, R.: Cell-RRT: decomposing the environment for better plan. In: 2009 IEEE/RSJ International Conference on Intelligent Robots and Systems (2009) 9. Tianyu, L., Ruixin, Y., Guangrui, W., Lei, S.: local path planning algorithm for blind-guiding robot based on improved DWA algorithm. In: 2019 Chinese Control And Decision Conference (CCDC), 3–5 June 2019 10. Fox, D., Burgard, W., Thrun, S.: The dynamic window approach to collision avoidance. IEEE Robot. Autom. Mag. 4(1), 23–33 (1997). https://doi.org/10.1109/100.580977 11. Henkel, C., Bubeck, A., Xu, W.: Energy efficient dynamic window approach for local path planning in mobile service robotics**This work was conducted at the University of Auckland, Auckland, New Zealand. IFAC-PapersOnLine 49(15), 32–37 (2016). https://doi.org/10.1016/ j.ifacol.2016.07.610

Biotechnology, Life Sciences and Biosensing

Effect of Fluid Properties on Bone Scaffold Permeability Sook Wei Chan1 , Norhana Jusoh1,2(B) , and Adlisa Abdul Samad1 1 Department of Biomedical Engineering and Health Sciences, Faculty of Electrical

Engineering, Universiti Teknologi Malaysia, UTM Johor Bahru, 81310 Johor, Malaysia [email protected] 2 Medical Device Technology Center (MEDiTEC), Institute Human Centred Engineering (iHumen), Universiti Teknologi Malaysia, UTM Johor Bahru, 81310 Johor, Malaysia

Abstract. Good permeability of the scaffold is critical to allow for inflow of cells, nutrients, as well as for waste product transfer. However, during designing the scaffold, the fluid properties of growth factor is always not being highlighted even though this component is one of fundamental is tissue engineering principles. Therefore, the purpose of this study is to determine the effects of fluid properties and viscosity on permeability of different 3D printed bone scaffold design. In this study, six 3D printed scaffold with different pore shape (circular and hexagonal) and different pore size (250 µm, 450µm and 650 µm) were designed by using Computer-aid Design software, SolidWorks. The scaffold design was simulated by using Computational Fluid Dynamic (CFD) simulation to analyze on how the fluid viscosity and inlet velocity affect permeability across the scaffold. The simulation results showed the hexagonal scaffold model encompassed more in the upper limit of natural bone permeability when compared to circular scaffold model. Moreover, based on analysis results, the favorable scaffold design was scaffold with hexagonal pore shape, pore size 650 µm and inlet velocity 0.0005 m/s since it has higher permeability value compared to circular scaffold. Hence, the permeability value of hexagonal scaffold was closer to the maximum value of natural bone permeability which is critical for successfulness of the long-term implant. Keywords: Bone Scaffold · Inlet Velocity · Viscosity · Permeability

1 Introduction In bone tissue engineering, development of bone scaffold allows the improvement of biological performance and regeneration of complicated bone tissue which is crucial phase in the sector [1]. However, creation of scaffold can be considered as a complicated procedure which involves the proper analysis and controlling of many parameters such as mechanical properties and biodegradation [2]. One of the significant restrictions of the bone implant after the surgery is vascularization for the diffusion of nutrients supply and elimination of waste [3]. Influenced by the mechanism of bone growth, faster © The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 F. Ibrahim et al. (Eds.): ICIBEL 2022, IFMBE Proceedings 107, pp. 21–29, 2024. https://doi.org/10.1007/978-3-031-56438-3_3

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internal vascularization potential with efficient osteoinduction bioactivity feature in 3D printed scaffolds are possible for the perfect bone replacement [4]. Bone biomaterial are still not enough for generating vascularization [5]. Therefore, Vascular Endothelial Growth Factor (VEGF) is needed as a primary angiogenic factor for the generation of microvascular network and vascular sprouts [6, 7]. Permeability prediction when designing the scaffold is helpful for scaffold optimization in order to balance mechanical properties and permeability as it would affect biological activities [8]. The scaffold permeability is a determinant factor as it plays a major role in the ability for cells to penetrate the porous media and for nutrients to diffuse [9]. Scaffold permeability can be affected by scaffold pore size, pore shape and fluid flow viscosity and velocity. Cell growth is highly dependent on the nutrients and waste product transfer through the porous structure [10]. High flow rate which causes washout of cells that leads to lower bone growth may happen due to higher permeability. Nevertheless, if the permeability of the scaffolds is lower, it will lead to low nutrient supply to cells which create hindrance for the growth of the bones [2]. Furthermore, scaffold’s permeability prediction is necessary to ensure that the permeability is in the range of human bones. Therefore, this study was conducted to simulate and analyse the effects of viscosity and velocity by considering different type of fluids which were Dulbecco modified Eagle’s minimal essential medium (DMEM), VEGF and blood plasma. The 3D-printed scaffold permeability also was compared based on different pore shape and size.

2 Methodology In this experiment, the process involved design selection, computational design, and design analysis. SolidWorks was used in designing the scaffold according to the desired dimension and material properties. Measurement and applied the material on the scaffold. Three scaffold design with different pore sizes (250 µm, 450µm and 650 µm) with two different pore shapes (circular and hexagonal). In this study, the material used was Polycaprolactone (PCL). The PCL material was created under Custom Material in SolidWorks. The properties of PCL included Elastic Modulus (E), 1200 MPa; Mass Density, 1100 kg/m3 ; tensile strength, 16 MPa [11]; Poisson’s ratio (v), 0.442 [12]; Shear modulus, 416.1 N/mm2 . Once the scaffold was created, fluid modelling was started with defined volume interest (VOI) minus the scaffold model by Volume Extract Feature. Then, the model of fluid was converted into one of the neutral file formats, iges file before imported into the CFD solver package. Before the analysis of CFD, fluid properties were inserted, and boundary condition were defined. Once these parameters and conditions were identified, the meshing was done, the simulation was conducted. Pressure and velocity distribution for all scaffold design were obtained. By applying the parameter of pressure difference across the fluid model, permeability was calculated using Darcy’s formula [2]. Figure 1 show the representative images for scaffold design, fluid flow model, meshed model and boundary conditions for circular and hexagonal pore shape. The inlet velocity applied were 0.0005 m/s, 0.001 m/s and 0.035 m/s, whereas the outlet velocity applied was assumed to be zero and a consistent flow condition was assumed within the scaffold [2].

Effect of Fluid Properties on Bone Scaffold Permeability

23

Fig. 1. Scaffold design (a,e), fluid flow model (b,f), meshed model (c,g) and boundary conditions (d,h) for circular (a-d) and hexagonal (e-h) pore shape.

The viscosity of DMEM at 37 °C is 0.45 × 10−3 Pa.s. Based on previous literature, the VEGF concentration is said to be in between 0.1–1.0 mg/ml and the blood plasma at normal human temperature 37 ◦ C is said to be 1.8 times more viscous compared to water [13]. Furthermore, many cardiovascular handbooks consider blood viscosity values between 0.0035 Pa.s and 0.0055 Pa.s to be normal [14]. The choosen viscosities were 0.00145 Pa.s, 0.00255 Pa.s, and 0.00495 Pa.s to represent DMEM, DMEM + VEGF and human blood, respectively.

3 Result and Discussion The value of pressure drops across the fluid flow model was obtained through the colour scale of the ANSYS Fluid Simulation results. Figure 2 show the representative image of pressure distribution for circular and hexagonal pore shape. The findings showed that pressure drops of both fluid models increased when the inlet velocity increased from 0.001 m/s to 0.035 m/s. When taking into account pore size and viscosity, both models with pore size 250 µm and viscosity 0.00495 Pa.s recorded the highest pressure drop, whilst both models with pore size 650 µm and viscosity 0.00145 Pa.s recorded the lowest pressure drop. This also indicated that the pressure drop increased when pore size decreased. When comparing both pore shapes, circular fluid model with pore size 250 µm and viscosity 0.00495 Pa.s exerted a higher pressure drop compared to hexagonal model. Moreover, hexagonal model with pore size 650 µm and viscosity 0.00145 Pa.s showed lower pressure drop compared to circular model. Based on the pressure drop value, permeability of both circular and hexagonal fluid model was calculated by using the Darcy formula at different pore sizes, viscosities and inlet velocities as shown in Table 1 and Table 2, respectively.

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Fig. 2. Pressure distribution for circular (a,b,c) and hexagonal (d,e,f) pore shape at 250 µm (a,d), 450 µm (b,e) and 650 µm (c,f) pore size. Table 1. Permeability of circular fluid model Viscosity (Pa.s)

Inlet Velocity (m/s)

Permeability (m2 ) 250 µm

450 µm

650 µm

0.00145

0.0005

1.76E-09

5.11E-09

9.86E-09

0.00145

0.001

1.77E-09

5.16E-09

9.24E-09

0.00145

0.035

1.81E-09

4.83E-09

8.28E-09

0.00255

0.0005

1.77E-09

5.11E-09

9.89E-09

0.00255

0.001

1.77E-09

5.17E-09

9.23E-09

0.00255

0.035

1.83E-09

5.01E-09

8.75E-09

0.005

0.0005

1.78E-09

5.11E-09

9.90E-09

0.005

0.001

1.86E-09

5.17E-09

9.24E-09

0.005

0.035

1.85E-09

5.08E-09

8.98E-09

Figure 3 and 4 shows the relationship between permeability and pore size at different inlet velocities and at 0.00255 Pa.s for circular and hexagonal scaffolds respectively. Generally, when pore size increases while viscosity remains constant, permeability of both circular and hexagonal scaffolds increases linearly from pore size 250 µm to 650 µm. Based on the graphical representation, when both scaffolds have fixed viscosity, both circular and hexagonal scaffolds with pore size 650 µm and velocity 0.0005 m/s recorded the highest permeability whereas scaffolds with pore size 250 µm with inlet velocity 0.035 m/s recorded the smallest permeability. Figure 5 and Fig. 6 shows the relationship between permeability and pore size at different viscosities and at inlet velocity of 0.001 m/s for circular and hexagonal scaffold,

Effect of Fluid Properties on Bone Scaffold Permeability

25

Table 2. Permeability of hexagonal fluid model Viscosity (Pa.s)

Inlet Velocity (m/s)

0.00145

Permeability (m2 ) 250

450

650

0.0005

2.70E-09

6.97E-09

1.17E-08

0.00145

0.001

2.59E-09

6.84E-09

1.10E-08

0.00145

0.035

2.51E-09

6.33E-09

9.80E-09

0.00255

0.0005

2.70E-09

6.98E-09

1.17E-08

0.00255

0.001

2.59E-09

6.86E-09

1.10E-08

0.00255

0.035

2.55E-09

6.59E-09

1.03E-08

0.005

0.0005

2.70E-09

6.97E-09

1.17E-08

0.005

0.001

2.59E-09

6.87E-09

1.10E-08

0.005

0.035

2.57E-09

6.72E-09

1.06E-08

Fig. 3. Permeability of circular scaffold at different pore sizes and inlet velocities.

respectively. Generally, when pore size increased and inlet velocity remained constant, permeability of both circular and hexagonal scaffolds increased from pore size 250 µm to 650 µm. When both scaffolds have same inlet velocity, both the circular and hexagonal scaffolds with pore size 650 µm recorded the highest permeability whereas scaffolds with pore size of 250 µm recorded the smallest permeability. Overall, when comparing between circular and hexagonal scaffolds, the hexagonal scaffold shows a higher permeability compared to circular scaffolds. Table 3 shows the comparison of permeability value between current study and previous study. The calculated permeability of hexagonal scaffold was higher than circular scaffold. The permeability of circular scaffolds was ranging from 1.76E-09 m2 to 9.90E-09 m2 . On the other hand, the hexagonal scaffold permeability was ranging from 2.51E-09 m2 to 1.17E-08 m2 . Therefore, the permeability of hexagonal scaffold was encompassed more

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Fig. 4. Permeability of hexagonal scaffold at different pore sizes and inlet velocities.

Fig. 5. Permeability of circular scaffold at different pore sizes and viscosities.

in the range of upper limit of permeability of natural human bone, which is between 0.5E-08 to 5E-08 [2]. The three-dimensional imaging, flow modality, and numerical stimulation of the scaffold physical property all influence the scaffold’s threshold permeability [19]. In order to induce vascularization and mineralization of scaffolds, an adequate threshold permeability is 3 × 10 − 11 m2 [19]. Thus, the permeability results of both circular and hexagonal scaffolds were higher than the threshold permeability. When designing a scaffold, threshold permeability and range of permeability of natural human bone must take into consideration. Overall, the permeability results of both hexagonal and circular modal were comparable with other findings.

Effect of Fluid Properties on Bone Scaffold Permeability

27

Fig. 6. Permeability versus of hexagonal scaffold at different pore sizes and viscosities.

Table 3. Comparison of permeability value between current study and previous study. Authors

Permeability (m2 )

Mode of study

Minimum

Maximum

Kohles et al. [15]

1.00E-10

1.00E-09

Experimental

Grimm and Williams [16]

4.00E-11

1.10E-08

Experimental

Ma et al. [8]

2.90E-10

3.91E-09

Simulation and Experimental

Beaudoin et al. [17]

4.67E-10

1.48E-08

Simulation

Nauman et al. [18]

2.68E-11

2.00E-08

Experimental

Singh et al. [2]

5.00E-09

5.00E-08

Simulation

Current study (Circular Pore)

1.76E-09

9.90E-09

Simulation

Current study (Hexagonal Pore)

2.51E-09

1.17E-08

Simulation

4 Conclusions In conclusion, the 3D-printed bone scaffold unit cell was designed by using SolidWorks with circular pore and hexagonal pore shape with size of 250 µm, 450 µm and 650 µm. ANSYS Fluid Simulation software was used to study the effects of fluid viscosity, inlet velocity, pore size and pore shape towards the permeability of scaffold. Hexagonal scaffolds demonstrated higher permeability range compared to circular scaffolds. When pore size and inlet velocity increased, the permeability across scaffolds was increased. This indicated that there was a relationship between pore size, inlet velocity and permeability. However, viscosity did not have a significant effect on permeability. Thus, hexagonal scaffold with pore size 650 µm, inlet velocity 0.0005 m/s was the suitable design since it has higher permeability value compared to circular scaffold. The permeability value of hexagonal scaffold is closer to the maximum value of natural bone permeability. Scaffold

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with good permeability allows cell and nutrient inflow as well as waste product transfer via blood vessel system which is critical for successfulness of the long-term implant. Therefore, this study gave new insight on the important of blood fluid properties in designing a bone scaffold. Acknowledgement. This work was supported by Universiti Teknologi Malaysia and Fundamental Research Grant Scheme (FRGS) grant (FRGS/1/2020/STG05/UTM/02/10) from Ministry of Education, Malaysia.

References 1. Wang, C., et al.: 3D printing of bone tissue engineering scaffolds. Bioact. Mater. 5(1), 82–91 (2020) 2. Singh, S.P., Shukla, M., Srivastava, R.K.: Lattice modeling and CFD simulation for prediction of permeability in porous scaffolds. Mater. Today Proc. 5(9), 18879–18886 (2018) 3. Liu, X., et al.: Vascularization of natural and synthetic bone scaffolds. Cell Transplant. 27(8), 1269–1280 (2018) 4. Yan, Y., et al.: Vascularized 3D printed scaffolds for promoting bone regeneration. Biomaterials 190–191, 97–110 (2019). https://doi.org/10.1016/j.biomaterials.2018.10.033 5. Marrella, A., et al.: Engineering vascularized and innervated bone biomaterials for improved skeletal tissue regeneration. Mater. Today 21(4), 362–376 (2018). https://doi.org/10.1016/j. mattod.2017.10.005 6. Abe, Y., Watanabe, M., Chung, S., Kamm, R.D., Tanishita, K., Sudo, R.: Balance of interstitial flow magnitude and vascular endothelial growth factor concentration modulates three-dimensional microvascular network formation. APL Bioeng. 3(3), 036102 (2019) 7. Kaigler, D., Wang, Z., Horger, K., Mooney, D.J., Krebsbach, P.H.: VEGF scaffolds enhance angiogenesis and bone regeneration in irradiated osseous defects. J. Bone Miner. Res. 21(5), 735–744 (2006) 8. Ma, S., et al.: Manufacturability, mechanical properties, mass-transport properties and biocompatibility of triply periodic minimal surface (TPMS) porous scaffolds fabricated by selective laser melting. Mater. Des. 195, 109034 (2020) 9. Dias, M.R., Fernandes, P.R., Guedes, J.M., Hollister, S.J.: Permeability analysis of scaffolds for bone tissue engineering. J. Biomech. 45(6), 938–944 (2012) 10. Lipowiecki, M., et al.: Permeability of rapid prototyped artificial bone scaffold structures. J. Biomed. Mater. Res. Part A 102(11), 4127–4135 (2014) 11. Cameron, R.E., Kamvari-Moghaddam, A.: Synthetic bioresorbable polymers. In: Durability and Reliability of Medical Polymers, pp. 96–118. Elsevier (2012). https://doi.org/10.1533/ 9780857096517.1.96 12. Lu, L., et al.: Mechanical study of polycaprolactone-hydroxyapatite porous scaffolds created by porogen-based solid freeform fabrication method. J. Appl. Biomater. Funct. Mater. 12(3), 145–154 (2014) 13. Klabunde, R.E.: Viscosity of Blood. Cariovascular Physiology Concepts ((2013) 14. Nader, E., et al.: Blood rheology: key parameters, impact on blood flow, role in sickle cell disease and effects of exercise. Front. Physiol. 10, 1–14 (2019) 15. Kohles, S.S., Roberts, J.B., Upton, M.L., Wilson, C.G., Bonassar, L.J., Schlichting, A.L.: Direct perfusion measurements of cancellous bone anisotropic permeability. J. Biomech. 34(9), 1197–1202 (2001)

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16. Grimm, M.J., Williams, J.L.: Measurements of permeability in human calcaneal trabecular bone. J. Biomech. 30(7), 743–745 (1997) 17. Beaudoin, A.J., Mihalko, W.M., Krause, W.R.: Finite element modelling of polymethylmethacrylate flow through cancellous bone. J. Biomech. 24(2), 127131–129136 (1991) 18. Nauman, E.A., Fong, K.E., Keaveny, T.M.: Dependence of intertrabecular perme- ability on flow direction and anatomic site. Ann. Biomed. Eng. 27(4), 517–524 (1999) 19. Prasadh, S., Wong, R.C.W.: Unraveling the mechanical strength of biomaterials used as a bone scaffold in oral and maxillofacial defects. Oral Sci. Int. 15(2), 48–55 (2018). https://doi. org/10.1016/S1348-8643(18)30005-3

Antimicrobial Properties of Nanoporous Hydroxyapatite Doped with Polyphenols Extracted from Euphorbia tirucalli L. (Pokok Tetulang) Alwani Ibrahim1 , Tun Iqmal Haziq Tun Rashdan Arief1 , Nur Farahiyah Mohammad1,2(B) , Nashrul Fazli Mohd Nasir1,2 , Khairul Farihan Kasim3 , Siti Shuhadah Md Saleh3,4 , and Farah Diana Mohd Daud5 1 Faculty of Electronic Engineering and Technology, Universiti Malaysia Perlis, Pauh Putra,

02600 Arau, Perlis, Malaysia [email protected] 2 Medical Device and Life Science Cluster, Sport Engineering Research Centre (SERC), Universiti Malaysia Perlis, Pauh Putra, 02600 Arau, Perlis, Malaysia 3 Faculty of Chemical Engineering and Technology, Universiti Malaysia Perlis, Jejawi 2, 02600 Arau, Perlis, Malaysia 4 Biomedical and Nanotechnology Research Group, Centre of Excellence Geopolymer and Green Technology (CEGeoTech), Universiti Malaysia Perlis, Jejawi, 02600 Arau, Perlis, Malaysia 5 Manufacturing and Materials Engineering Department, Kulliyyah Engineering, International Islamic University Malaysia (IIUM), Jalan Gombak, 53100 Kuala Lumpur, Malaysia

Abstract. Biomaterials such as hydroxyapatite (HA) have been widely studied for their biocompatibility and osteoconductive properties as implant materials in modern medical science. Due to its lack of antimicrobial behavior as pure HA, doping is a necessity to improve its function. This study focused on the synthesis of nanoporous HA doped with polyphenols obtained by methanol extraction from Euphorbia tirucalli L. plant using Soxhlet method. Characterization of HA using FTIR spectrum revealed the absorption of HA functional groups. Meanwhile, FTIR spectra of HA doped with polyphenols proved the successful doping. Phytochemical tests such as TPC and TFC measured 91.05 mg GAE/ml and 71.37 mg QE/ml respectively. A 20 mg/ml extract concentration resulted in 132 mm2 of inhibition growth and 80 mg/ml extract concentration found both 162 mm2 and 120 mm2 of inhibition growth for bacteria densities respectively. The crude extract possesses antioxidant activity and HA doped with polyphenols exhibits antimicrobial properties due to the presence of the polyphenols. Keywords: Biomaterials · Hydroxyapatite · antimicrobial · polyphenols · Euphorbia tirucalli L.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 F. Ibrahim et al. (Eds.): ICIBEL 2022, IFMBE Proceedings 107, pp. 30–37, 2024. https://doi.org/10.1007/978-3-031-56438-3_4

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1 Introduction Hydroxyapatite (HA) is a popular biomaterial due to its biocompatibility and osteoconductive quality [1–4]. Hydroxyapatite is an apatite mineral with a chemical formula of (Ca10 (PO4 )6 (OH)2 ) consisting of a major inorganic constituent, up to 60–70% of the bone matrix [5–7]. However, some drawbacks such as lacking antimicrobial properties occurred for pure HA to be implanted which made this necessary for doping especially plant-derived organic compounds doping instead of metal ions doping due to the cytotoxicity [8]. The association of biomaterials as implant materials can lead to chances of implant-associated infection on their surfaces which creates a crucial requirement for antimicrobial properties studies [9]. Besides, there are less comprehensive scientific studies discussing plant-derived compound doping for HA. These conditions have paved the way for researchers to study particularly antimicrobial properties of doping using bioactive compounds. On the other hand, bioactive constituents in the plant have been proven to possess crucial biological activities such as antimicrobial, anti-inflammatory, antioxidant, antiviral and antidiabetic properties [10–12]. This leads to the broad studies of phytochemicals in plants including Euphorbia tirucalli L. (Pokok Tetulang) as some studies have revealed its pharmacological activities such as antimicrobial and antioxidant [13–15]. Polyphenols content in Euphorbia tirucalli L. extracts possesses a considerable antimicrobial effect and antioxidant properties [16]. In this study, nanoporous hydroxyapatite was synthesized and doped with polyphenols extracted from Euphorbia tirucalli L. using Soxhlet method with 80% methanol as the solvent.

2 Methods 2.1 Materials Euphorbia tirucalli L. (Pokok Tetulang) fresh branch/stem samples from a local village in Kemaman, Terengganu, Malaysia were collected for crude extraction using methanol as the solvent. Precursors for calcium and phosphate which were used for the study were calcium nitrate tetrahydrate (Ca (NO3 )2 .4H2 O) and diammonium hydrogen phosphate ((NH4 )2 .HPO4 ) respectively. Pluronics® P123 as a non-ionic triblock co-polymer was used to guide the construction of the pore template. In addition, sodium hydroxide (NaOH) was used to keep the pH of the mixtures at 11. 2.2 Crude Extraction of Euphorbia tirucalli L. Stems Euphorbia tirucalli L. samples were washed under tap water and then rinsed with distilled water. The samples were dried under sun heat between 4–7 days. Dried grinded powders were obtained by an electrical blender and they were filtered with a plastic sieve to obtain fine powders. Next, Soxhlet extraction method was used to extract the crude from 30 g of fine powders, wrapped in nylon fabric, by using 300 ml of 80% methanol with 1:10 ratio of samples to solvent. The solvents containing the extract were then evaporated by a rotary evaporator for one hour. The crude extract obtained was kept in the refrigerator at 4 °C until further use.

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2.3 Synthesis of HA Powders A non-ionic surfactant, Pluronics® P123 was diluted in 100 ml of distilled water before 9.446 g of Ca (NO3 )2 .4H2 O was added to make the translucent micellar solution. The surfactant-calcium solution was obtained with 30 min of stirring. Subsequently, 3.169 g of (NH4 )2 .HPO4 was dissolved in 60 ml of distilled water becoming a phosphate solution. The phosphate solution was moderately dripped into the surfactant-calcium solution in continuous stirring until forming a milky suspension. Throughout the mixing step, 1M of NaOH was used to maintain the liquid pH at 11 before aging the solution for 24 h at room temperature. The white precipitate was been washed with distilled water and centrifuged five times in 10 min before oven drying for 24 h at 100 °C. Then, a mortar and pestle were used to pulverize them into fine powders and the white powders obtained were calcined for 6 h at 550 °C. This method was selected based on widely used HA powders synthesis process from previous study using wet precipitation method [17]. 2.4 Preparation of HA Pellets Doped with Crude Extract Two different concentrations of crude extracts were prepared by mixing 20 ml (crude extracts) and 80 ml (distilled water) and another 80 ml (crude extracts) and 20 ml (distilled water), 20% and 80% crude extracts concentrations respectively. The samples were named ET20 and ET80 accordingly. 3 g of HA fine powders were then added into each mixture and mixed. Both mixtures were left for the soaking process in the shaker water for 24 h. After filtering, the remaining powders were dried in the desiccator within the next 24 h and ground using mortar and pestle into dried powders. For each mixture concentration, 0.25 g was weighted for producing pellets using a hydraulic press machine with 185 MPa pump pressure setting within 4–6 s. Lastly, the pellet was measured to get 1.6 mm thick and 10 mm diameter dimensions. Processes were repeated for 12 pellets. 2.5 Characterizations of Nanoporous HA Powders FTIR spectra were recorded using an FTIR instrument for the determination of the functional group such as the presence of polyphenols in the samples. 2.6 Phytochemicals Analysis Total phenolic content (TPC) in the extracts was determined using Folin-Ciocalteu calorimetric method. 0.1 ml of the sample was added to 0.2 ml and 8 ml of Folin reagents and distilled water respectively. After mixing well, they were incubated for 3 min. 1 ml of sodium carbonate Na2 CO3 (20%) was then added and the mixtures were left in a dark place for 30 min. The absorbance was measured with UV-VIS spectrophotometer with a wavelength of 765 nm. TPC is expressed in mg Gallic acid equivalent (GAE)/g extract. Total flavonoid content (TFC) in Euphorbia tirucalli L. extracts was measured using aluminium chloride colorimetric method. 0.5 ml of the extracts was added into 2 ml of distilled water. 0.15 ml of NaNO3 (50%) was then added into the mixture followed by 5 min of incubation. Then, 0.15 ml of aluminium chloride (AlCl3) was added and incubated

Antimicrobial Properties of Nanoporous Hydroxyapatite Doped

33

for another 15 min. The absorbance was measured at 415 nm, TFC was expressed in mg which is equivalent to quercetin per gram of extract. The antioxidant activity test of the crude extracts was carried out using 2, 2-diphenyl1-picrylhydrazyl (DPPH) method. 60 µM of DPPH solution in ethanol was prepared. 1 ml of extract for different concentrations (0.1, 0.5, 0.625, 1.25, 2.5, 3.5, and 5 mg/ml) were dissolved in 0.1 ml of 95% ethanol. 200 µl of extracts were added to 2500 µl of 60 µM of DPPH solution before incubating in the dark for 30 min. For control, 200 µl of 80% methanol was added with 2500 µl of 60 µM of DPPH and incubated in dark for 30 min at room temperature. All samples measured the absorbance at 517 nm in a spectrophotometer. 2.7 Antimicrobial Activity Agar disc diffusion method was carried out to test the antimicrobial activity of HA pellets against bacteria. Initially, the general procedures such as medium preparation, bacteria cultivation and inoculum of bacteria were employed before the test was done. The media used for culturing the organism was nutrient agar (NA) for Staphylococcus aureus (S.aureus). It was prepared by dissolving 10 g of NA powder into 500 ml of distilled water and autoclaved at 121 °C for 20 min. NA was allowed to cool to room temperature before dispensing into petri dishes. S.aureus provided from Biomaterial Laboratory in UniMAP were subcultured on a sterilized nutrient agar plate and incubated at 37 °C for 24 h. They were then stored in the refrigerator at 4 °C for maintaining stock culture. S.aureus which has been harvested was micro pipetted into the test tube after 1–2 ml of distilled water was dropped into the subculture plate. The concentration of bacteria went for serial dilution to reach approximately 1.5 × 10 CFU/ml of inoculum. After comparing with McFarland turbidity standard (0.5 McFarland turbidity standard), the concentration was adjusted to 2 × 10 CFU/ml. Then, 100 µL of adjusted S.aureus suspension was spread onto the surface of the agar plate using a cotton swab. Minimum Inhibitory Concentration (MIC) was used for determining the antimicrobial activity of HA pellets against bacteria. Utilized nutrient agar from the petri dishes was poured into another petri dish in the range of 4.0 mm depth and HA pellets were immersed immediately before the agar hardened. The petri dish was kept capped and labeled and incubated at 37 °C for 24 h before the zone of inhibition was observed. In addition, 10 µL of 50 mg/ml of antibiotics were added into a well for positive control. As for negative control, two petri dishes were set for both 1:1000 and 1:100 S.aureus density. All samples of 20% and 80% mixed doped extracts were prepared separately.

3 Results and Discussion 3.1 Characterization of Nanoporous HA FTIR analysis referred in Figure 1 (a) determined the functional group present in HA. The characteristic bands for nanoporous HA were present in the samples. The stretching vibration (V3) of phosphate groups in HA presented at absorption peaks 1089.6 cm−1 ,

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1022.1 cm−1 , 599.8 cm−1 and 561.7 cm−1 . Meanwhile, absorption peaks at 1414.1 cm−1 and 3574.9 cm−1 were attributed to carbonate groups’ vibration and stretching of the hydroxyl groups of HA respectively. All three functional groups of HA were present proving HA was successfully synthesized. FTIR analysis for HA doped with polyphenols of the crude extract was carried out to confirm the presence of the functional groups consistently. Figure 1 (b) shows the spectrum of the characteristic bands for polyphenols doping of nanoporous HA. The bands appeared at wavelength = 1089 cm−1 , 1088 cm−1 , and 963 cm−1 are the characteristics for the CH functional group. This FTIR spectrum between 1089 cm−1 to 963 cm−1 represents the molecular fingerprint of the aromatic C-H stretching related to phenolic compounds which shows the doping of polyphenols into HA [18].

Fig. 1. (a). FTIR spectra of HA (b). FTIR spectra of HA doped with polyphenols with 20% (ET20) and 80% (ET80) of Euphorbia tirucalli L. extracts.

3.2 Phytochemical Analysis Euphorbia tirucalli L. extracted by 80% methanol solvent using the Soxhlet method produced a percentage yield of 50.12%. From the crude extracts, some phytochemical tests were carried out to determine total phenolic, total flavonoid and antioxidant activity. Total phenolic content (TPC) was determined using Folin-Ciocalteu method in gallic acid equivalent (mgGAE/g) of dry matter. Standard curve equation (y = 0.5194x–0.0695, R2 = 0.9999) showed that absorbance increases directly proportional to extract solution concentration. It was found that TPC in the crude was 91.05 mg GAE/ml. Meanwhile, the aluminum chloride complex formation assay which determined the total flavonoid content (TFC) of the extracts produced 71.37 mg QE/ml with quercetin as the standard. The same graph pattern was determined (standard curve equation: y = 0.8637x + 0.0071, R2 = 0.9992). This study revealed that Euphorbia tirucalli L. methanol extraction with Soxhlet method extracted higher TPC than TFC with a slight difference. The antioxidant activity of the crude extracts using 80% methanol extraction was tested with DPPH assay. DPPH scavenging activity of Euphorbia tirucalli L. extracts recorded the antioxidant properties having the potential to scavenge the DPPH free radicals. Table 1 demonstrates the DPPH scavenging activity of different Euphorbia

Antimicrobial Properties of Nanoporous Hydroxyapatite Doped

35

tirucalli L. extract concentrations using Soxhlet method. Scavenging activity percentage possesses an increasing trend with higher extract concentration. This can be explained by the antioxidant properties of polyphenols extracted from crude extracts as polyphenols are rich in antioxidants [19]. Table 1. DPPH scavenging activity of different Euphorbia tirucalli L. extract concentrations. Extract concentration (mg/ml) DPPH scavenging activity (%) 5

90.1852

3.5

89.2592

2.5

88.6111

1.25

76.2962

0.625

47.4074

0.5

35.8333

3.3 Antimicrobial Activity The agar disc diffusion method was carried out to test the antibacterial activity of HA doped with polyphenols extracted from Euphorbia tirucalli L. stems against S.aureus bacteria. Different concentrations of crude extract and different densities of S.aureus were recorded for antimicrobial properties. Refering to Table 2, this study revealed that 20 mg/ml extract concentration measured the inhibition growth of 132 mm2 tested with 104 of bacteria density. Meanwhile, 80 mg/ml extract concentration resulted in the inhibition growth of 162 mm2 and 120 mm2 for both density of bacteria 104 and 108 respectively. The methanolic extract was found to be effective for antimicrobial properties toward S.aureus. The phenolic compound was found to assist in breaking the cell wall of the bacteria [20]. Both extract concentration from the crude has been recorded to contain phenolic compounds and flavonoid which explained the presence of antimicrobial activity. Antimicrobial activity can be ascribed to the behavior of polyphenols [21].

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Table 2. Antibacterial activity (Inhibition growth) of various extract concentrations of Euphorbia tirucalli L. Extract concentration (mg/ml) 20

Density of bacteria (104)

80

132

Inhibition growth (mm2) -

162

120

Inhibition growth (mm2)

Density of bacteria (108)

4 Conclusion Nanoporous HA doped with polyphenols extracted from Euphorbia tirucalli L. using Soxhlet method was well synthesized. The FTIR spectra revealed the absorption bands’ presence of all HA functional groups. Phytochemical tests such as TPC, TFC and antioxidant activity of the crude revealed the presence of polyphenols compounds. Higher concentration of extracts recorded higher DPPH scavenging activity. HA doped with polyphenols showed a good antimicrobial property when tested with S.aureus bacteria. Acknowledgements. The authors would like to thank the Ministry of Higher Education Malaysia for the support from the Fundamental Research Grant Scheme (FRGS) under a grant number of FRGS/1/2021/TK0/UNIMAP/02/59 and the Universiti Malaysia Perlis for the Materials Fund (RESMATE) under a grant number of 9001-00626.

References 1. Raimi Razali, K., Fazli Mohd Nasir, N., Ee Meng, C.: Preliminary analysis of nHA based tissue engineering scaffold dielectric characteristics relative permittivity and permeability measurements for materials using microwave techniques view project dielectric measurement view project. ARPN J. Eng. Appl. Sci. 11, 4987–4990 (2016) 2. Mohammad, N.F., Muhammed, M.H., Zakaria, Z., Abdullah, A.A., Mohammad, I.S.: Characterization of calcium phosphate bioceramic from Paphia Undulata shells. In: 2012 International Conference on Biomedical Engineering (ICoBE) (2012) 3. Mohammad, N.F., Othman, R., Yeoh, F.Y.: Pore characteristics of mesoporous carbonated hydroxyapatite synthesized with different nonionic surfactant and carbonate concentration. Mater. Sci. Forum, 353–360 (2015)

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4. Mohammad, N.F., Zahid, M.A., Awang, S.A., Zakaria, Z., Abdullah, A.A.: Synthesis and characterization of bioceramic from Malaysian cockle shell. In: 2010 IEEE Symposium on Industrial Electronics and Applications (ISIEA 2010), pp. 413–416. Penang, Malaysia (2010) 5. Fiume, E., Magnaterra, G., Rahdar, A., Verné, E., Baino, F.: Hydroxyapatite for biomedical applications: a short overview. Ceramics 4, 542–563 (2021). https://doi.org/10.3390/cerami cs4040039 6. Razali, K.R., et al.: The effect of gelatin and hydroxyapatite ratios on the scaffolds’ porosity and mechanical properties. In: IEEE Conference on Biomedical Engineering and Sciences, pp. 256–259. Miri, Sarawak, Malaysia (2014) 7. Mohd Roslan, M.R., et al.: The state of starch/hydroxyapatite composite scaffold in bone tissue engineering with consideration for dielectric measurement as an alternative characterization technique (2021) 8. Ciobanu, C.S., Massuyeau, F., Constantin, L.V., Predoi, D.: Structural and physical properties of antibacterial AG-doped nano-hydroxyapatite synthesized at 100 °C. Nanoscale Res. Lett. 6, 1–8 (2011). https://doi.org/10.1186/1556-276X-6-613 9. Mirzaee, M., Vaezi, M., Palizdar, Y.: Synthesis and characterization of silver doped hydroxyapatite nanocomposite coatings and evaluation of their antibacterial and corrosion resistance properties in simulated body fluid. Mater. Sci. Eng. C 69, 675–684 (2016). https://doi.org/10. 1016/j.msec.2016.07.057 10. Seo, D.J., Choi, C.: Antiviral bioactive compounds of mushrooms and their antiviral mechanisms: a review (2021) 11. Altemimi, A., Lakhssassi, N., Baharlouei, A., Watson, D.G., Lightfoot, D.A.: Phytochemicals: extraction, isolation, and identification of bioactive compounds from plant extracts. Plants 6 (2017). https://doi.org/10.3390/plants6040042 12. Tran, N., Pham, B., Le, L.: Bioactive compounds in anti-diabetic plants: from herbal medicine to modern drug discovery (2020) 13. Jahan, N., Rehman, K.U., Ali, S., Bhatti, I.A.: Antimicrobial potential of Gemmo-modified extracts of Terminalia Arjuna and Euphorbia tirucalli. Int. J. Agric. Biol. 13, 1001–1005 (2011) 14. Upadhyay, B., Singh, K.P., Kumar Biotechnology, A.: Ethno-medicinal, phytochemical and antimicrobial studies of Euphorbia tirucalli L. J. Phytol. 2, 65–77 (2010) 15. de Araújo, K.M., et al.: Identification of phenolic compounds and evaluation of antioxidant and antimicrobial properties of Euphorbia tirucalli L. Antioxidants 3, 159–175 (2014). https:// doi.org/10.3390/antiox3010159 16. Munro, B., Vuong, Q.V., Chalmers, A.C., Goldsmith, C.D., Bowyer, M.C., Scarlett, C.J.: Phytochemical, antioxidant and anti-cancer properties of Euphorbia tirucalli methanolic and aqueous extracts. Antioxidants 4, 647–661 (2015). https://doi.org/10.3390/antiox4040647 17. Mohd Pu’ad, N.A.S., Abdul Haq, R.H., Mohd Noh, H., Abdullah, H.Z., Idris, M.I., Lee, T.C.: Synthesis method of hydroxyapatite: a review. In: Materials Today: Proceedings. pp. 233–239. Elsevier Ltd (2019) 18. Lucarini, M., et al.: Grape seeds: chromatographic profile of fatty acids and phenolic compounds and qualitative analysis by FTIR-ATR spectroscopy. Foods 9 (2020). https://doi.org/ 10.3390/foods9010010 19. Abbas, M., et al.: Natural polyphenols: an overview. Int. J. Food Prop. 20, 1689–1699 (2017). https://doi.org/10.1080/10942912.2016.1220393 20. Oliveira, R.N., et al.: FTIR analysis and quantification of phenols and flavonoids of five commercially available plants extracts used in wound healing. Revista Mater. 21 767–779 (2016). https://doi.org/10.1590/S1517-707620160003.0072 21. Alshuniaber, M.A., Krishnamoorthy, R., AlQhtani, W.H.: Antimicrobial activity of polyphenolic compounds from Spirulina against food-borne bacterial pathogens. Saudi J. Biol. Sci. 28, 459–464 (2021). https://doi.org/10.1016/j.sjbs.2020.10.029

Fundamental Research for Leg Band Type Wearable Electrocardiogram Monitor-Examination of Electrode Position and Construction of Basic System Reina Kobayashi(B) and Akinori Ueno Tokyo Denki University, Tokyo 120-8551, Japan [email protected], [email protected]

Abstract. With a view to future development of wearable leg-band device for daytime electrocardiogram (ECG) monitoring, we explored viable electrode configurations on the surface of the left thigh for three human subjects. A configuration composed of outer two segments in the top and the third stages for negative and positive electrodes, and of inner one segment in the second stage for indifferent electrode (we call this configuration as the site_c1 c3 f2 ) were compared with another configuration composed of outer two segments in the second and forth stages for negative and positive electrodes, and of inner one segment in the third stage for indifferent electrode (we call this as the site_c2 c4 f3 ) in terms of signal quality of measured ECG. By virtue of a self-produced bioamplifier with high common mode rejection (>90 dB), mean detection rate of sensitivity, accuracy and positive predictive value of ECG R wave during rest were 84.3%, 79.6% and 88.3%, respectively, for the site_c1 c3 f2 . Whereas, those for the site_c2 c4 f3 were 5.2%, 2.1% and 4.1%, respectively. In the use of the site_c1 c3 f2 and a constructed interface circuit, left-thigh ECG were wirelessly transmitted and successfully displayed on a tablet computer. Keywords: Electrocardiogram · Wearable ECG Monitor · Single Leg Lead

1 Introduction Ischemic heart disease (IHD) is the leading cause of death worldwide [1], and can coexist arrhythmia [2]. Since electrocardiogram (ECG) is available for detection of IHD as well as less-frequently occurring arrythmia, wearable devices for long term ECG monitoring are considered useful. Smartwatch, such as Apple Watch 4 (Apple, Cupertino, USA), is rational and feasible device with wide user’s acceptance, however, there exist a drawback due to a requirement for bimanual contact to the watch for the ECG measurement. In view of extending measurable time length for ECG monitoring and increasing opportunities for arrythmia detection, re-examination of fixing site for wearable devices seems meaningful for ECG monitoring with both hands free. One of promising approaches without the aforementioned drawback may be that leverages © The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 F. Ibrahim et al. (Eds.): ICIBEL 2022, IFMBE Proceedings 107, pp. 38–45, 2024. https://doi.org/10.1007/978-3-031-56438-3_5

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armband device [3]. However, the armband device can be vulnerable to arm and/or hand motion during the tasks. Therefore, we focused on the leg, particularly the left thigh considering human cardiac vector, with a view to future application to thigh-band device. Then, we explored viable electrode configurations on the surface of the left thigh.

2 Materials and Methods 2.1 Segmentation of the Left Thigh Surface for Electrode Placement As shown in the Fig. 1(a), we divided the surface of the left thigh according to the following procedure: (i) We asked the subject to stand in a natural position with their feet shoulder-width apart, (ii) To set the top horizontal line as shown in the Fig. 1(a), we taped the thigh horizontally to the floor so that the tape passed through the crotch, (iii) To set the bottom horizontal line, we taped the thigh horizontally to the floor so that the tape passed through the top of the patella, (iv) To set the center horizontal line, we taped the thigh horizontally to the floor between the upper and lower lines. (v) To quarter the surface of the left thigh into four rows, we taped the thigh horizontally between the top and the center lines, and between the center and the bottom lines, respectively, (vi) We taped longitudinally the thigh so as to connect between the head of the fibula and the anterior superior iliac spine, (vii) We taped longitudinally the thigh so that the five horizontal lines (i.e. circumferences of the thigh) were divided into six equal lengths as can be seen in Fig. 1(b).

(a) Example photographs of the segmentation

(b) Top view cross-section diagram

Fig. 1. Segmentation of the left thigh surface for electrode placement.

2.2 Constructed System for Measuring Electrocardiographic Potential We constructed experimental setup for simultaneous measurements of the left thigh ECG (ECGL-thigh ) and reference ECG (ECGref ), as shown in Fig. 2. The reference lead II ECG

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was measured with a commercial electrocardiograph (BIOPAC Systems, BN-ECG2), whereas ECGL-thigh was measured with a self-produced bioamplifier in Fig. 3. The selfproduced bioamplifier includes a fully-differential analogue front end which exerted high input impedance and high common-mode rejection ratio (CMRR) [4, 5]. The gain and passband of the bioamplifier were set respectively to 56 dB and to 0.7–70 Hz, as shown in Fig. 4(a). The CMRR of the bioamplifier exceeded 90 dB in the passband (see Fig. 4(b)).

Fig. 2. Experimental setup for simultaneous measurements of ECGL-thigh and ECGref , and labels for divided segments on the thigh surface for electrode placement.

Fig. 3. Circuit diagram of the self-produced bioamplifier.

ECGL-thigh signal was not only digitized with a commercial A/D converter, but also transmitted wirelessly to a tablet computer via an interface circuit and a wireless transmission circuit in Fig. 5. The interface circuit adjusted the amplitude and DC

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(a) Gain

41

(b) CMRR

Fig. 4. Frequency characteristics of the self-produced bioamplifier.

Fig. 5. Circuit diagram of the interface circuit and the wireless transmission circuit.

bias of ECGL-thigh signal. The wireless transmission circuit consisted of a microcontroller (Microchip, PIC24EP256MC202-I/SP) and a Bluetooth® module (Microchip, RN42XVP-I/RM). The microcontroller sampled the adjusted ECGL-thigh signal at 500 Hz, then communicated with a USART in the Bluetooth® module according to the flow in Fig. 6. The tablet computer equipped with another Bluetooth® module received the transmitted ECG data and displayed its waveform according to the flow in Fig. 7. 2.3 Experimental Methods ECGL-thigh measurement was conducted for three adults in a supine position. All subjects provided informed consent prior to participating in the experiment. We conducted a preliminary experiment by choosing 3 of 24 segments in Fig. 1(a), then selected two contrasting configurations. One is the configuration composed of outer two segments in the top and the third stages for negative and positive electrodes, and of inner one segment in the second stage for indifferent electrode (we call this configuration as the site_c1 c3 f2 ). The other is that composed of outer two segments in the second and forth stages for negative and positive electrodes, and of inner one segment in the third stage for indifferent electrode (we call this as the site_c2 c4 f3 ). In the qualified experiment, five minutes measurement was conducted for each site in the order.

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Fig. 6. Flowchart for wireless transmission in the wireless transmission circuit.

Fig. 7. Flowchart of data receiving and wave displaying program in the tablet computer.

2.4 Methods of Analysis Quality of the obtained ECGL-thigh signal was analyzed in terms of detection rate of R wave in the middle 1-min recording by comparing R wave of ECGref signal [6]. As preprocessing, moving average and bandpass filtering were performed. Possible candidates of R wave were detected by a threshold method. When no corresponding R wave exists within a timing of the reference R wave ±30 ms, we deemed R wave of ECGL-thigh was “undetected”. Then, when the difference between reference R-R interval (RRI) and left thigh RRI was ≤4 ms, the latter R wave of ECGL-thigh was deemed as “correctly detected”. When the difference between the reference RRI and the left thigh RRI was >4 ms, the latter R wave of ECGL-thigh was deemed as “falsely detected”. After counting

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the number of “correctly detected” (N TP ), “falsely detected” (N FP ) and “undetected” (N FN ), detection rates of sensitivity (PSNS ), accuracy (PACC ) and positive predictive value (PPPV ) were calculated using the Eq. (1), (2) and (3), respectively. N TN was set to zero because it did not exist in the present analysis method [7]. In addition, mean peak-to-peak amplitude of the filtered R wave was computed. NTP × 100 [%] NTP + NFN NTP + NTN = × 100 [%] NTP + NTN + NFN + NFP NTP PPPV = × 100 [%] NTP + NFP PSNS =

PACC

(1) (2) (3)

3 Experimental Results Figure 8(a) shows example recordings of ECGL-thigh from the site c1 c3 f2 and of simultaneously measured ECGref . In the recording of ECGL-thigh , periodic spikes were observed and synchronized with the R wave of ECGref . Therefore, the recording from the site c1 c3 f2 is deemed to be electrocardiographic signal. Figure 8(b) is a screenshot of the tablet computer displaying wirelessly transmitted ECGL-thigh . By comparing waveforms in the screenshot and in the dotted rectangle in Fig. 8(a), we could confirm that the measured ECGL-thigh was successfully digitized and transmitted wirelessly to the tablet computer using the constructed system. As contrasted to Fig. 8(a), no synchronized spikes were observed in the recording from the site c2 c4 f3 in Fig. 8(c).

(a) Simultaneous recording with reference (Site_c1c3f2)

(b) Tablet computer display (Site_c1c3f2)

(c) Simultaneous recording with reference (Site_c2c4f3)

Fig. 8. Electrocardiogram measured from the left thigh of Subject #C.

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Fig. 9. Mean amplitude and standard deviation of the electrocardiographic R wave of the left thigh at each site.

Table 1. Sensitivity (PSNS ), accuracy (PACC ), positive predictive value (PPPV ) for the R wave of ECGL-thigh at electrode sites of c1 c3 f2 and c2 c4 f3 . Subject ID

PSNS [%] c1 c3 f2

c2c4f3

PACC [%]

PPPV [%]

c1 c3 f2

c1c3f2

c2 c4 f3

N TP + N FN c2c4f3

c1 c3 f2

c2c4f3

#A

97.3

5.3

94.7

3.4

97.3

8.6

73

71

#B

100.0

6.1

100.0

1.7

100.0

2.2

68

66

#C

55.7

4.1

44.0

1.1

67.7

1.5

79

73

Mean

84.3

5.2

79.6

2.1

88.3

4.1

73.3

70.0

Figure 9 shows comparison of mean R-wave amplitude between the sites c1 c3 f2 and c2 c4 f3 . The amplitude from the site c1 c3 f2 was significantly higher than that from the site c2 c4 f3 in all subjects (p < 0.001 by Wilcoxon rank-sum test). As can be seen in Table 1, all of detection rates of R wave indicated markedly higher value when the signal was measured from the site c1 c3 f2 than from the site c2 c4 f3 in all subjects.

4 Discussions As can be seen in Fig. 8(a), (c), and Table 1, the use of the electrode site c1 c3 f2 showed superior detectability of the R wave to the site c2 c4 f3 . These sites were selected in this study based on the results of preliminary experiments, in which thorough measurements were performed for a single subject using the upper sites of c1 c3 a2 , c1 c3 b2 , c1 c3 c2 , c1 c3 d2 , c1 c3 e2 , c1 c3 f2 and lower sites of c2 c4 a3 , c2 c4 b3 , c2 c4 c3 , c2 c4 d3 , c2 c4 e3 , c2 c4 f3 , respectively. The contrastive results in this study agreed with the preliminary results. Compared with the input referred amplitude the R waves of the conventional ECG in Fig. 8(a), that of ECGL-thigh was apparently smaller even in the use of the electrode site c1 c3 f2 .This successful detection from the thigh is attribute to the employment of the recently proposed analogue front end [5] and the instrumentation amplifier with

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high CMRR (>90 dB), which is higher than standard regulation (60 dB) for general electrocardiographs [8].

5 Conclusion In this study, we divided surface area of the left thigh and explored viable electrode configuration for the measurement of ECG R wave. The use of the electrode site c1 c3 f2 showed markedly higher detection ratio (PSNS = 84.3%, PACC = 79.6%, PPPV = 88.3%) than the use of the site c2 c4 f3 (PSNS = 5.2%, PACC = 2.1%, PPPV = 4.1%). By leveraging self-produced bioamplifier and interface circuit with a Bluetooth® module, left-thigh ECG was wirelessly transmitted and successfully displayed on a tablet computer.

References 1. The top 10 causes of death. https://www.who.int/en/news-room/fact-sheets/detail/the-top-10causes-of-death. Accessed 15 Sept 2022 2. National Cerebral and Cardiovascular Center | Knowledge about the Disease | Ischemic Heart Disease. https://www.ncvc.go.jp/hospital/pub/knowledge/disease/ischemic-heart-dis ease/. Accessed 30 Sept 2002. In Japanese 3. Rachim, V.P., Chung, W.: Wearable noncontact armband for mobile ECG monitoring system. IEEE Trans. Biomed. Circuits Syst. 10(6), 1112–1118 (2016) 4. Aihara, T., Ueno, A.: Physical model-based study on non-contact electrocardiogram measurement of low birth weight infants in incubators through a cloth. In: 13th Biomedical Engineering International Conference (BMEiCON) (2021) 5. Nakamura, H., Sakajiri, Y., Ishigami, H., Ueno, A.: A novel analog front end with voltagedependent input impedance and bandpass amplification for capacitive biopotential measurements. Sensors 20(9), 2476 (2020) 6. Takano, A., Ishigami, H., Ueno, A.: Non-contact measurements of electrocardiogram and cough-associated electromyogram from the neck using in-pillow common cloth electrodes: a proof-of-concept study. Sensors 21(3), 812 (2021) 7. Lee, J.S., Heo, J., Lee, W.K., Lim, Y.G., Kim, Y.H., Park, K.S.: Flexible capacitive electrodes for minimizing motion artifacts in ambulatory electrocardiograms. Sensors 14(8), 14732–14743 (2014) 8. Medical electrical equipment - Part 2-47: Particular requirements for the basic safety and essential performance of ambulatory electrocardiographic systems JIST60601-2-47, p. 24 (2018). http://www.kikakurui.com/t6/T60601-2-47-2018-01.html. Accessed 27 Oct 2022. In Japanese

Glutathione Precursors Supplementation Effects on Renal Function, Lipid Profile and Body Composition Nur Rasyidah Hasan Basri1,2 , Mas Sahidayana Mohktar1,2(B) , Wan Safwani Wan Kamarul Zaman1,2 , and Selvam Rengasamy3 1 Department of Biomedical Engineering, Faculty of Engineering, University of Malaya, 50603

Kuala Lumpur, Malaysia [email protected] 2 Center for Innovation in Medical Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia 3 Chakra We Care Resources Sdn. Bhd., A-G-15, Pusat Perniagaan Metro, Metro Square, Jalan PJS 2B/3, 46150 Petaling Jaya, Selangor, Malaysia

Abstract. Glutathione (GSH) helps to reduce the production of oxidative stress in our body caused by metabolic syndrome (MS) condition. While oral GSH is bioavailable in laboratory animal models, its efficacy effect in humans has vast opportunities to be explored. We aim to study the significance of GSH precursor’s oral supplement on volunteers for 8 weeks of daily consumption. The dosage differential of GSH precursor’s oral supplements on human blood GSH levels is also investigated. The study was conducted on 300 volunteers from Kuala Lumpur and Petaling Jaya, Malaysia. Volunteers were randomized into control and intervention groups; Group 1 (consumed 1.6 g of Immune Formulation 200®) and Group 2 (consumed 3.2 g of Immune Formulation 200®). Blood samples were collected for GSH analysis, renal function, and lipid profile. A bio-impedance analysis is conducted for body composition assessment. Statistical analysis was performed to evaluate the significant difference in the aforementioned blood component between groups. Results showed a significant increment in total GSH level after consuming oral GSH precursors supplementation, especially for subjects in Group 1. Significant changes were observed in some of the renal function parameters namely, creatinine for subjects in group 1 and sodium, carbon dioxide and creatinine for group 2 subjects. Subjects who consumed two doses of oral Immune Formulation 200® for eight weeks show substantial changes in their total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterols levels. Meanwhile, no changes were observed in body composition parameters for both group. Keywords: Glutathione precursors · Supplementation · Renal Functions · Lipid Profile · Body Composition

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 F. Ibrahim et al. (Eds.): ICIBEL 2022, IFMBE Proceedings 107, pp. 46–56, 2024. https://doi.org/10.1007/978-3-031-56438-3_6

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1 Introduction Glutathione (GSH) is synthesized from glutamate, cysteine, and glycine and the process is catalyzed sequentially by two cytosolic enzymes, c-glutamylcysteine synthetase and GSH synthetase. Studies have examined the impact of the supplementation of glutathione (GSH), γ-l-glutamyl-l-cysteinyl-glycine, on human blood GSH levels. A study shows that GSH levels in the blood of 54 non-smoking adults increased after six months versus baseline [1]. Another study, by Park, Shimura et al. (2014) on healthy human volunteers who were orally supplemented with GSH showed the GSH contents in the protein-bound fraction of plasma significantly increased from 60 to 120 min after GSH supplementation [2]. In glutathione-deficient patients with advanced HIV infection, short-term oral supplementation with whey proteins increases plasma glutathione levels [3]. The results suggest that oral and transdermal glutathione supplementation may have some benefit in improving some of the transsulfuration metabolites in subjects diagnosed with an autism spectrum disorder [4]. The GSH precursor supplement tested in the current study contains three amino acids that are needed for GSH synthesis to happen; cystine replacing cysteine, glutamine, and glycine. L-cysteine is reduced from L-cystine in the cells, and it is one of the rate-limiting precursor amino acids for GSH synthesis. However, no significant changes were observed in biomarkers of oxidative stress, including glutathione status, in a clinical trial of oral glutathione supplementation in 40 healthy adults who took the oral supplement for 4 weeks [5]. To assess the feasibility of supplementing oral glutathione, a study by Witschi et al. (1992) was reported to determine the systemic availability of glutathione in seven healthy volunteers. The study found that it was not possible to increase circulating glutathione to a clinically beneficial threshold by the oral administration of only a single dose of 3 g of glutathione [6]. Therefore, the study aims to observe the effects of GSH oral supplementation on renal functions, lipid profile and body composition parameters.

2 Materials and Methods 2.1 Data Collections Three hundred (300) volunteer subjects were recruited from Malaysia. The subject’s inclusion criteria are body mass index (BMI) ≥24 and age from 18–65 years old. However, volunteers with chronic illnesses or pregnant/lactating mothers were excluded. Volunteers visited the clinic assigned for data collection and a written consent form was obtained. The study protocol was approved by the University of Malaya Research Ethics Committee with reference number: UM.TNC2/RC/H&E/UMREC – 152. Most of the subjects who volunteered for this study are between 30–39 years of age which are 35% of the total participants. While 30% of the subjects are between 20–29 years old. There was 24% of subjects whose ages were around 40–49 years old, and 11% were 50–59 years old. Out of all subjects who participated in the study, 34% of them were male, and the remaining were female.

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2.2 Supplementation Volunteer subjects were randomized into a control group and two intervention groups that consumed the supplement. The Control group consisted of volunteers who proceeded with their routine lifestyle and came for measurements for the baseline and 8 weeks from the first measurement. While other two intervention groups were scheduled to come for baseline measurement and once a four weeks assessment during the supplementation period. The first group of supplementations (Group 1) were assigned to consume 1.6 g of Immune Formulation 200® supplement while the second group (Group 2) with doses of 3.2 g supplement of Immune Formulation 200® supplement daily. The supplementation period took 8 weeks to complete for each subject. Every 4 weeks, the intervention group’s subjects are required to come for assessment. 2.3 Blood Withdrawal for GSH Analysis and Blood Test Approximate of 15 ml blood was withdrawn from each subject for GSH analysis, haematology and lipid profile test. 3 ml blood was collected in purple, grey and yellow TUD blood tube (with gel and clot activator) each was sent to the Clinical Haematology Laboratory at University Malaya Medical Centre (UMMC) for tests. The blood withdrawal was carried out by professional medical staff with the help of the research assistants. The flow of the procedure is shown in Fig. 1. Sample preparation for GSH analysis was conducted in the laboratory. Another 5 ml of blood was collected in a plain blood collection tube with a clot activator and was centrifuged at 3000 rpm for about 15 min to get the plasma serum. The plasma serum was pipetted into a 1.5 ml microcentrifuge tube PP (Tarsons Products Pte. Ltd., Kolkata, India) and stored in a 2–8 °C freezer before further GSH assay analysis. GSH analysis and calculation were carried out according to the manufacturer’s recommended protocol (Sigma-Aldrich (M) Pte Ltd, Kuala Lumpur, Malaysia). 2.4 Body Composition Measurements Bio-impedance analysis (BIA) was conducted to measure the body composition of the subjects. Using the BODYSTAT QuadScan 4000 device, subjects were required to lie down in the supine position, electrodes were placed on the right hand and their foot was connected to the device by wires. The anthropometric measurements such as the subject’s weight, height, and waist circumference were entered then proceeded with measurement. The results displayed on the device screen were recorded. 2.5 Parameters The renal functions, lipid profile and body composition parameters are tabulated in Table 1, together with the normal reference range and the units.

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.

.

Fig. 1. Flow chart of blood investigation analysis. Table 1. Normal reference range of Renal Functions, Lipid Profiles and Body Composition Parameters Parameters

Normal Ref range

Renal Function Glucose, μmol/L

800ppm) due to lack of ventilation (see Fig. 7 (b)). The result in 7(a) shows that the lowest sleep quality was in Case 4. Although there is a very small correlation between CO2 concentration and the percentage of deep sleep as shown in Fig. 7(c), Case 5 shows a 42% improvement of deep sleep compared to Case 4. The finding agrees with the statistical t-test result that shows the difference is significant at P < 0.05. This demonstrates that the CO2 level in the bedrooms may affect the sleep quality of the participants, supported by other parameters such as type of aircon, temperature and air circulation. Other than that, this study also reveals the improvement of deep sleep percentage in inverter air-conditioning compared to non-inverter air-conditioning by 17.8% (P < 0.05), ceiling fan control ON compared to ceiling fan turn OFF by 26.3% (P < 0.05) and aircon set temperature of 26 °C compared to 24 °C by 19.5% (P < 0.05).

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(a)

(b)

(c)

Fig. 7. (a) Percentage of deep sleep by cases, (b) CO2 concentration (ppm) by cases, (c) Correlation of percentage of deep sleep and CO2 concentration

4 Conclusion This paper demonstrates that the type of aircon, aircon set temperature, air circulation, and CO2 concentration may affect the quality of sleep. It was found that Case 5 experienced the best sleep quality using inverter air-conditioning at a set temperature of 26 °C with both ceiling fan control and ventilator turned on. Inverter provides better sleep quality due to its ability to control temperature and humidity better than Non-inverter aircon. The different conditions tested in this study can be a reference sleep condition to increase sleep quality, especially for those who experience difficulty in sleeping.

References 1. Dobing, S., Frolova, N., Mcalister, F., Ringrose, J.: Sleep quality and factors influencing self-reported sleep duration and quality in the general internal medicine inpatient population (2016). https://doi.org/10.1371/journal.pone.0156735 2. Emer, P.D., et al.: F35 sleep monitoring in Huntington’s disease using Fitbit compared to polysomnography. J. Neurol., Neurosurg. Psychiatry 92(1) (2021) 3. Lan, L., Tsuzuki, K., Liu, Y.F., Lian, Z.W.: Thermal environment and sleep quality: a review. Energy Build. 149, 101–113 (2017) 4. Moreno-Pino, F., Porras-Segovia, A., López-Esteban, P., Artés, A., Baca-García, E.: Validation of Fitbit Charge 2 and Fitbit Alta HR against polysomnography for assessing sleep in adults with obstructive sleep apnea. J. Clin. Sleep Med. 15(11), 1645–1653 (2019) 5. Nersisson, R., Mekala, M., Ruban, N., Kumar Panda, S., Prakash Muduli, S., Mary Mekala, A.: Sleep quality monitor using stress analysis and REM sleep detection. J. Theor. Appl. Inf. Technol. 20(2) (2013)

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6. Okamoto-Mizuno, K., Mizuno, K.: Effects of thermal environment on sleep and circadian rhythm (2012). http://www.jphysiolanthropol.com/content/31/1/14 7. Osterbauer, B., Koempel, J.A., Ward, S.L.D., Fisher, L.M., Don, D.M.: A comparison study of the Fitbit activity monitor and PSG for assessing sleep patterns and movement in children. J. Otolaryngol. Adv. 1(3), 24–35 (2016) 8. Patel, A.K., Araujo, J.F.: Physiology Sleep Stage. StatPearls Publishing, St. Petersburg (2022) 9. Ramar, K., et al.: Sleep is essential to health: an American academy of sleep medicine position statement. J. Clin. Sleep Med. 17(10), 2115–2119 (2021) 10. Shahab, H., Sepideh, K., Michael, H.S., Diller, K.R.: Accuracy of wristband Fitbit models in assessing sleep: systematic review analysis. J. Med. Internet Res. 21(11), e16273 (2019) 11. Walker, M.P., Liston, C., Hobson, J.A., Stickgold, R.: Cognitive flexibility across the sleepwake cycle: REM-sleep enhancement of anagram problem solving. Cogn. Brain Res. 14 (2002). www.elsevier.com/locate/bres

Author Index

A Abdul Samad, Adlisa 21 Aeria, Christopher James 215 Ahmad, Mohd Yazed 89, 126, 158 Alkhalaf, Hussein Yahya 158, 169 Azlan, Norsinnira Zainul 65 Azzan, Sulaiman 135 B Basri, Nur Rasyidah Hasan

46

C Chan, Sook Wei 21 Chua, Meng Kiat 144 Chun, Boon Jian 144 D Dan, Min Kai 201 Do, Tien-Dung 114 Ðo´coš, Miroslav 73, 81 Durairajah, Vickneswari 135, 144, 181, 191, 201, 215 F Fazlur, Farah Amira Binti 181 G Gobee, Suresh 135, 144, 181, 191, 201, 215 H Hamzah, Norhamizan 89 Hamzaid, Nur Azah 89 Hashim, Fatimah Fawzi 158, 169 Hossen, Sajjad 65 Hou, Cheong Soon 191 Hui, Kenny James Ling Neng 253 Husnain, Anees Ul 8

I Ibrahim, Alwani 30 Ibrahim, Fatimah 230, 253 Ismail, Aniza 3 Ismail, Arif Mawardi 114 Ismail, Muhammad Isyraf 8 J Jaitoo, Vedanta 135 Jamal, Shamsuriani Md 3 Jamali, Annisa 242 Jamaluddin, Nurul Fauzani 230, 253 Jeoti, Varun 57 Jian, Ting Kwong 181 Jovanovi´c, Nikola 73 Juhari, Siti Hajar 126 Jusoh, Norhana 21 K Kalamkovi´c, Jovana 73 Kasim, Khairul Farihan 30 Kipli, Kuryati 242 Kobayashi, Reina 38 Koji´c, Sanja 73, 81 L Latef, Tariq Bin Abdul Lee, Kai Sheng 144 Low, Chun You 201

169

M Mahadi, Wan Nor Liza Binti 169 Mahdy, Zaleha Abdullah 3 Mazalan, Mazlee 114 Md Saleh, Siti Shuhadah 30 Mili´c, Lazar 57, 73, 81 Min, Schubert Tan Su 191 Ming, Lim Lai 65 Mohammad, Nur Farahiyah 30 Mohammed, Nawaf 135

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 F. Ibrahim et al. (Eds.): ICIBEL 2022, IFMBE Proceedings 107, pp. 263–264, 2024. https://doi.org/10.1007/978-3-031-56438-3

264

Mohd Daud, Farah Diana Mohd Nasir, Nashrul Fazli Mohktar, Mas Sahidayana Mokhtar, Mas Sahidayana Mokhtar, Norrima 8

Author Index

30 30 46 253

N Naeem, Jannatul 89 Noor, Anas Mohd 114 Nordin, Anis Nurashikin 65 O Osman, Noor Azuan Abu 106 Othman, Mohamadariff Bin 169 P Petrovi´c, Bojan 73, 81 Q Qi, Lim Wei 191 Qureshi, Saima 57 R Rahim, Rosminazuin Ab 65 Rahman, Rahana Abd 3 Ramiah, Harikrishnan 158 Rao, Harinivas Rao Suba 89 Razack, Azad Hassan Abdul 230 Razalli, Mohammad Shahrazel 114 Rengasamy, Selvam 46 Riza, Mohd Saiful 65 Rosdi, Alif Aiman Bin 181 Roslan, Lidyana 242

S Sabudin, Raja Zahratul Azma Raja 3 Saiboon, Ismail Mohd 3 Samsudin, Zambri 65 Shah, Noraisyah Mohamed 8 Ship, Chern Wei 201 Shun, Kum Shou 181 Soin, Norhayati 242 Stefanovi´c, Sofija 73, 81 Stojanovi´c, Goran 73, 81 Stojanovi´c, Goran M. 57 Suhaimi, Muhammad Irsyad 65 T Tee, Yik Seng 201 Tun Rashdan Arief, Tun Iqmal Haziq

30

U Ueno, Akinori 38 Ulia, Muhd Nazreen 242 Usman, Juliana 106 V Vejin, Marija

73, 81

W Wahab, Yufridin 114 Wen, Woo Kai 230 Wong, Yi Chen 144 Z Zahudi, Azrena Zaireen Ahmad 106 Zailani, Mohamad Afiq Hidayat 3 Zakaria, Mohd Rosydi 114 Zakaria, Nor Farhani 114 Zaman, Wan Safwani Wan Kamarul 46, 114