Nanoindentation of natural materials : hierarchical and functionally graded microstructures

Nanoindentation of Natural Materials Hierarchical and Functionally Graded Microstructures

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Nanoindentation of Natural Materials Hierarchical and Functionally Graded Microstructures

Arjun Dey Anoop Kumar Mukhopadhyay

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2019 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed on acid-free paper International Standard Book Number-13: 978-1-4987-8405-4 (Hardback) International Standard Book Number-13: 978-1-315-15554-8 (eBook) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright​ .com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

My beloved wife, Manju, daughter, Roopkatha (Brishti), and daughter, Sanjbati (Tutum)

—Anoop My beloved mother, Minati, father, Dilip, wife, Omprita, and son, Abahon (Mishuk)

—Arjun

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Contents Preface.................................................................................................................... xiii Acknowledgments.................................................................................................xv Authors.................................................................................................................. xix Contributors........................................................................................................ xxiii Common Abbreviations..................................................................................... xxv 1. Basics of Hierarchical and Functionally Graded Structures and Mechanical Characterization by Nanoindentation: A Paradigm Shift for Nano/Microstructural Length Scale..................... 1 Arjun Dey, Deeksha Porwal, Nilormi Biswas, Aniruddha Samanta, Manjima Bhattacharya, Mohammed Adnan Hasan, A. K. Gupta, and Anoop Kumar Mukhopadhyay 1.1 Introduction.............................................................................................. 1 1.2 Some Truths and Interesting Facts........................................................ 1 1.2.1 Bone: A Tough Hybrid Composite.............................................1 1.2.2 Teeth: A Hard but Tough Hybrid Functionally Graded Composite......................................................................................5 1.2.3 Shell and Scale: A Hard-Tough Layered Functionally Graded Hybrid Composite.......................................................... 9 1.2.4 Hair Fiber: A Tough Hierarchical Layered Architecture...... 11 1.2.5 Wonder of Mother Nature and Mimicking It......................... 13 1.3 Mechanical Characterization by Nanoindentation: A Paradigm Shift for Nano/Microstructural Length Scale............ 15 1.4 What Can Nanoindentation Do?......................................................... 16 1.5 Hardness and Modulus: The Vital Properties of Materials and Their Measurement Limitations at Nano/Microstructural Length Scale.................................................. 16 1.6 Nanoindentation: A Boon for Mechanical Characterization at the Scale of Microstructure.............................................................. 17 1.7 Basic Theory of Nanoindentation........................................................ 18 1.8 The Need for Finite Element (FE) Modeling of Nanoindentation.... 21 1.9 Summary................................................................................................. 23 References........................................................................................................ 23 2. Nanoindentation of Bone: A Tough Hybrid Composite........................ 27 Manjima Bhattacharya, Anoop Kumar Mukhopadhyay, and Arjun Dey 2.1 Introduction............................................................................................ 27 2.2 Structure of Bone................................................................................... 27 vii

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2.2.1 Structural Anisotropy at Different Length Scales................. 28 2.3 Importance of Nanoindentation..........................................................30 2.4 Factors Influencing the Nanomechanical Properties of Human Cortical Bone....................................................................... 32 2.4.1 Influence of Location on Nanomechanical Properties of Bone............................................................................................33 2.4.2 Influence of Direction of Orientation on Nanomechanical Properties of Bone.................................. 38 2.4.3 Influence of the Density Variation on the Nanomechanical Properties of Bone.................................43 2.4.4 Influence of Age on the Nanomechanical Properties of Bone.......................................................................................... 45 2.4.5 Influence of Gender on the Nanomechanical Properties of Bone...................................................................... 49 2.4.6 Influence of State of Hydration on the Nanomechanical Properties of Bone...................................................................... 50 2.4.7 Influence of Experimental Conditions on the Nanomechanical Properties of Bone........................... 50 2.5 Summary.................................................................................................54 References........................................................................................................54 3. Nanoindentation of Teeth: A Hard but Tough Hybrid Functionally Graded Composite................................................................ 59 Nilormi Biswas, Anoop Kumar Mukhopadhyay, and Arjun Dey 3.1 Introduction............................................................................................ 59 3.2 Formation and Structure of Tooth.......................................................63 3.2.1 Formation and Structure of Enamel........................................64 3.2.1.1 Differences between Enamel and HAp..................... 66 3.2.2 Formation and Structure of Dentine and DEJ........................ 66 3.3 Nanomechanical Property Evaluation of Tooth................................ 67 3.3.1 Nanomechanical Property Evaluation of Enamel Tissue..... 68 3.3.1.1 Young’s Modulus and Hardness of Sound Enamel.... 68 3.3.1.2 Young’s Modulus and Hardness of Carious Enamel.... 68 3.3.1.3 Young’s Modulus and Hardness of Sound Enamel in Terms of Age and Location��������������������� 69 3.3.1.4 Young’s Modulus and Hardness of Hypomineralized Enamel�������������������������������������� 69 3.3.1.5 Effect of Bleaching on Nanomechanical Property of Enamel������������������������������������������������������ 71

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3.3.1.6 Effect of Flouride Treatment on Nanomechanical Property of Enamel����������������� 72 3.3.1.7 Dissolution and Erosion of Enamel............................ 72 3.3.2 Nanomechanical Property Evaluation of Dentine and DEJ.... 73 3.3.2.1 E and H of Sound Dentine........................................... 73 3.3.2.2 E and H of Carious Dentine........................................ 74 3.4 Summary................................................................................................. 74 References........................................................................................................ 75 4. Fracture Toughness of Enamel Region: Role of DEJ and Modeling of Fracture Toughness.......................................................83 Nilormi Biswas, Arjun Dey, and Anoop Kumar Mukhopadhyay

4.1 Introduction............................................................................................83 4.2 Structural Hierarchy and Fracture Toughness..................................84 4.3 Role of DEJ............................................................................................... 88 4.4 Micro-mechanical Modeling for Prediction of Fracture Toughness................................................................................................ 93 4.5 Summary................................................................................................. 97 References........................................................................................................ 97 5. Nanoindentation Creep of Enamel and Dentine Tissues................... 103 Nilormi Biswas, Latika Khurana, Arjun Dey, and Anoop Kumar Mukhopadhyay

5.1 Introduction.......................................................................................... 103 5.2 Creep Behavior of Enamel.................................................................. 106 5.3 Creep Behavior of Dentine.................................................................. 113 5.4 Summary............................................................................................... 121 References...................................................................................................... 122

6. Nanoindentation of Hair Fiber: A Tough Hierarchical Layered Architecture.................................................................................. 125 Aniruddha Samanta, Manjima Bhattacharya, Anoop Kumar Mukhopadhyay, Shekhar Nath, and Arjun Dey 6.1 Introduction.......................................................................................... 125 6.2 Nanomechanical Properties of Hair Fiber: What Literature Says.... 129 6.3 Macromechanical and Nanotribological Properties of Human Hair..................................................................................... 131 6.4 Summary............................................................................................... 137 References...................................................................................................... 138

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7. Nanoindentation of Shell and Scale: A Hard-Tough Layered Functionally Graded Hybrid Composite................................................ 141 Manjima Bhattacharya, Anoop Kumar Mukhopadhyay, and Arjun Dey 7.1 7.2 7.3 7.4

Introduction.......................................................................................... 141 Structure................................................................................................ 141 Importance of Nanoindentation........................................................ 143 Nanomechanical Properties of Different Varieties of Molluscan Shells.............................................................................. 144 7.4.1 Nanomechanical Properties of Gastropods........................... 145 7.4.1.1 Limnetic Shell.............................................................. 145 7.4.1.2 Pteropod Shell............................................................. 146 7.4.1.3 Abalone Shell............................................................... 150 7.4.2 Nanomechanical Properties of Bivalve Shells....................... 155 7.4.2.1 Farreri Shell................................................................. 155 7.4.2.2 Saxidomus Purpuratus Shell..................................... 157 7.4.2.3 Green Mussel Shell..................................................... 158 7.4.2.4 Brachiopod Shells....................................................... 160 7.4.2.5 Oyster Shells................................................................ 163 7.5 Nanomechanical Response of Other Varieties of Shells................ 164 7.5.1 Nanomechanical Response of Arthropoda............................ 164 7.5.1.1 Mantis Shrimp............................................................. 164 7.5.1.2 American Lobster....................................................... 165 7.6 Nanomechanical Response of Fish Scales........................................ 167 7.6.1 Arapaima Gigas........................................................................ 168 7.6.2 Alligator Gar Scales.................................................................. 169 7.6.3 Polypteridae Fish Scales.......................................................... 171 7.7 Summary............................................................................................... 171 References...................................................................................................... 173

8. Combined Nanoindentation and Finite Element Approach in Natural Hierarchical Structures.......................................................... 177 Deeksha Porwal, Arjun Dey, A. K. Gupta, Kallol Khan, Anand Kumar Sharma, and Anoop Kumar Mukhopadhyay 8.1 Basics of Finite Element Method....................................................... 177 8.2 Nonlinear Analysis.............................................................................. 178 8.2.1 Geometric Nonlinearity........................................................... 179 8.2.2 Material Nonlinearity.............................................................. 180 8.2.3 Boundary Condition Nonlinearity........................................ 182 8.3 Finite Element Analysis and Nanoindentation............................... 182 8.3.1 Development in the Field of Nanoindentation and Its FE Modeling................................................................. 183 8.3.2 Material Models Used in Nanoindentation Simulation...... 184

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8.3.3 Evaluation of Substrate Effect from Nanoindentation Simulation.................................................................................. 185 8.3.4 Optimization Technique Used in Nanoindentation Simulation................................................................................. 185 8.4 Description of the FE Modeling of Nanoindentation..................... 186 8.5 Combined Nanoindentation and FE Approach.............................. 189 8.6 Combined Nanoindentation and FEM Study of Silicon................ 192 8.7 Validation of the Present FE Based Model from Literature Data.... 193 8.8 Issues of Computation Time, Accuracy, and Error of the FE Based Simulation Work....................................................................... 194 8.9 Case Study: Finite Element Modeling of Nanoindentation of a Fish Scale....................................................................................... 196 8.10 Summary............................................................................................... 199 References...................................................................................................... 199 9. Conclusions................................................................................................... 203 Arjun Dey, Nilormi Biswas, Aniruddha Samanta, Manjima Bhattacharya, and Anoop Kumar Mukhopadhyay Index...................................................................................................................... 207

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Preface Hierarchical and functionally graded microstructures in natural materials are the creation of Mother Nature, and we admit that we understand only a little about them. Hierarchical and functionally graded structures found in bone, teeth, hair, scale, and shell are covered in this book. Further, their detailed microstructure, composition, etc. are presented in detail. The basics of the well-established nanoindentation technique and its advantages in the purview of characterizing natural hybrid bio-composites are also analyzed. Further, the basics of combined nanoindentation and finite element approach for characterization of functionally graded structures have been put into perspective. In addition, the detailed mechanical properties of bone, dentine, enamel, hair, scale, and shell evaluated by micro/nanoindentation technique, especially at the scale of micro/nanostructure, are analyzed in terms of the correlations of mechanical properties with microstructures. Further, attempts have been made to model the experimentally measured mechanical properties on the basis of microstructures. Moreover, the combined nanoindentation and finite element approach has been briefly introduced to hint at the demonstration of its capability for further characterizations of deformation and damage scenarios at the nano/micro scale of the relevant microstructure. The major highlight of this book is provision of a thorough knowledge base about natural hybrid bio-composite structures and correlation of the same with mechanical properties at the micro/nano structural level, thereby establishing how such correlations could act as precursors for developing bioinspired engineered structures for structural as well as functional ceramics. There are nine chapters in this book. Chapter 1 presents the overview and basics of hierarchical and functionally graded natural structures and mechanical characterization by nanoindentation technique. Chapter 2 deals with nanoindentation of bone, while Chapters 3, 4, and 5 provide thorough mechanical properties investigations of dentine and enamel tissues. Nanoindentation responses of hair fiber as well as shell and scale are revealed in Chapters 6 and 7, respectively. Chapter 8 presents the utilization of the combined nanoindentation and finite element approach in characterization of the hierarchical and functionally graded structures. Finally, Chapter 9 offers a summary of the major findings that emerged out of the information presented in the foregoing chapters.

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Acknowledgments At the onset, we express our sincere thanks to honorable directors of CSIRCentral Glass and Ceramic Research Institute (CSIR-CGCRI), Kolkata, and U. R. Rao Satellite Centre (formerly known as ISRO Satellite Centre), Bangalore, for their constant encouragement and infrastructural facilities. We are grateful to all technical and support staff of Advanced Mechanical and Materials Characterization Division (AMMCD), CSIR-CGCRI, for assistance received during this work. We also express our sincere thanks for incredible help and support received from the former students and research scholars of the Nanoindentation Laboratory at AMMCD of CSIR-CGCRI.

What Arjun says…. It is now my turn to thank the people who were involved behind the scenes for the completion of this book. First, I am very grateful to my beloved parents, Mr. Dilip Kumar Dey and Ms. Minati Dey, and to my dear wife, Omprita Chatterjee, who took on all the responsibilities for family matters and gave me the encouragement to publish this book. I have also experienced a hidden support from our newborn son Master Abahon (Mishuk). I am also most thankful to my dear boudi (sister-in-law) Ms. Manjulekha Mukherjee (spouse of Dr. Mukhopadhyay, my coauthor) for providing continuous emotional support and caring for almost every need during the publication of this book. Special thanks are due to Dr. A. K. Sharma, Distinguished Scientist, Deputy Director, Mechanical Systems Area, URSC-ISRO; Professors Bikramjit Basu and Vikram Jayram from IISc Bangalore; Professors Tapas Laha and Rabibrata Mukherjee from IIT Kharagpur; Professors Srinivasa Rao Bakshi and Pijush Ghosh from IIT Madras; Dr. Harish C. Barshilia and Dr. Parthosarathi Bera from CSIR-NAL; Professor Niloy K. Mukhopadhyay, IIT-BHU; Professors Kuruvilla Joseph and K. Prabhakaran from IIST, Trivundrum; Professor Subroto Mukherjee, IPR, Gandhinagar; Dr. Arvind Sinha, CSIR-NML; Professor Indranil Lahiri and Debrupa Lahiri from IIT Roorkee; Professor Dinesh Rangappa, VTU; Professor Debalina Bhattacharjee from NIT Surathkal; Professor Ajoy Kumar Pandey from NIT Warangal; Dr. Ankur Kumar Gupta, URSC-ISRO; Professor Amitava Basu Mullick, IIEST; Professors Kallol Khan and Shibendu Shekhar Roy from NIT Durgapur; Professor Ajoy Kumar Ray, Ex-Director, IIEST, Shibpur; and Professor Gerhard Wilde, Director, Institute of Materials Physics, University of Muenster. xv

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Many thanks are also offered to my doctoral and master’s students I. Neelakanta Reddy, A. Carmel Mary Esther, Deeksha Porwal, Dipta Mukherjee, Prajwal Kolambe, Md. Adnan Hasan, Prudhivi Yashwantkumar Bhavanisankar, S. Rashmi, Manish Kumar Nayak, Debajyoti Palai, Shubham Mishra, Mourya Sandeep Pradeepkumar, Kunal Banerjee, and G. L. Priyanka, who always gave moral support. Moreover, I have always received enormous support from Professors Nil Ratan Banyopadhyay and Subhabrata Datta from IIEST, Shibpur, without whose encouragement my academic and research career would not, perhaps, have grown to where it is today. Before I finish, I am most fortunate to have met Dr. Anoop, who is not only my master’s and PhD guide and coauthor of this book, but also the pathfinder of my research life.

What Anoop says…. At the very outset I thank my late, beloved parents Mr. Girija Bhusan Mukherjee and Mrs. Kamala Mukherjee and my late in-laws Mr. Beni Madhab Bhattacharya and Mrs. Bijaya Bhattacharya for making me what I am today. I wish with tearful eyes and sweet memories that all of them were alive today! The huge support received from Mr. and Mrs. S. K. Mukherjee, Ex-General Manager, Hindustan Aeronautics Limited, Bangalore, Professor S. Bhattacharya of Institute of Management Technology, Ghaziabad, and Dr. Mrs. A. Bhattacharya of RKDIT, Ghaziabad, Mr. and Mrs. A. Bhattacharya of Silvasa, Mumbai, is also gratefully acknowledged. I would recognize the tremendous contributions of all four of my living sisters-in-law, four late brothers and one late sister, two late uncles and aunts to my upbringing and their encouragement of my academic efforts. I am also more than grateful to my dear, beloved wife Mrs. Manjulekha Mukherjee and to my two sweet daughters, Miss Roopkatha Mukhopadhyay, age 20, and Miss Sanjhbati Mukhopadhyay, age 15; but for whose tremendous patience, love, care, and emotional support, this book would not have seen the light of day. It is a joy for me to recognize the kind encouragement received from my nephew Mr. N. Mukherjee and his wife Mrs. M. Mukherjee, my granddaughter Miss N. Mukherjee, my niece, Miss L. Mukherjee, and their mother, Mrs. S. Mukherjee. I also appreciate the kind contributions made by Professors Y-W. Mai, M. V. Swain, and Mark Hoffman of Australia; Dr. Brian Lawn of NIST, USA; Professor S. Priyadarshy of USA; Dr. R. W. Steinbrech of Germany; Mr. I. Dean of South Africa; Professor M. K. Sanyal, Ex-Director, SINP; Dr. D. Chakraborty, my PhD guide; Dr. S. Kumar, Dr. B. K. Sarkar, Dr. C. Ganguly, Dr. H. S. Maity, Dr. K. K. Phani, Dr. D. K. Bhattacharya; Dr. K. Muraleedharan, our present director; late Drs. A. P. Chatterjee, D. Basu, and Mr. D. Chakraborty, all of

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CSIR-CGCRI; Dr. S. Tarafder of NML, Jamshedpur; Professor B. Basu of IISc, Bangalore; Professor N. R. Bandyopadhyay of IIEST; Professor A. N. Basu of Jadavpur University; and of course, Professor I. Manna of IIT Kanpur, to my developments as a researcher. I especially thank Professor R. Mukherjee of IIT Kharagpur and late Mr. P. Basu Thakur of Icon Analytical Pvt. Ltd., India, without whose active support the Nanoindentation Laboratory would not have been created at all at CSIR-CGCRI. Thanks are also due to Mr. A. Rao, Mr. S. Vaidya, and Mr. P. Rastogi of Bruker Nanosurfaces Division. I also thank my colleagues Mr. D. Sarkar, Mr. I. Biswas, Drs. D. Bandyopadhyay and G. Banerjee, Miss S. Datta, Mr. S. Dey, Mrs. R. Chakraborty, and Mr. S. Biswas of the Project Management Division, and particularly all my other colleagues from the Publication Section and the Non-Oxide Ceramic and Composite Division of CSIR-CGCRI for their all-around help and cooperation during the writing of this book. I am also grateful to my dear brothers Mr. S. Acharya and Mr. D. Moitra of CSIR-CGCRI and to Mr. R. Das as well as Dr. Anup Khetan of RTIICS, but for whom I would not have been simply alive today, to write even the first page of this book. I also thank Dr. S. K. Bhadra, Chief Scientist, and Mr. K.  Das Gupta, Ex-Director of CSIR-CGCRI, for their kind encouragement, support, and advice at all stages. Very special thanks are due to my colleagues Dr. R. N. Basu, Dr. P. S. Devi, Dr. D. Kundu, Dr. G. De, Dr. H. S. Tripathy, Dr. S. Dasgupta, Dr. S. Bandyopadhyay, and Mr. A. K. Chakraborty of CSIR-CGCRI. I also thank the Almighty to have kindly provided me a student like Dr. Arjun Dey, the coauthor of this book. He, his wife, and son A. Dey are truly a part of my existence, and he is definitely much more than a mere student or junior colleague to me. I feel proud to be his thesis supervisor, just by chance, may be, as they say, by the Decree of God. I acknowledge all the near and distant relatives, teachers, friends, colleagues, former and present students, and well-wishers, for their huge support and kind encouragement received at every bend of my life and particularly during the execution of this book writing project. Last but not least, I express my gratitude to my dear friend poet Joy Goswami, who taught me how to live through the hours of grief and pain, through the hours of torture and torment, just to see through the apparently endless night that ends in a new whisper of love, a new breath of life, and a new day of sunrise. Arjun Dey Anoop Kumar Mukhopadhyay

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Authors Dr. Arjun Dey is a scientist at U. R. Rao Satellite Centre (formerly known as ISRO Satellite Centre), Indian Space Research Organisation, Bengaluru. Dr. Dey earned his bachelor’s degree in mechanical engineering in 2003 from Biju Patnaik University of Technology; followed by a master’s degree in materials engineering from Indian Institute of Engineering Science and Technology (IIEST), Shibpur, Howrah (formerly Bengal Engineering and Science University) in 2007. Working as CSIRSRF at CSIR-Central Glass and Ceramic Research Institute (CSIR-CGCRI), Kolkata, he earned his doctoral degree in materials science and engineering in 2011 from IIEST, Shibpur. Dr. Dey has already garnered many prestigious awards, such as “Satellite Technology Day 2018 Award,” for both Best Technical Paper and Innovative Idea in “Spark” Event from URSC-ISRO in 2018; Dr. R. L. Thakur Memorial Award for young scientists working in the field of ceramics from The Indian Ceramic Society in 2012; DST-Fast Track Young Scientist Scheme Project Grant Award from the Department of Science and Technology, India, in 2011; Young Engineers Award and Metallurgical and Materials Engineering Division Prize from The Institution of Engineers (India) in 2011 and 2016; and Young Scientist Award from Materials Research Society of India, Kolkata chapter, in 2009 for significant contributions in the field of Materials Science and Engineering. Dr. Dey also earned the prestigious CSIR-Senior Research Fellowship Award from the Council of Scientific and Industrial Research (CSIR) during the completion of his PhD. The research work of Dr. Dey culminated in more than 240 publications, including approximately 105 in SCI journals including from NATURE Group. He has coauthored two books—Nanoindentation of Brittle Solids (2014) and Microplasma Sprayed Hydroxyapatite Coatings (2015) from CRC Press/ Taylor & Francis Group—and one book chapter (“Micromechanical and Finite Element Modeling for Composites”) published by IGI Global, USA. He has guided 3 PhD (2 ongoing) and 13 MTech (2 ongoing) students. He has delivered several invited talks in international conferences, IITs, and academic universities. For the 2016–18 period he has been an executive committee member in Karnataka Chapter of The Indian Ceramic Society. Dr. Dey serves as reviewer of 40 different peer reviewed journals including Advanced Materials, Advanced Materials Interfaces, Advanced Engineering xix

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Materials, ACS Applied Materials & Interfaces, Materials & Design, Composite Part B, Solar Energy, Applied Surface Science, Ceramics International, Materials Science and Engineering B, Surface and Coatings Technology, Metallurgical and Materials Transactions A etc. Dr. Anoop Kumar Mukhopadhyay is a chief scientist and head of the Advanced Mechanical and Materials Characterization Division of CSIRCGCRI, Kolkata, India. He earned his bachelor’s degree with honors in physics from Kalyani University, Kalyani, in 1978 followed by a master’s degree in physics from Jadavpur University, Kolkata, in 1982. Way back in 1978, he had initiated in India the research work on evaluation, analysis, and microstructure mechanical properties correlation of non-oxide ceramics for high temperature applications, prior to joining CSIR-CGCRI, Kolkata, India, in 1986, as a staff scientist. Working on the critical parameters that control the high temperature fracture toughness of silicon nitride and its composites, he earned his PhD in science in 1988 from the Jadavpur University, Kolkata. During 1990–92 he was awarded the prestigious Australian Commonwealth Post Graduate Research Fellowship and made pioneering contributions on the role of grain size in wear of alumina ceramics during his postdoctoral work on development of wear and fatigue resistant oxide ceramics, with world-renowned Professor Yiu-Wing Mai and Professor Mike Swain at the University of Sydney, Australia. At CSIR-CGCRI, Kolkata, India, Dr. Mukhopadhyay has established an enthusiastic research group on evaluation and analysis of mechanical and nanomechanical properties of glass, ceramics, bioceramic coatings and biomaterials, thin films, and natural biomaterials. Dr. Mukhopadhyay has an impressive total of close to 300 publications including SCI journals (including NATURE Group), national and international conference proceedings/book of abstracts, etc., with h-index of 26, i10 index of 52, and a total citation of more than 2200 (www.scholar.google.co.in) to his credit. He also has seven patents, with three of them already granted; four book chapters published, and two books coauthored with Dr. A. Dey of ISAC, ISRO, Bangalore, India, and published by Taylor & Francis Group/CRC Press, USA to his credit. He has supervised 11 doctoral students, including 6 candidates who have already earned their PhDs from Indian Institute of Engineering, Science and Technology in 2011 and from Jadavpur University in 2014, 2015, and 2016. He has also guided more than 20 MTech dissertation theses and 30 BTech theses. He contributed three chapters to Handbook of Ceramics edited by Dr. S. Kumar, internationally

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famous glass technologist and former director of CSIR-CGCRI, Kolkata, India, and published by Kumar and Associates, Kolkata. Along with coauthors Dr A. K. Gupta and Dr A. Dey of ISAC, ISRO, Bangalore, India, he recently contributed a chapter to Materials Design Using Computational Intelligence Techniques edited by Professor S. Dutta of SRM University, India, and published by IGI Global, USA in 2016. He serves on the editorial board of Soft Nanoscience Letters. In 2008, he won the Best Poster Paper Award at the 53rd DAE Solid State Physics Symposium. He also won in 2000 the Sir C V Raman Award of the Acoustical Society of India. In the same year he also won the Best Poster Paper Award of the Materials Research Society of India. He was awarded in 2000 the Visiting Scientist Fellowship to work on the fracture and nanoindentation behavior of ceramic thermal barrier coatings with worldrenowned scientist Dr. R. W. Steinbrech at the Forschungszentrum, Juelich, Germany. He was awarded in 1997 the Outstanding Young Person Award for Science and Innovation by the Outstanding Young Achievers Association, Kolkata. He won the Lions Club of India Award in 1996. His work was recognized in 1995 through the Best Poster Paper Award of the Materials Research Society of India. In 2010, his paper won the Best Research Paper Award at the Diamond Jubilee Celebration Ceremony of CSIR-CGCRI, Kolkata. His current research interests cover a truly diverse span, for example, physics of nano scale deformation for brittle solids, high strain rate shock physics of ceramics, tribology of ceramics, nanotribology of ceramic coatings and thin films, microstructure mechanical and/or functional property correlation as well as ultrasonic characterization and fatigue of (a) structural and bioceramics, bio-ceramic coatings, bio-materials; (b) multilayer composites; and (c) thick/thin hard ceramic coatings. He also has active interest in microwave processing of ceramics, ceramic composites, and ceramic metal or ceramic/ ceramic joining.

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Contributors Manjima Bhattacharya Senior Research Fellow (CSIR) CSIR-Central Glass and Ceramic Research Institute Kolkata, India Nilormi Biswas Woman Scientist (DST) CSIR-Central Glass and Ceramic Research Institute Kolkata, India A. K. Gupta Scientist U. R. Rao Satellite Centre (URSC) Bangalore Indian Space Research Organisation India Mohammed Adnan Hasan Project Trainee U. R. Rao Satellite Centre (URSC) Bangalore Indian Space Research Organisation India Kallol Khan Assistant Professor National Institute of Technology Durgapur, India Latika Khurana Bachelor Student Department of Ceramics Engineering Indian Institute of Technology (BHU) Varanasi, India

Shekhar Nath Ex-Scientist Applied Interfacial and Materials Science CavinKare Research Centre CavinKare Pvt. Ltd. Chennai, India and Ex-Head, R&D Jyoti Ceramic Industries Private Limited Nashik, India Deeksha Porwal Doctoral Research Scholar National Institute of Technology Durgapur, India Aniruddha Samanta Doctoral Research Scholar CSIR-Central Glass and Ceramic Research Institute Kolkata, India Anand Kumar Sharma Distinguished Scientist Deputy Director, Mechanical Systems Area U. R. Rao Satellite Centre (URSC) Bangalore Indian Space Research Organisation India

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Common Abbreviations FGM SEM FESEM ESEM TEM AFM SPM XRD EDX FTIR HAp FAP H E Er σy K n KIC (P–h) ν γ ISE Ra DEJ CaP MEMS NEMS CMCs MMCs FE FEM BC FEA BM BMH CDR APDL CMC MEA

functionally graded hierarchical microstructure scanning electron microscopy field emission scanning electron microscopy environmental scanning electron microscopy transmission electron microscopy atomic force microscopy scanning probe microscopy X-ray diffraction energy dispersive X-ray spectroscopy Fourier transform infra-red spectroscopy hydroxyapatite fluorapatite hardness/nanohardness Young’s modulus reduced Young’s modulus yield stress strength coefficient work hardening exponent fracture toughness load versus depth Poisson’s ratio work of fracture indentation size effect surface roughness dentine enamel junction calcium phosphate micro electro mechanical systems nano electro mechanical systems ceramic matrix composites metal matrix composites finite element finite element method boundary condition finite element approach bilinear model bilinear model with hardening critical depth ratio ANSYS parametric design language cell membrane complex methyl eicosanoic acid xxv

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IF KAPs HS UHS HGT UV ROS UTM UMIS BSE CP HP FIB HSBs IPM K m BSU ECM SAZ LHA CU TN PC PCT SBF NMH INTL HBSS WO BO TOF-SIMS

Common Abbreviations

intermediate filament keratin-associated proteins high sulfur ultra high sulfur high glycine-tyrosine ultra violet reactive oxygen species universal testing machine ultra-micro-indentation system back scatter electron carbamide peroxide hydrogen peroxide focused ion beam Hunter–Schreger bands interprismatic matrix bioprotein matrix bone structural unit extracellular matrix sub-Antarctic zone Limacina helicina antarctica Cavolinia uncinata Trochus niloticus pore canals protruding pore canal tubes simulated body fluid nonlinear model with hardening interstitial lamellae Hanks’ balanced salt solution white osteons black osteons Time-of-Flight Secondary Ion Mass Spectrometry

1 Basics of Hierarchical and Functionally Graded Structures and Mechanical Characterization by Nanoindentation: A Paradigm Shift for Nano/ Microstructural Length Scale Arjun Dey, Deeksha Porwal, Nilormi Biswas, Aniruddha Samanta, Manjima Bhattacharya, Mohammed Adnan Hasan, A. K. Gupta, and Anoop Kumar Mukhopadhyay

1.1 Introduction Natural nano-bio-hybrid composite materials, for example, bone, teeth, scale, shell, hair [1–10], etc., provide a different arrangement of material structures at various length scales that assist to perform various mechanical, biological, and chemical functions. These multifunctional or multi-objective properties of the natural hybrid composite structures often lie in a functionally graded fashion locally or in the entire body. The science behind these structures and their correlation with the properties are not yet fully understood. Understanding the science would certainly be of tremendous technological and societal importance.

1.2 Some Truths and Interesting Facts 1.2.1 Bone: A Tough Hybrid Composite Bone is a hybrid-composite material composed of organic compounds (mainly collagen) reinforced with inorganic compounds (CaP compounds, HAp and other minerals). Thus, we can say that bone is a natural ceramicpolymer hybrid composite that consists of HAp/CaP ceramics, collagen fiber, 1

2

Nanoindentation of Natural Materials

organic proteins, and water. Generally, the amount of inorganic species is 70%, and the rest is the organic fiber and proteins. However, the precise composition of bone differs depending on its variant (cortical or spongy), gender, species, era, age, anatomical location, nutrition intake, etc. If someone wants to evaluate mechanical properties of bone, then at a different length scale the measured data would be different as the bone possesses hierarchical structural organization or it shows different structural architecture at various length scale levels. The same is shown schematically in Figure 1.1. The macrostructure of a typical bone (comprising of cancellous or porous and cortical or hard bone) would be size of a few millimeters, and it can be seen by a naked eye. Now, if we look into the microstructure (from 1 to 500 μm), that is, beyond a naked eye, we shall be able to find out lamellar structure, Haversian systems, osteons, single trabeculae, etc. with the help of an optical or electron microscope. Subsequently, a high-resolution electron microscope can help to detect the subsequent nanostructures ­(features below 1 micron and up to few nanometers) of collagen fiber, non-collagenous organic proteins, and CaP crystals. Further, detail of collagen (triple helix structure in the order of less than 10 nm) can be also investigated by transmission electron microscopy. Weiner and Wagner [11] showed seven hierarchical levels of bone structure. They demonstrated that nanoscale HAp crystals are aligned within self-assembled collagen fibrils. Then collagen fibrils are arranged with specific layered orientation to form osteons. The osteons ultimately form either spongy or dense bone. Liu and Webster [12] report in detail about assembly of collagen fibers and bone mineral crystals. If we put a critical look on collagen fibril structure, we can find the bundle of collagen fiber typically of 300 nm length and of 1.2 nm diameter. The hexagonal HAp nanostructure crystals of ~50 × 25 × 3 nm3 are arranged between the gap (called a “hole-zone” with a dimension of around 40 nm) of two vertically oriented collagen fibers. On the other hand, Type I collagens exist as self-assembled triple helix bundles with a periodicity of ~67 nm [12]. For a biological function (such as formation of new bone), the non-collagenous proteins have a great importance. SEM photomicrographs of the plan section and cross section of a typical human cortical bone are shown in Figure 1.2a–f, respectively [13]. The major components of the human cortical bone, that is, lamella, Haversian canal, and osteons are shown in Figure 1.2. The osteons are generally circular and/ or elliptical in nature. Microcracks are also observed in the lamellar structure (Figure 1.2f). Thus, bone has a hierarchical structure with different types of materials with different orientation/arrangement that make it globally ­heterogeneous and anisotropic yet tough and durable. The question we would like to address is, how do such nano- and microlevel structures contribute to the macroscale toughness of bone? How do the mechanical properties at nanoscale vary at different parts of bone, and how does that contribute to sharing of load

Lamellar structure

~3–7 µm

Microstructure

Osteons ~10–500 µm

HC

Ostiocytes

HC-haversian cannal

Cancellous or spongy bone Cortical bone

FIGURE 1.1 Schematic of the hierarchical structure of bone.

M a c r o s t r u c t u r e

Nanostructure

~40 nm Hexagonal HAp crystals

~0.5–1 µm

Collagen fibril

~1.5 nm ~8.5 nm Triple helix structure of collagen

Basics of Hierarchical and Functionally Graded Structures 3

4

Nanoindentation of Natural Materials

×100 SE

500 µm

(a)

100 µm

(b)

×500 SE

100 µm

(c)

×1.00 k SE

50.0 µm

(d)

×1.50 k SE

(e)

×500 SE

30.0 µm

×2.50 k SE

20.0 µm

(f )

FIGURE 1.2 SEM photomicrographs of the bone [13]: (a) lower magnification, plan section; (b) higher magnification, plan section; (c) lower magnification, cross section; (d) a single ostions, cross section; (e) higher magnification showed microcracks; and (f) ostiocytes in the lamella.

during high strain rate loading? In connection with this, we would like to also address the issue of how the inherent microstructural anisotropy beneficially affects toughness. The toughness component of the bone comes from the fiber and organic phase, while the hardness component arises from minerals and HAp crystals. The relative density and macro-mechanically measured elongation, modulus, and strength data are summarized in  Table 1.1  [12].

5

Basics of Hierarchical and Functionally Graded Structures

TABLE 1.1 Relative Density and Macro-Mechanical Properties of Healthy Human Bone Properties Relative density Elongation (%) Elastic modulas (GPa) Ultimate tensile strength (MPa)

Cancellous Bone

Cortical Bone (Longitude)

Cortical Bone (Transverse)

0.005–0.7 5–7 0.1–0.5 2–20

0.7–1.8 1–3 17–30 130–150

0.7–1.8 1–3 7–13 50–60

Source: Liu, Huinan, and Thomas J. Webster, World Scientific Publishing ISBN: 978-981-447563-1, pp. 1–51, 2007.

However,  in  subsequent chapters we can find that the macro-­ properties are entirely different than those of the local microstructural and/or sub­ microstructural properties. 1.2.2 Teeth: A Hard but Tough Hybrid Functionally Graded Composite Another interesting natural nano-bio-hybrid composite is teeth. Mother Nature provides the incredible beauty of this subtle, beautiful, yet complex microstructure that almost never gives up unless it is uprooted or broken in an accident. What gives it this tenacity and durability? It must have its genesis at the nanoscale of the microstructure that needs to be revealed to understand the underlying basic scientific issues. It is well known now that teeth are ceramic-polymer nano-hybrid composites comprised of the hard enamel, the more ductile dentine, and a soft connective tissue, the dental pulp. While the outer part of the enamel shows the higher hardness and modulus as the percentage of calcium drops from the outer to the inner enamel region, the role of the dentine enamel junction and its nanomechanical properties are far from well understood. Therefore, it is important to address the aforesaid issues. Basically, tooth is a microscopically functionally graded CaP based natural bio-composite material (Figure 1.3). Furthermore, tooth has also a hierarchical architecture, for example, from macrostructure to microstructure to nanostructure (Figure 1.3), like bone structure discussed in the previous section. In tooth architecture, the outermost layer has hard enamel, then ductile dentine, and the innermost layer has soft dental pulp. It is well known that enamel is the hardest structure in the human body with approximate 96 wt% HAp. The comparatively softer yet tough dentine has a porous structure and is made up of ~70% inorganic material (i.e., HAp), ~20% organic materials (i.e., collagen fiber) and ~10% water by weight. In enamel microstructure, closely packed (dense) enamel prisms or rods of diverse orientations are found. The aforesaid enamel prisms or rods are encapsulated within an organic protein called as enamel sheath. Now, if we go further down the length scale, we shall find that the prisms or rods basically consist of nanosized CaP crystals.

DEJ Dentine Pulp

cro

re

u uct

cro

–s

~40 nm Hexagonal HAp crystals

t ~1.5 nm

N ~8.5 nm Triple helix structure of collagen

Fibril

–s e ur ct u r

FIGURE 1.3 Schematic of the hierarchical structure of human teeth.

Ma

r –st

Mi

~10–20 µm Intratubular dentine ~67 µm

Dentine tubule Peritubule ~2–5 µm

M

re ctu tr u

–s

no

a M

Na

re ctu tru

DEJ Dentine Pulp

e ur ct u str o– r ic

Enamel

~5–7 µm

Hexagonal HAp crystal

~40 nm

Enamel

e ur ct u r

Different part of enamel prism or rod: H-head. C-center, N-neck, T-tail, ES-enamel sheath

Different alignment of enamel prisms

ES

N T

C

st

o– cr

~25 mm

H

o an

~25 mm

6 Nanoindentation of Natural Materials

7

Basics of Hierarchical and Functionally Graded Structures

On the other side, dentine has a hybrid composite structure, that is, a collagen matrix reinforced with nanosized CaP crystals. Dentine also has c­ hannel-like microstructures (which supply the nutrition from the pulp region to the crown part of the teeth) called dentine tubules. Further, the interface between the dentine enamel junction (DEJ) is made with dome-shaped excavations (the irregular interface interlocks with two tissues, e.g., the hard enamel and the soft dentine). It is interesting to note that the enamel never delaminates in real life from the dentine despite millions or billions of recurring masticatory loading/unloading during piercing or cutting or grinding or chewing or crushing of any food items, or even during unexpected accidental collision. The scanning electron photomicrographs of different regions of the teeth are shown in Figure 1.4a–c. The detailed microstructure of enamel prisms along with enamel sheath with different orientation are shown in Figure 1.4a–b [7]. In contrast, the detailed microstructure of the dentine region comprising of dentine

IR R IR Acc.V Magn Dot 20.0 kV 0000× SE

R

IR

R Acc.V Magn Dot 20.0 kV 10000× BSE

10 µm

(a)

(b)

00

2.

10 µm (c)

5 µm

(d)

00 4. µm

00

6.

00

8.

.0 10

10 .00

0

0

4.0

0

6.0

0

8.0

0.0

2.0

µm

0

FIGURE 1.4 Scanning electron photomicrographs of enamel: (a) cross sectional view and (b) plan sectional view. (Reprinted from He, L. H., and M. V. Swain, Journal of the Mechanical Behavior of Biomedical Materials 1:18–29, 2008. With permission.) Photomicrographs of dentine: (c) SEM (Reprinted from Roy, S., and B. Basu, Materials Characterization 59:747–756, 2008. With permission.) and (d) AFM image. (R = enamel rod, IR = Inter-rod).

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Nanoindentation of Natural Materials

tubules is shown in Figure 1.4c [14]. The further detail of the dentine structure is illustrated with an atomic force photomicrograph shown in Figure 1.4d [13]. The analysis of the mechanical properties, for example, hardness, elastic modulus, fracture toughness, etc., are always complex for the different regions of teeth due to their hierarchical structure. Today, the nanoindentation technique is being used frequently on metals, metallic thin films, bulk ceramics, ceramic thin films, dense and porous ceramic coatings, quasicrystals, MEMS, NEMS, bio-materials, CMCs, MMCs, biological materials (e.g., tissue, bone, and dentine), polymers, polymer-clay nanocomoposites, ceramic-ceramic and/or metal-ceramic nanocomposites, etc. for the evaluation of mechanical properties, for example, hardness and Young’s modulus, including the fracture toughness at a scale compatible with the microstructural length scale, because it is at this scale that the issues of structural integrity are determined [15]. Therefore, it will be of extreme scientific importance and technological significance to understand how the mechanical probe interacts with the hierarchical microstructure at different regions, for example, enamel, dentine, and DEJ having different orientations (Figure  1.5). The interpretation of the mechanical behavior of teeth, a nano-bio-composite with different structural levels, shall need to be performed in correlation to its structural and compositional characteristics. The elaborative discussions and experimental results are appended in Chapters 3 to 5. Further, enamel is hard and brittle in nature. It possesses higher hardness (3–5 GPa c.f. 50 μN.s–1), the work value slightly decreased (Figure 2.24a). The plastic effects that denote the work done during indentation actually exist in the unloading curve [64]. Due to the varying plastic effect [64] the Young’s modulus values derived from the load function without any holding time might not be consistent. Thus, to get rid of this varying nature, a holding time of 180 s was applied to the same load function [63]. The work done during the indentation was greater compared to those of the previous condition; however, it did not follow any particular trend of variation with the loading rates (Figure 2.24b) [64]. The effect of holding time could minimize the plastic contribution to the unloading data of nanoindentation, but the hardness values were supposed to be affected by the creep effect. Therefore, to get a proper solution of the problem, holding times were varied in the range of ~10–1000 s at a fixed loading rate ~300 μN.s–1 for the maximum peak load of 6000 μN. The load function and the typical P-h plot for the same are reported in [63]. The Young’s modulus increased slightly (~23–26 GPa) with the holding times (Figure 2.25a) [63]. The corresponding work done also increased (~980– 1150 μN.μm) with the holding times but did not follow any particular increasing trend of variation (Figure 2.25b). The insignificant trend of variation was attributed to the constant loading rate for this load function. However, the little bit of variations in the E and work done with the holding times resulted due to the local variations within the osteonal lamella of the bone specimen [63]. 1200

2000

1100 1600

Work (µN.µm)

Work (µN.µm)

1000 900

1200

800 700

800

600 500

(a)

0

200 400 600 800 Loading rate (µN.s–1)

1000

0

(b)

400 600 800 200 Loading rate (µN.s–1)

1000

FIGURE 2.24 Variations of work done with the loading rates for loading functions (a) without holding time and (b) with holding time. (Data taken from Zaifeng, F., and J. Y. Rho, Journal of Biomedical Materials Research 67A:208–214, 2003.)

53

Nanoindentation of Bone

40

1400

35

Work (µN.µm)

1200

E (GPa)

30 25

1000

20 15 10

(a)

800 600

0

200

400 600 800 Holding time (s)

0

1000

200

(b)

400 600 800 Holding time (s)

1000

FIGURE 2.25 Variations of (a) Young’s modulus (E) and (b) work done with the holding time. (Data taken from Zaifeng, F., and J. Y. Rho, Journal of Biomedical Materials Research 67A:208–214, 2003.)

The Young’s modulus and hardness of a human cortical bone also exhibited significant load dependencies when indentations were performed on the thin and thick lamellae of cortical bone at varying loads of ~0.4–5 mN [59]. Interestingly, the Young’s modulus of the thick lamellar region decreased with increasing load whereas the thin lamellar region did not show any significant dependency on the load (Figure 2.26). Similar trend of variation was observed for hardness [59] with increasing load in the thin and thick lamellar regions of the cortical bone. This discrepancy in the mechanical properties of the thin and thick lamellae of bone might be plausibly due to the difference in the intrinsic structure, direction of orientation of the collagen fiber, etc. For thick lamellae, the 40

Thick lamellae Thin lamellae

E (GPa)

30 20 10 0

0

1

2

3 P (mN)

4

5

FIGURE 2.26 Variation of Young’s modulus (E) with load (P) in the osteonal thin and thick lamellae. (Data taken from Hengsberger, S. et al., Bone 30:178–184, 2002.)

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Nanoindentation of Natural Materials

relative density of the HAp crystals is higher compared to that of the thin lamellae. This provided higher resistance to polishing and, hence, higher Young’s modulus for thick lamellae of the bone. Again, the decreasing trend of Young’s modulus with increasing load (Figure 2.26) might be correlated to the orientation of the collagen fibers. It is plausible that with the increase in load, the longitudinal fiber orientation in the thick lamellae generated the probability of damage accumulation process compared to the transverse lying fibers present in the thin lamellae of the cortical bone, which ultimately resulted in the altogether decreasing trend but higher Young’s modulus and hardness of the thick lamellae of the cortical bone.

2.5 Summary The nanomechanical properties of human cortical bone are strong functions of (a) locations, (b) direction of orientation, (c) density, (d) state of hydration, and (e) loading conditions. The nanohardness and Young’s modulus of human cortical bone, on the other hand, are almost insensitive to the variations in age and gender. However, the fracture properties and the toughness of the cortical bone are significant functions of human age. The variations in the mechanical properties of bone with the above mentioned factors are mainly attributed to the variation in mineral density and its concentration as well as the differences in the orientation of the collagen fibers of the human cortical bone.

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59. Hengsberger, S., A. Kulik, and P. H. Zysset. 2002. Nanoindentation discriminates the elastic properties of individual human bone lamellae under dry and physiological conditions. Bone 30:178–184. 60. Hengsberger, S., A. Kulik, and P. H. Zysset. 2001. A combined atomic force microscopy and nanoindentation technique to investigate the elastic properties of bone structural units. European Cells and Materials 1:12–17. 61. Carter, D. R., and W. C. Hayes. 1976. Bone compressive strength: The influence of density and strain rate. Science 194:1174–1176. 62. Carter, D. R., and W. C. Hayes. 1977. The compressive behavior of bone as a two-phase porous structure. Journal of Bone and Joint Surgery: American Volume 59:954–962. 63. Zaifeng, F., and J. Y. Rho. 2003. Effects of viscoelasticity and time-dependent plasticity on nanoindentation measurements of human cortical bone. Journal of Biomedical Materials Research 67A:208–214. 64. Oliver, W. C., and G. M. Pharr. 1992. An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. Journal of Materials Research 7:1564–1583. 65. Oyen, M. L. 2006. Nanoindentation hardness of mineralized tissues. Journal of Biomechanics 39:2699–2702. 66. Franzoso, G., and P. K. Zysset. 2009. Elastic anisotropy of human cortical bone secondary osteons measured by nanoindentation. Journal of Biomechanical Engineering 131:021001. 67. Vanleene, M. 2006. Caractérisation multi-echelle des propriétés mécaniques de l’os. Université de Technologie de Compiègne.

3 Nanoindentation of Teeth: A Hard but Tough Hybrid Functionally Graded Composite Nilormi Biswas, Anoop Kumar Mukhopadhyay, and Arjun Dey

3.1 Introduction Tooth microstructure is an important factor in determining resilience against damage accumulation. It has specialized hierarchical structure starting from the smallest level to its final macrostructure. The load-bearing capacity of teeth is limited by the susceptibility of the enamel to fracture. However, tooth enamel is far from homogeneous and isotropic. The outer covering of the tooth is the enamel, the hardest substance of the human body. The second layer of tissue is the dentine. It is comparatively a much softer layer than enamel. It is bound to the hard enamel by a specialized layer, the dentineenamel junction. Deeper inside, the tooth is composed of vascularized soft connective tissue—dental pulp (Figure 3.1) [1]. The surface layer of tooth root is a thin layer of bony material, the cementum. Functionally, the tooth enamel sustains a wide range of imposed loads and contact induced stresses without failure, and teeth retain their shape while doing so [2]. In addition to the normal loads, enamel will be exposed to shear forces because of the various masticatory forces at different angles. Recent reports indicate that masticatory loading forces of teeth could be as low as 28 to more than 1200 N [3,4]. Hence, it is of extraordinary research interest to investigate how enamel sustains and survives such high forces for millions of cycles. A comparative study has been made of human and great ape molar tooth enamel (Figure 3.2). Nanoindentation techniques are used to map profiles of elastic modulus and hardness across sections from the dentine-enamel junction to the outer tooth surface. The measured data profiles overlap between species, suggesting a degree of commonality in material properties. Using established deformation and fracture relations, critical loads to produce function-threatening damage in the enamel of each species are calculated for characteristic tooth sizes and enamel thicknesses. The results suggest that differences in load-bearing

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Enamel

Crown

Dentine Gingiva Pulp Periodontal ligament

Root

Cementum

FIGURE 3.1 Schematic diagram of the transeverse section of a molar tooth. (Reprinted from Hsieh, Y. S. et al., Sensors 13:8928–8949, 2013. With permission.) Homo

Pan 2 mm

(a)

Gorilla

(b)

Pongo

(c)

(d)

FIGURE 3.2 Longitudinal sections of the molar tooth, (a) human (Homo), lower third; (b) chimpanzee (Pan), lower second; (c) orangutan (Pongo), lower second; (d) gorilla (Gorilla), upper first. (Reprinted from Lee, J. J. W. et al., ActaBiomaterialia 6:4560–4565, 2010. With permission.)

capacity of molar teeth in primates are less a function of underlying material properties than of morphology. In relation to this, it is important to know the mechanical properties and the deformation mechanisms of enamel in relation to its compositional and hierarchical microstructure. In comparison to enamel, the dentine has much higher organic component (Figure 3.3). Structurally and compositionally, as

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Nanostructure H C

Enamel prisms Enamel DEJ Dentine Pulp

N T ES

~10–20 µm

Microstructure

~40 nm HAp Crystals

Different part of enamel ~5–7 µm prism or rod: H-Head, C-Center, N-Neck, T-Tail, ES-Enamel sheath

Dentine tubule Peritubule ~2–5 µm Microstructure

Intertubular dentine ~67 nm Fibril

Nanostructure ~1.5 nm ~40 nm HAp Crystals

~8.5 nm Triple helix structure of collagen

FIGURE 3.3 Hierarchical structure of teeth.

odontoblasts penetrate into dentine tubules, dentine and pulp are usually considered as a vital complex in response to bacterial invasion. Hence, the major role of dentine is to protect the pulp rather than mastication, and so the mechanical property requirements of dentine are for toughness rather than hardness as for enamel. The microstructural hierarchy (Figure 3.3) and precise distribution of organic components endow dental hard tissues with excellent anisotropic mechanical properties, which ensures their lifetime survival in the mouth as a load bearing organ.

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Acc. V Magn 20.0 kV 10,000×

Dot BSE Enamel

8 µm

(a)

Acc. V Magn 20.0 kV 10,000×

(b)

Acc. V Magn 20.0 kV 5000×

Amalgam

Acc. V Magn 20.0 kV 5000×

Amalgam

10 µm

(c)

Dot SE

HAP

6 µm

10 µm

(d)

FIGURE 3.4 SEM photomicrographs of the nanoindentation impressions on (a) enamel (250 mN loading), (b) HAp (200 mN loading), (c) amalgam (150 mN loading), and (d) amalgam (250 mN plus 900 s holding), respectively. (Reprinted from He, L. H., and M. V. Swain, Journal of Dentistry 35:431– 437, 2007. With permission.)

The residual nanoindentation impression as shown in Figure 3.4 compares the surrounding deformations for different materials. Figure 3.4a showed residual deformation in enamel without any prominent cracks, whereas HAp exhibited typical brittle behavior under sharp indentation as shown in Figure 3.4b. Amalgam, due to its low hardness, had larger indents at lower forces, and no cracks were detected around the indents in amalgam (Figure 3.4c and d) [6]. The single HAp crystallites are the basic structural element in enamel [7–10]. These crystallites form bundles, which frame the micro-scale building blocks of enamel. The crystallites bundles have rod-like structure. These are often referred to as prisms (p) interspersed in a biopolymer that exist as the interprismatic matrix (IPM). The next level of structural hierarchy in enamel is determined on the basis of two factors: (1) their spatial arrangement and (2)  the respective spatial orientations of these arrangements. Incidentally there can be two varieties of such structure. One is called the radial enamel structure, and the other is called the decussating enamel structure. The three-dimensional distribution of different enamel varieties in a tooth structure actually forms the next level of the hierarchical structure. The basic idea behind this model [7] is simple. As depicted in ref. [7–9],

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it is a quasi-self-similar development of different levels of hierarchical microstructure. The most common element in all of these levels is the fact that at any given level there exists a hard mineral phase and/or an assembly of hard mineral phase. Depending on the level of hierarchical structure, in each level the hard phase is always enveloped in a soft polymeric protein matrix even starting from the lowest level of hierarchy in the microstructure of a given tooth. Thus, the gradual development to a level (n) of such a hierarchical microstructure translates out to existence of (n+1) hard particles embedded in a soft matrix. In other words, under the assumption of a constant mineral volume fraction of each hierarchical level, the more the level of hierarchy, the larger the amount of biopolymeric protein matrix. This in turn leads to localized enhancement in damage tolerance. Thus, the simplified model considers that it is the hard particles that necessarily always carry the entire tensile load. It also assumes that it is the soft biopolymeric matrix phase that itself shears out and in the process transfers the tensile load onto the hard particles. However, it is obvious that the presence of a higher amount of the biopolymeric protein phase will induce a general decrease in Young’s modulus. It follows further that there is almost a certain chance that classical tensile strength of such a composite will be much lower than the composite that contains, for example, the least of the biopolymeric matrix phase [7]. For developing biomimetic restorative materials and for improving the execution of clinical dental preparations, it is extremely important to understand the mechanisms and mechanics of both stress redistribution and dissipation in the tooth. To develop this kind of understanding, in turn, the knowledge of the mechanical properties and microstructural features of enamel and dentine evolves as matters of extraordinary research importance. Recently, with the development of nanoindentation techniques, small volume samples such as dental hard tissues can be measured more accurately on a small selected region of the specimen, non-destructively, and the specimen preparation is less time consuming as the test can be done on a bulk polished specimen [4]. The aim of this chapter is to review the application of nanoindentation technique to dental hard tissue studies and discuss the relevant implications in respect to the microstructural and compositional characteristics of these natural biocomposites. Nanoindentation studies on enamel and dentine are reviewed. Further, on the basis of our recent investigations, enamel is used to illustrate the importance of interrelation between the local microstructure and organic components on the mechanical behavior of these natural biocomposites.

3.2 Formation and Structure of Tooth Although tooth development is a continuous process, the developmental history of teeth is divided here into several morphologic “stages” for

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descriptive purposes. While the size and shape of individual teeth are different, they pass through similar stages of bilogical evolutional development. Tooth development or odontogenesis is the complex process by which teeth form from embryonic cells, grow, and erupt into the mouth. For human teeth to have a healthy oral environment, all parts of the tooth must develop during appropriate stages of fetal development. The tooth germ is an aggregation of cells that eventually forms a tooth. These cells are derived from the ectoderm of the first pharyngeal arch and the ectomesenchyme of the neural crest. The tooth germ is organized into three parts: the enamel organ, the dental papilla, and the dental sac or follicle [11]. 3.2.1 Formation and Structure of Enamel The enamel tissue is formed in two stages: the secretory and maturation stages [11]. Initially during the secretion stage, the ameloblasts develop at their apical (secretory) surface, by the Tomes’ process. The secretory stage comes to an end when an ameloblast has completed its principal secretory activities and the developing enamel has reached its full thickness locally. The ameloblast cells undergo a series of cytological changes in preparation for enamel maturation resulting in a sharp increase in matrix degradation and replacement by tissue fluid. At the final stage of development, however, the mineral volume percentage increases steeply from 10–20% to 80–90%. One of the most important steps in the enamel maturation stage is the organization of HAp crystals into bulk enamel, which is achieved by extracellular protein matrices secreted from the Tomes’ processes of ameloblasts (Figure 3.5). Amelogenin undergoes self-assembly to form nano-spheres possibly favoring crystallite elongation along the c-axis, resulting in long and thin crystals arranged in parallel [12]. The nanocrystallites grow in thickness as enamel mineralization proceeds and the organic matrix is progressively removed. This allows the crystallites to come into contact and may induce mechanical stresses distorting the atomic arrangement and forming dislocations. Moreover, the external surfaces of the crystallites are not smooth but display frequent step-like irregularities [13]. The top of the fish-scale-like structure of enamel has a rounded discontinuity that is basically some protein fragments retained primarily at the incisal edges and proximal sides of rod bound­ aries. These discontinuities play two important mechanical functions. First, they define three-dimensional cleavage planes that will deflect cracks [14,15], thus preventing them from advancing straight through enamel and causing catastrophic macromechanical failure. The crack instead spreads laterally over a larger volume. Second, the presence of minute quantities of protein remnants allows limited differential movement between adjacent rods [16], which also reduces stresses without crack growth. Enamel is known to be much more flexible and tough than its major component [15,17]. This is due to the minor components of enamel, namely the protein remnants and water, which have a profound plasticizing effect [18].

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The smallest structural units are HAp crystallites. In a cross-­sectional view [19] they approximately resemble roughly flattened hexagons. Typically the hexagons are about 68 nm wide with a mean thickness of 26 nm [20]. The HAp crystallites are arranged and bundled together by protein molecules into larger-scale “keyhole-shaped” structures (Figure 3.5a), called enamel rods. Inside a single rod, the directional arrangement of the hydroxyapatite crystallites varies. The crystallites lie parallel to the rod axis in the central part of the rod and those near the edge of the rod usually have an angle of ~45° to the longitudinal axis of the rods (see Figure 3.5b) [21]. The arrangement of rods determines the next level unit, namely enamel types, for example, middle enamel (Figure 3.5c) and inner enamel (Figure 3.5d). The rods have a diameter of ~5 μm and are encapsulated by thin protein rich sheaths that are arranged parallel in a direction perpendicular to the ­dentine-enamel

2 µm (a)

(c)

(b)

2 µm

2 µm

2 µm (d)

FIGURE 3.5 SEM photomicrographs of different regions of enamel: (a) outer enamel showing keyhole shaped and (b) sausage shaped almost 45° alignment of enamel rods or prisms, (c) middle enamel and (d) inner enamel showing hybrid and longitudinal alignment of enamel rods or prisms, respectively. (Reprinted from Biswas, N. et al., Journal of the Institution of Engineers (India): Series D Metallurgical & Materials and Mining Engineering 93:87–95, 2012. With permission.)

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junction (DEJ) from dentine to the outer enamel surface. In some areas, the enamel rods are seen to twist together or change their direction slightly to reinforce the whole structure [22]. 3.2.1.1 Differences between Enamel and HAp The first major difference between HAp and enamel is that HAp is a typical monolithic crystalline ceramic while the enamel is nanobio hybrid composite. It is thus obvious that like any other brittle ceramic, HAp is also a brittle and hard solid. Thus, the indentation response of HAp is analogous to those of other brittle ceramics [24]. The major distinctive feature of enamel structure, as discussed above, is the existence of the biopolymeric protein films in between the crystallites and in between the enamel rods. These films provide by their viscoelastic deformation nature a stress cushioning effect that is otherwise absent in sintered HAp ceramics. This viscoelastically deformable film redistributes and dissipates the externally applied stresses at the microstructural level and in the process saves the HAp crystallites from getting fractured [25]. That is why enamel has relatively higher fracture toughness than that of pure bulk HAp [17]. In enamel, it is believed that the layered crystallites may play a major role in crack deflection and ligamentary crack bridging [17]. In addition, it has been already reported that nanoscale hardness and Young’s moduli measured in the head area of enamel rods registered magnitudes higher than those evaluated in the tail area of enamel rod and in the inter-prism region [24,26]. It is suggested [24,26] that higher amount of soft organic tissue in the tail and inter-prism regions could be a cause behind the relatively lower magnitude of nanoscale mechanical properties. It is also believed that a simultaneously important factor in this aspect could be the localized change in crystal orientations in the tail and inter-prism regions in comparison to that in the head region of enamel rods [24–26]. 3.2.2 Formation and Structure of Dentine and DEJ As with enamel, the formation of dentine starts with the activation of cells, namely odontoblasts [11]. As the dental mesenchyme cells begin to differentiate into odontoblasts, they begin to secrete several molecules that promote mineralization of the extracellular matrix. These proteins include tenascin, bono-1, dental sialophosphoprotein, and alkaline phosphatase [11]. Finally, as the odontoblast phenotype emerges, osteonectin and type I collagen are secreted as components of the extracellular matrix and form an approxi­ mately 10–30 μm wide unmineralized zone between the mineralized dentine and odontoblasts, namely predentin. Thus, the mineralization pattern of dentine is different from that of enamel. Here, the mineralization is devel­ oped layer by layer immediately after the secretion of the protein matrix. Similarly, as described above, the matrix proteins in predentin (newly ­secreted p ­ rotein-rich unmineralized dentine) regulate the growth and deposition of the HAp crystals.

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Moreover, odontoblasts synthesize and secrete type I collagen to the predentin layer. The details of the hierarchical structure for a typical collagen are described in [27–30]. In predentin, collagen molecule fibers aggregate with their long axes in parallel into fibrils, which further arrange to bundles, possibly with the help of proteoglycans [27] to form the collagen component of mature dentine. The dentine structurally consists of tubular, peritubular, and intertubular dentine (Figure 3.3). As a result of cell activity of odontoblasts and due to the insertion of the odontoblasts process into the mineralized structure, dentin forms its tubular pattern characteristic feature (Figure  3.3). From the outer surface of the dentine to the area nearest the pulp, these tubules follow an S-shaped path. The diameter and density of the tubules are greatest near the pulp [27]. Tapering from the inner- to the outermost surface, they have a diameter of 2.5 nm near the pulp, 1.2 nm in the middle of the dentine, and 900 nm at the DEJ [27]. Their density is 59,000 to 76,000 per square millimeter near the pulp, whereas the density is only half as much near the enamel. Dentinal tubules give high permeability to the dentine. In addition to an odontoblast process, the tubule contains dentinal fluid. This dentinal fluid is a complex mixture of proteins, such as albumin, transferrin, tenascin, and proteoglycans [28]. In addition, there are branching canalicular systems that connect to each other to form a complex network. Dentine tubules are surrounded by highly calcified peritubular dentine, which is more radio-opaque and electron-dense than intertubular dentine. The less calcified intertubular structure, which contains more organic material than peritubular dentine, comprises the remaining dentine body and lies between regions of peritubular dentine [28]. The ratios (Rpt/HAp) of protein to HAp are different in different regions of tooth [29]. For instance, (Rpt/HAp)Dentine> (Rpt/HAp)DEJ> (Rpt/HAp)Enamel. Particularly, it occurs as one transit from enamel through the DEJ to the dentine zone. The width of the transition region can vary depending on the teeth concerned. Measured for five teeth [29] the average widths of the occlusal DEJ was ~13 μm, which was nearly twice as wide as the cervical DEJ width of about 6 μm. This large difference indicates diverse structural differences between the microstructures of the DEJ and the organic matrix in the dentine. At the early stages of tooth morphogenesis the formation of the DEJ begins. The process is complex and far from well understood. However, the general viewpoint is that this process is linked to a mixture of dentinal proteins. These proteins are secreted by odontoblasts and enamel proteins from ameloblasts [29].

3.3 Nanomechanical Property Evaluation of Tooth Nanoindentation has become a common technique for the determination of local mechanical properties of structural features in biological hard

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tissues [31–36]. Although nanoindentations only examine a thin surface layer (