An Introduction to Biomaterials - Second Edition Textbook

The BIOMEDICAL ENGINEERING Series Michael R. Neuman, Series Editor Second Edition AN INTRODUCTION TO BIOMATERIALS Edited by Jeffrey O. Hollinger Michael R. Neuman, Series Editor Published Titles Electromagnetic Analysis and Design in Magnetic Resonance Imaging, Jianming Jin Endogenous and Exogenous Regulation and Control of Physiological Systems, Robert B. Northrop Artificial Neural Networks in Cancer Diagnosis, Prognosis, and Treatment, Raouf N.G. Naguib and Gajanan V. Sherbet Medical Image Registration, Joseph V. Hajnal, Derek Hill, and David J. Hawkes Introduction to Dynamic Modeling of Neuro-Sensory Systems, Robert B. Northrop Noninvasive Instrumentation and Measurement in Medical Diagnosis, Robert B. Northrop Handbook of Neuroprosthetic Methods, Warren E. Finn and Peter G. LoPresti Angiography and Plaque Imaging: Advanced Segmentation Techniques, Jasjit S. Suri and Swamy Laxminarayan Analysis and Application of Analog Electronic Circuits to Biomedical Instrumentation, Robert B. Northrop Biomedical Image Analysis, Rangaraj M. Rangayyan Foot and Ankle Motion Analysis: Clinical Treatment and Technology, Gerald F. Harris, Peter A. Smith, Richard M. Marks Introduction to Molecular Biology, Genomics and Proteomic for Biomedical Engineers, Robert B. Northrop and Anne N. Connor Signals and Systems Analysis in Biomedical Engineering, Second Edition, Robert B. Northrop An Introduction to Biomaterials, Second Edition Jeffrey O. Hollinger Michael R. Neuman, Series Editor AN INTRODUCTION TO BIOMATERIALS Second Edition Edited by Jeffrey O. Hollinger Boca Raton London New York CRC Press is an imprint of the Taylor & Francis Group, an informa business CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2012 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 Version Date: 20111010 International Standard Book Number-13: 978-1-4398-1257-0 (eBook - PDF) 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. 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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 Contents Preface...............................................................................................................................................ix Editor�� xiii Contributors�� xv Chapter 1 Consensus Definitions, Fundamental Concepts, and a Standardized Approach to Applied Biomaterials Sciences��1 Jeffrey O. Hollinger Section I Biology, Biomechanics, Biomaterial Interactions: Wound Healing Biology Chapter 2 Cutaneous Wound Pathobiology: Raison d’etre for Tissue Engineering......................7 Lauren K. Macri and Richard A. F. Clark Chapter 3 Osseous Wound Healing............................................................................................. 23 Ayesha Nawab, Mark Wong, Daniel Kwak, Lyndsey Schutte, Aditi Sharma, and Jeffrey O. Hollinger Section I Biology, Biomechanics, Biomaterial Interactions: Cellular Mechanics Chapter 4 Cell and Tissue Mechanobiology................................................................................ 43 Wei-hui Guo, Pedro Alvarez-Urena, and Yu-li Wang Section I Biology, Biomechanics, Biomaterial Interactions: Materials–Host Interactions Chapter 5 Cell–Material Interactions: Fundamental Design Issues for Tissue Engineering and Clinical Considerations........................................................ 57 Gregory M. Harbers and David W. Grainger Chapter 6 Host Response to Biomaterials.................................................................................... 91 Dolly J. Holt and David W. Grainger Chapter 7 Protein Adsorption at the Biomaterial–Tissue Interface........................................... 119 Philip LeDuc, Lina Gonzalez, Britta M. Rauck, and Yadong Wang v vi Contents Section II Biomaterials Testing, Statistics, Regulatory Considerations, Intellectual Property: Standardized Materials Testing Chapter 8 In Vitro Testing of Biomaterials................................................................................ 137 Jinku Kim, Abiraman Srinivasan, and Jeffrey O. Hollinger Chapter 9 Assessment of Biomaterials: Standardized In Vivo Testing...................................... 157 Vivek P. Raut, Thomas E. Patterson, Joseph C. Wenke, Jeffrey O. Hollinger, and George F. Muschler Section II Biomaterials Testing, Statistics, Regulatory Considerations, Intellectual Property: Statistics Chapter 10 Basic Principles of Statistics: Considerations for Biomaterials Engineers�� 175 Robert T. Rubin, Jessica McKlveen, Amanda S. Fultz, and Michael E. Rhodes Chapter 11 Therapy Development, Animal Testing, and Regulatory Issues............................... 191 Charles E. Hart, Mark Citron, Andrea Loewen-Rodriguez, and Jeffrey O. Hollinger Chapter 12 Fundamentals of Patenting for the Biomaterials Scientist........................................207 Earl M. Douglas Section III Biomaterials Compositions Chapter 13 Proteins and Amino Acid–Derived Polymers........................................................... 227 Joshua C. Haarer, Beau D. Inskeep, and Kay C. Dee Chapter 14 Three-Dimensional Fibrin Constructs in Tissue Engineering.................................. 249 Bill Tawil and B. Wu Chapter 15 The Poly(α-esters)..................................................................................................... 263 Dennis D. Jamiolkowski and Edward J. Dormier Chapter 16 Biomedical Polyurethanes......................................................................................... 281 Michael Szycher vii Contents Chapter 17 Polymers Derived from l-Tyrosine........................................................................... 301 Joachim Kohn, Aniq Darr, and Jaap Schut Chapter 18 Poly(Propylene Fumarate)......................................................................................... 317 Xinfeng Shi, Allan Henslee, Diana Yoon, F. Kurtis Kasper, and Antonios G. Mikos Chapter 19 Hyaluronan................................................................................................................ 331 John H. Brekke, Gregory E. Rutkowski, and Kipling Thacker Chapter 20 Complex Polysaccharides: Chitosan and Alginate.................................................... 359 Frank Rauh, Michael Dornish, Reva Street, and Arun R. Shrivats Chapter 21 Collagen: A Natural Biomaterial for Tissue Engineering......................................... 379 A. Hari Reddi and Pedro Alvarez-Urena Chapter 22 Polyphosphazenes..................................................................................................... 385 Lakshmi S. Nair, Yusuf M. Khan, and Cato T. Laurencin Chapter 23 Biologically Active Glasses....................................................................................... 401 Hoda M. Elgendy, Dennis E. Curtin, and Ahmed El-Ghannam Chapter 24 Silk-Based Biomaterials: Biology, Properties, and Clinical Applications................ 421 Guokui Qin and David L. Kaplan Chapter 25 Calcium-Based Bioceramics: Biology, Properties, and Clinical Applications......... 433 Stefanie Crumlett, Brian Singleton, Sunho Oh, and Joo L. Ong Section IV Biomaterials Applications Chapter 26 Tissue Engineering of Skin....................................................................................... 447 Heather M. Powell and Steven T. Boyce Chapter 27 Polymeric Biomaterials for Drug and Nucleic Acid Delivery.................................. 461 Yihua Loo and Kam W. Leong Chapter 28 Orthopedic Prostheses and Joint Implants................................................................ 487 Shanfeng Wang, Lichun Lu, Bradford L. Currier, and Michael J. Yaszemski viii Contents Chapter 29 Bone and Biomaterials..............................................................................................509 Syam P. Nukavarapu, James S. Wallace, Hoda M. Elgendy, Jay R. Lieberman, and Cato T. Laurencin Chapter 30 Functional Regeneration of Synovial Joints In Vivo: The Role of Biomaterials and Scaffold Design............................................................................. 529 Chang Hun Lee and Jeremy J. Mao Chapter 31 Tissue Engineering, Biomaterials, and the Nervous System.................................... 543 Lauren E. Kokai, Lizzie Y. Santiago, and Kacey G. Marra Chapter 32 Ligaments, Biomaterials, and Tissue-Engineering Opportunities............................ 565 Joseph Freeman, Valerie I. Walters, and Albert L. Kwansa Chapter 33 Cardiovascular Tissue Engineering and Biomaterials�� 583 Priya R. Baraniak and William R. Wagner Index�� 613 Preface The second edition of An Introduction to Biomaterials consists of 33 chapters that have been conceived and organized as a practical educational road map to provide a fundamental understanding of key families of biomaterials and underscore their potential applications in clinical therapeutics. These 33 chapters are organized into four sections: Section I: Biology, Biomechanics, Biomaterials Interactions Section II: Biomaterials Testing, Statistics, Regulatory Considerations, Intellectual Property Section III: Biomaterials Compositions Section IV: Biomaterials Applications The organizational format for the first 12 chapters provides basic instructional guidance on the seminal steps that must be addressed as a biomaterial transitions from an idea to the laboratory bench, and the design and development mature through specific testing paradigms and comply with regulatory guidance to the clinical patient. Consequently, two dedicated chapters in this edition emphasize wound healing. What does the “body” do when a biomaterial is implanted? When the “body” incurs a wound, how does it heal? What are the cellular and humoral responses to biomaterial implantation? A unique feature of this edition is the emphasis on biological principles. This emphasis is purposeful: Biology will direct and determine the performance of biomaterials in the body. Consequently, it is crucial that those who design biomaterials for applied clinical purposes are adequately educated on “host biology.” Chapters 2–7 provide the biological fundamentals for the biomaterials scientist. The skin and its hierarchal organization, healing, and regeneration are addressed in Chapter 2. The logic for a chapter on skin is that it may be considered a prototypic “soft tissue”; it is the largest organ in the body and the first “barrier” crossed by the biomaterial as it enters the body. The second most commonly transplanted tissue is bone (blood is the most commonly transplanted tissue); it is detailed in Chapter 3 in terms of organization, repair, healing, remodeling, and regeneration. Both Chapters 2 and 3 are new to the second edition. Chapter 4 is an exciting new chapter for the second edition, and the significance of cell biomechanics and biomaterials is discussed. Underscored in this chapter are the crucial relationships across the individual cells and consortia of cells that provide key cues in determining whether biomaterials will have a successful host relationship, that is, whether the biomaterial will be biocompatible. The interface between the biomaterial and the host is lucidly detailed in Chapter 5, and Chapters 6 and 7 on cell–cell interactions and cell-signaling and the inflammatory and complement cascades have been updated. The interaction of biomaterials is described in detail in Chapter 6, in which the biomaterial–host interface is eloquently presented and the importance of proteins underscored, especially with regard to the biomaterial–host interface. The adsorption of proteins is explained with respect to the biological constraints, including solid–liquid interfaces, diffusion, protein types, and the specific interactions of protein-adsorbed biomaterials. Throughout the text, chapter authors introduce standard, accepted terminology and explain and define nomenclature in a didactic, tutorial approach. Fundamental concepts and the standardization in biomaterials design, development, and testing for applied indications are emphasized. Further, the authors diligently define terminology and provide citations and resources for an expanded explanation. For example, the key to successful applied biomaterials technology is that the biomaterial is biocompatible. As such, biocompatibility is defined as the ability of a material to perform with an appropriate host response in a specific application (Williams 1987). It is therefore noteworthy that the second edition emphasizes and details host responses in dedicated chapters, including ix x Preface Chapters 5 through 7, with highly instructional text and supportive figures. Moreover, it follows that with the goal of biomaterials transitioning to the clinic, the student must be aware of and knowledgeable in the conceptual biological basis of “an appropriate host response in a specific application.” Consequently, the second edition includes both new and revised chapters on cells, tissues, and signaling molecules in wound healing cascades. The second edition includes two highly revised chapters (Chapters 8 and 9) that underscore standardized materials testing exploiting in vitro and in vivo paradigms consistent with regulatory guidelines. Moreover, many regulatory standards for materials assessments are provided as tables in the chapters, including the International Standard Organization (ISO) and the American Society for Testing of Materials (ASTM). Thus, the text is an indispensible resource for planning a road map for applied biomaterials development, testing, and biocompatibility validation. Bringing an idea from the laboratory to the clinic requires the execution of a systematic series of stringent, standardized laboratory assays and preclinical experimentation. The data generated from this body of work must be tested and scrutinized using specific statistical methodology. Therefore, a new chapter (Chapter 10) in the second edition provides an introductory, yet sufficiently comprehensive scope on the basic principles of statistics. Chapter 11 is an updated chapter with new U.S. Food and Drug Administration (FDA) information on the highly complex biomaterials– biological issues relevant to patient applications. Chapter 12 is unique to a biomaterials textbook; it emphasizes an element that is omitted in classic biomaterials texts: intellectual property. Terms such as patent disclosure are introduced and described, as well as functional roles of the Patent and Trademark Organization (PTO), the agency that issues patents to inventors. This new chapter lucidly and instructionally describes how an idea may be translated into an issued patent with specific claims that will protect the inventor’s biomaterial sciences and technology and could lead to commercialization. It was beyond the scope of this textbook to address an inestimable variety of possible clinical opportunities matched to an equally vast number of classes and types of biomaterials. Therefore, this textbook emphasizes the path forward from the laboratory through regulatory avenues that would most likely be encountered or that are most frequently exploited by scientists and clinicians who design and develop biomaterials intended for clinical applications. Chapters 13 through 25 present and discuss 13 important classes of biomaterials where fundamental and applied research are active and clinical applications compelling. Performance properties, synthesis and testing, as well as applied opportunities are also described in these chapters. Commonly matched clinical applications to many of these biomaterials families are addressed in Chapters 26 through 33. The second edition of the textbook has been significantly improved over the first edition that was published in 2006. The improvements include 9 completely new chapters and 24 chapters that have been updated and revised with new accomplishments and contemporary data. The authors include extensive references, and therefore, the inquisitive student has every opportunity to explore a particular subject of interest in more detail; this is a valuable educational tool. The second edition is a comprehensive, yet manageable didactic work that will be an invaluable desk reference and instructional textbook for undergraduates, graduate students, postdoctoral fellows, and seasoned investigators working in the exciting field of applied biomaterials sciences. The second edition of the textbook includes a question and answer supplement. Each chapter has either questions or problems that emphasize key chapter learning objectives. The answers provided to academic instructors in the supplement are derived from the chapter. Questions and problems will re-enforce chapter educational themes, specific concepts and fundamental objectives presented by the authors. Following reading of the chapter, there will be a synergistic instructional benefit for the students in responding to the questions and problems. As the editor of the second edition, I am extremely grateful to my editorial sidekick and chief nitpicker, Amy Donovan, who painstakingly scrutinized every word of every chapter, and to the coauthors of each chapter who committed extensive time and effort in writing their chapters and Preface xi complied with my gentle urging that their chapters had to be an educational jewel for undergraduate, graduate students, and workers in the exciting field of biomaterials sciences. My goal as an editor is to inspire keen young minds, invigorate seasoned ones, and put a compelling resource on the desk of biomaterials scientists and clinicians. REFERENCE Williams, D. F. 1987. Definitions in biomaterials. In Proceedings of a Consensus Conference of the European Society of Biomaterials (1986). Chester: Elsevier. Editor Dr. Jeffrey O. Hollinger graduated from Hofstra University in 1969 and received a dental degree and PhD from the University of Maryland in 1973 and 1981, respectively. In addition, he completed a dental residency program and craniofacial fellowship in the U.S. Army Dental Corps. Since 2000, Dr. Hollinger has been a tenured professor at Carnegie Mellon University (CMU) in the departments of biomedical engineering and biological sciences. He is the director of the Bone Tissue Engineering Center at CMU. From 1993 to 2000, he was a tenured professor at the Oregon Health Sciences University in the departments of surgery and developmental biology, and he directed the Northwest Wound Healing Center. In 1993 Dr. Hollinger retired from the U.S. Army as a colonel after serving 20 years of active duty. During that period, he was the director of the Army’s Bone Program, as well as the director of the Department of Physiology and Biochemistry at the U.S. Army Institute of Dental Research at the Walter Reed Army Medical Center in Washington, DC. He has over 35 years of experience in bone regeneration using biological factors, biomaterials, and preclinical animal models. Dr. Hollinger has received numerous federal grants as the principal investigator (NIH, NSF, DoD, and NIST) focusing on applied and fundamental sciences for bone regeneration and is engaged with several industrial groups emphasizing bone regenerative therapeutics, as well as serving on corporate boards. Dr. Hollinger has several patents and has licensed technology developed in his lab. He received the prestigious Clemson Award in biomaterials in 2008. He has over 250 peer-reviewed publications, abstracts, book chapters, and books. xiii Contributors Pedro Alvarez-Urena Department of Biomedical Engineering Carnegie Mellon University Pittsburgh, Pennsylvania Priya R. Baraniak Department of Biomedical Engineering Georgia Institute of Technology Atlanta, Georgia Steven T. Boyce Department of Surgery University of Cincinnati Cincinnati, Ohio John H. Brekke Bioactive Regenerative Therapeutics, Inc. Duluth, Minnesota Mark Citron Montclair, New Jersey Richard A. F. Clark Department of Biomedical Engineering Stony Brook University Stony Brook, New York Stefanie Crumlett Department of Biomedical Engineering University of Texas at San Antonio San Antonio, Texas Bradford L. Currier Department of Orthopaedic Surgery Mayo Clinic Rochester, Minnesota Dennis E. Curtin Fayetteville, North Carolina Aniq Darr New Jersey Center for Biomaterials Rutgers University Piscataway, New Jersey Kay C. Dee Department of Applied Biology and Biomedical Engineering Rose-Hulman Institute of Technology Terre Haute, Indiana Edward J. Dormier Ethicon, Inc. Somerville, New Jersey Michael Dornish FMC BioPolymer/NovaMatrix Sandvika, Norway Earl M. Douglas BioMimetic Therapeutics, Inc. Franklin, Tennessee Hoda M. Elgendy Eastern Virginia Medical School Norfolk, Virginia Ahmed El-Ghannam Department of Mechanical Engineering and Engineering Sciences University of North Carolina Charlotte, North Carolina Joseph Freeman Virginia Tech–Wake Forest School of Biomedical Engineering and Sciences Blacksburg, Virginia Amanda S. Fultz Ross University School of Veterinary Medicine St. Kitt’s and Nevis, West Indies Lina Gonzalez Department of Mechanical Engineering Carnegie Mellon Pittsburgh, Pennsylvania xv xvi Contributors David W. Grainger Department of Pharmaceutics University of Utah Salt Lake City, Utah Yusuf M. Khan Department of Orthopaedic Surgery University of Connecticut Farmington, Connecticut Wei-hui Guo Department of Biomedical Engineering Carnegie Mellon University Pittsburgh, Pennsylvania Jinku Kim Department of Biomedical Engineering Carnegie Mellon University Pittsburgh, Pennsylvania Joshua C. Haarer Boston Scientific St. Paul, Minnesota Gregory M. Harbers PercuPort Santa Clarita, California Charles E. Hart Advanced BioHealing Brentwood, Tennessee Allan Henslee Department of Bioengineering Rice University Houston, Texas Dolly J. Holt Department of Bioengineering University of Utah Salt Lake City, Utah Beau D. Inskeep Department of Bioengineering Clemson University Clemson, South Carolina Dennis D. Jamiolkowski Ethicon, Inc. Somerville, New Jersey Joachim Kohn New Jersey Center for Biomaterials Rutgers University Piscataway, New Jersey Lauren E. Kokai Allergan Medical Santa Barbara, California Daniel Kwak Department of Neonatal–Perinatal Medicine Primary Children’s Medical Center Salt Lake City, Utah Albert L. Kwansa Virginia Tech–Wake Forest School of Biomedical Engineering and Sciences Blacksburg, Virginia Cato T. Laurencin Department of Orthopaedic Surgery University of Connecticut Farmington, Connecticut Philip LeDuc Department of Mechanical Engineering Carnegie Mellon University Pittsburgh, Pennsylvania David L. Kaplan Department of Biomedical Engineering Tufts University Medford, Massachusetts Chang Hun Lee College of Dental Medicine Columbia University New York, New York F. Kurtis Kasper Department of Bioengineering Rice University Houston, Texas Kam W. Leong Department of Biomedical Engineering Duke University Durham, North Carolina xvii Contributors Jay R. Lieberman Department of Orthopaedic Surgery University of Connecticut Farmington, Connecticut Lakshmi S. Nair Department of Orthopaedic Surgery University of Connecticut Farmington, Connecticut Andrea Loewen-Rodriguez Advanced BioHealing La Jolla, California Ayesha Nawab Department of Oral and Maxillofacial Surgery University of Houston Houston, Texas Yihua Loo Institute of Bioengineering and Nanotechnology Singapore, Indonesia Lichun Lu Department of Tissue Engineering and Biomaterials Mayo Clinic Rochester, Minnesota Lauren K. Macri Department of Biomedical Engineering Stony Brook University Stony Brook, New York Jeremy J. Mao Columbia University New York, New York Kacey G. Marra Departments of Surgery and Bioengineering University of Pittsburgh Pittsburgh, Pennsylvania Jessica McKlveen Department of Psychiatry, Neuroscience Program University of Cincinnati College of Medicine Cincinnati, Ohio Antonios G. Mikos Departments of Bioengineering and Chemical and Biomolecular Engineering Rice University Houston, Texas George F. Muschler Department of Biomedical Engineering and Orthopaedic Surgery Cleveland Clinic Cleveland, Ohio Syam P. Nukavarapu Department of Orthopedic Surgery University of Connecticut Farmington, Connecticut Sunho Oh Department of Biomedical Engineering University of Texas at San Antonio San Antonio, Texa Joo L. Ong Department of Biomedical Engineering University of Texas at San Antonio San Antonio, Texas Thomas E. Patterson Department of Biomedical Engineering Cleveland Clinic Cleveland, Ohio Heather M. Powell Department of Materials Science and Engineering Ohio State University Columbus, Ohio Guokui Qin Department of Biomedical Engineering Tufts University Medford, Massachusetts Britta M. Rauck Department of Bioengineering University of Pittsburgh Pittsburgh, Pennsylvania Frank Rauh FMC BioPolymer/NovaMatrix Ewing, New Jersey xviii Contributors Vivek P. Raut Department of Biomedical Engineering Cleveland Clinic Cleveland, Ohio Arun R. Shrivats Department of Biomedical Engineering Carnegie Mellon University Pittsburgh, Pennsylvania A. Hari Reddi Department of Orthopedic Surgery University of California at Davis Sacramento, California Brian Singleton Department of Biomedical Engineering University of Texas at San Antonio San Antonio, Texas Michael E. Rhodes Department of Biology Saint Vincent College Latrobe, Pennsylvania Abiraman Srinivasan Department of Biomedical Engineering Carnegie Mellon University Pittsburgh, Pennsylvania Robert T. Rubin Department of Psychiatry and Mental Health Veterans Affairs Greater Los Angeles Healthcare System Los Angeles, California Reva Street Department of Biomedical Engineering Carnegie Mellon University Pittsburgh, Pennsylvania Gregory E. Rutkowski School of Chemical Engineering University of Minnesota Duluth Duluth, Minnesota Michael Szycher Sterling Biomedical Lynnfield, Massachusetts Lizzie Y. Santiago Freshman Engineering Program West Virginia University Morgantown, West Virginia Bill Tawil Department of Bioengineering University of California Los Angeles Los Angeles, California Jaap Schut New Jersey Center for Biomaterials Rutgers University Piscataway, New Jersey Kipling Thacker Lifecore Biomedical Chaska, Minnesota Lyndsey Schutte Department of Biomedical Engineering Carnegie Mellon University Pittsburgh, Pennsylvania Aditi Sharma Department of Biomedical Engineering Carnegie Mellon University Pittsburgh, Pennsylvania Xinfeng Shi Department of Bioengineering Rice University Houston, Texas William R. Wagner Department of Surgery University of Pittsburgh Pittsburgh, Pennsylvania James S. Wallace School of Medicine University of Connecticut Farmington, Connecticut Valerie I. Walters Virginia Tech–Wake Forest School of Biomedical Engineering and Sciences Blacksburg, Virginia xix Contributors Shanfeng Wang Department of Tissue Engineering and Biomaterials Mayo Clinic Rochester, Minnesota Yadong Wang Department of Bioengineering University of Pittsburgh Pittsburgh, Pennsylvania Mark Wong Department of Oral and Maxillofacial Surgery University of Texas School of Dentistry Houston, Texas B. Wu Department of Bioengineering University of California Los Angeles Los Angeles, California Yu-li Wang Department of Biomedical Engineering Carnegie Mellon University Pittsburgh, Pennsylvania Michael J. Yaszemski Department of Tissue Engineering and Biomaterials Mayo Clinic Rochester, Minnesota Joseph C. Wenke United States Army Institute of Surgical Research Fort Sam Houston, Texas Diana Yoon Department of Bioengineering Rice University Houston, Texas 1 Consensus Definitions, Fundamental Concepts, and a Standardized Approach to Applied Biomaterials Sciences Jeffrey O. Hollinger CONTENTS 1.1 1.2 Introduction............................................................................................................................... 1 Definitions.................................................................................................................................2 1.2.1 What Is a Biomaterial?.................................................................................................. 2 1.2.2 What Is Biocompatibility?............................................................................................. 2 1.3 Fundamental Concepts.............................................................................................................. 3 1.4 A Standardized Approach.........................................................................................................4 1.5 Summary...................................................................................................................................4 References and Suggested Reading.................................................................................................... 4 1.1 INTRODUCTION A textbook that focuses on biomaterials may take several pathways; however, there are two principal directions. One direction may emphasize a limited number of biomaterials that are used in clinical indications. In this case, an extensive and comprehensive discussion may be provided focusing on a natural polymer, such as collagen, or a family of laboratory-derived polymers that may include the poly(alpha-hydroxy acids). There are liabilities and advantages associated with this approach. Significantly, the limitation is to a select set of materials and their actual and potential applications. However, the benefit from such emphasis is that the extent of the information provided would be robust. Yet, there will be a restricted readership and student population to whom this narrow focus will benefit. Another direction for the textbook may entail a comprehensive presentation of any and all biomaterials that are either currently used or may be used in clinical situations. The virtue of this approach is the comprehensiveness of information collated in a single source; however, the distinct disadvantage is the massiveness of that single source. Moreover, there may simply be an overwhelming volume of inconsequential information for the student. Therefore, the direction taken for this biomaterials text emphasizes a compromise between an option that is narrowly focused and restricted, and one that attempts to capture everything about biomaterials. Compromises are susceptible to criticism. Despite this potential, the editor carefully selected biomaterials that have a longstanding, rich history of clinical utility as well as several exciting new materials that expand the conventional notion of clinical utility. The chapter content was inspired by the academic need for a textbook on biomaterials that either have profoundly impacted or will significantly influence the quality of clinical care. Furthermore, 1 2 An Introduction to Biomaterials to ensure that a didactic need was met in an instructional and comprehensive manner, recognized authorities who are leading experts with well-documented résumés prepared the chapters. Once the decision was made for the textbook content, fundamental concepts were identified and developed, for example, terminology, standards, and consensus definitions. A patient-centric approach for chapter content was taken by the editor. Consequently, the first section of the book underscores and addresses fundamental properties and processes of biological healing cascades in which uniform, key biological terminology is introduced. In addition, insightful logic is presented for the profound requirement of assessing clinically directed biomaterials using animal models that correlate with the intended clinical patient use. It is in that spirit, underscoring uniformity, that standards and consensus definitions are introduced. The argument for this approach, especially highlighted for the undergraduate student, is that standards and consensus enable meaningful dialog within and among laboratories and support discovery and progress. Consequently, conclusions can be derived that validate outcome. Decisions can be made and improvement realized, thus benefitting every aspect of patient heath care. 1.2 1.2.1 DEFINITIONS What Is a Biomaterial? This textbook focuses on biomaterials. It is quite logical and fair to ask if we have a consensus definition of the topic of biomaterial. One definition is “any substance (other than a drug) or combination of substances, synthetic or natural in origin, which can be used for any period of time, as a whole or part of a system, which treats, augments, or replaces any tissue, organ, or function of the body” (Dee et al. 2002, p. 2). Professors Dee, Puleo, and Bizios noted in their superb textbook on tissue–biomaterial interactions that “any current definition of biomaterials is neither perfect nor complete but … provide(s) an excellent reference or starting point for discussion” (p. 6). This is very good, assuming that the ensuing discussion leads to a consensus decision. A consensus definition for a biomaterial introduced in 1987 that has been referenced frequently in the literature may be traced back to Professor David F. Williams, a pillar of the biomaterials sciences: “a biomaterial is a nonviable material used in a medical device, intended to interact with biological systems” (Black 2006, p. 2). This definition is short and to the point, which are two properties especially enjoyed by legions of memorizing undergraduates. However, in reference to the above statement again (any current definition of biomaterials is neither perfect nor complete) is the following comment I wrote in the first edition of this textbook: “The recent development of materials containing living cells (e.g., artificial organs and tissue-engineered scaffolds) challenges the inclusion of the word nonviable in the traditional definition of a biomaterial” (Black 2006, p. 6). However, I remind the reader that Professors Dee, Puleo, and Bizios also stated that consensus definitions “provide an excellent reference or starting point for discussion” (Dee et al. 2002, p. 2). Consequently, it is noteworthy that in 1992, the term nonviable was deleted and the amended biomaterial consensus definition appeared: “A material intended to interface with biological systems to evaluate, treat, augment, or replace any tissue, organ, or function in the body” (Black 2006, p. 6). It is obvious that the consensus definition of 1992 preceded my comment in 2006. 1.2.2 What Is Biocompatibility? Biocompatibility is the ability of a material to perform with an appropriate host response in a specific application (Williams 1987). To this definition, thanking Professor Black, we clarify the phrase host response as follows: the reaction of a living system to the presence of a material (Black 2006). I will add further to the definition of biocompatibility by offering the descriptor predictable. Another minor modification that will be important for biocompatibility is to replace perform with the phrase function in situ. Consequently, a final working definition for the students using this Consensus Definitions, Fundamental Concepts, and a Standardized Approach 3 textbook is now as follows: biocompatibility is the ability of a material to function in situ with an appropriate and predictable host response in a specific application. The education of health care providers must include the following commandment: Do no harm to your patient. Biomaterials scientists must look beyond their analytical equipment and laboratory assays. They must see the patient who will benefit from their innovativeness, discovery, and clinical product. This logic being set forward, and as the editor for this textbook and mentor to my students, I emphasize that the corollary to the biocompatibility definition must include the following statements: A biocompatible biomaterial will do no harm. The biocompatible biomaterial will do no harm to the patient either in the implanted state, over time as it continues to interact in situ, or as the biomaterial biodegrades over time. This corollary is underscored in Section 1.3, and the topic is expanded upon in Chapter 6. 1.3 FUNDAMENTAL CONCEPTS In his penetrating textbook on the biological performance of materials, Professor Black reminded us of the 1992 consensus definition “that (a) biomaterial (is) a material intended to interface with a biological system” (p. 7). I will add to this definition the three words “do no harm.” The concept “do no harm” must be addressed. A profound example is the compelling orthopedic product InFuse Bone Graft (Medtronic, Minneapolis, Minnesota). A biomaterial may be used as a delivery system for a biological factor. The example of collagen (type I bovine) delivering recombinant human bone morphogenetic protein-2 (rhBMP-2) is a hallmark accomplishment in biomaterials sciences in which the natural polymer, collagen, combined with rhBMP-2 as the clinical product InFuse Bone Graft, first approved by the Food and Drug Agency (FDA), has been used successfully to treat single-level instrumented anterior lumbar interbody vertebral fusions. The remarkable benefit to the thousands of patients treated with the biomaterial-containing product, InFuse Bone Graft, is irrefutable. The selection of collagen as a component of that product was not serendipitous, rather, it was based on the essential property of collagen: biocompatibility and the notion that collagen would do no harm. The fundamental biological processes associated with a biomaterial implanted into a patient will be discussed in Chapters 2–7. The emphasis in these chapters is that the outcome of implantation of a biomaterial in a patient, that is, the consequence of the interface with the biological system, should be predictable, as intended, and beneficial, and that the resultant biomaterial-interface, biomaterial as a whole, and degradation products of the implanted biomaterial provide an improvement in the quality of life for the patient. The overarching theme for this fundamental is that the biomaterial will be biocompatible. Finally, regardless of whether you are a health care provider responsible for the direct, hands-on care of a patient or if you are a bench scientist, the patient receiving the biomaterial you designed and developed trusts you. That trust resides in the belief that the biomaterial being implanted will be helpful, not harmful. Consequently, certainty about biocompatibility must be absolute. In line with this fundamental is the requirement for standards. Standards are the alphabet scripting the common language that permits rational communication within a laboratory and among laboratories. Standards enable confirmation of outcome among laboratories through reproducible data. Confirmation is validation that an outcome is not serendipitous, but due to scientific design. When outcome data are reproducible and based on standardized procedures, decisions can be made: a decision to modify a biomaterial, accept it as is for the intended clinical use, or abandon it completely because it is unsafe and/or not effacious. The intent of adopting standards is to ensure that laboratories have an operational consensus and clear definition of acceptable property thresholds for biomaterials. For example, if a biomaterials property must comply with a standard that is safe, what is the safety threshold? What is meant by safe? How do we determine safety? What are the criteria that can be measured that will validate safety? (See Chapters 8 and 9.) 4 1.4 An Introduction to Biomaterials A STANDARDIZED APPROACH Standard procedures for testing biomaterials during design and development stages, as well as in conditions duplicating the intended clinical application, permit outcome validation of performance properties of a biomaterial. Outcome validation that can be repeated in the same laboratory as well as in other laboratories underscores confidence and certainty of the biomaterial characterization. Procedural standards (e.g., methods and techniques) and outcome standards (i.e., performance properties) identify discrete elements that will enable confirmation among laboratories. Toward this objective, the passage of the Medical Device Bill (1976) placed an emphasis on biomedical device testing. Guidelines for biomaterials testing were promulgated by the National Heart, Lung, and Blood Institute (1980, 1985) that underscored reliable (i.e., repeatable and reproducible) approaches for assessing and standardizing the assessment of biomaterials. The International Standards Organization (ISO) first published in 1992 a reference guide that has subsequently been supplemented and is one of the hallmarks for biomaterials testing standards. In addition, the American Society for Testing and Materials (ASTM) provides a similar function. Comprehensive descriptions of ISO and ASTM are provided in Chapters 8 and 9, in which in vitro and in vivo biomaterials paradigms are presented. 1.5 SUMMARY Biomaterials, as emphasized in this textbook, are intended for clinical applications. Therefore, rigorous, systematic, and standardized testing protocols must be adhered to that ensure the biomaterial will function effectively for the intended application and that the outcome of that application does not harm the patient. To do no harm, and therefore ensure safety, must be the hallmark characteristic of any biomaterial that is implanted into a patient. Toward that goal, standardized testing procedures must carefully transition through in vitro and in vivo methodologies and must provide accurate and reproducible outcome data. Moreover, standardized testing procedures must duplicate as closely as possible the actual intended clinical application. Furthermore, an end use state, that is, the biomaterial produced as a product in which manufacturing and sterilization procedures are included, must be validated. REFERENCES AND SUGGESTED READING Guidelines for Physicochemical Characterization for Biomaterials. 1980. National Heart, Lung and Blood Institute. Dearborn, MI: University of Michigan Library. Black, J. 2006. Biological performance of materials. In Fundamental of Biocompatibility, ed. J. Black, 4th ed. Boca Raton, FL: CRC Press-Taylor & Francis. Boretos, J. W., and W. Eden. 1984. Contemporary Biomaterials, Material and Host Response, Clinicial Applications, New Technology and Legal Aspects. 232–3. Park Ridge, NJ: Noyes Publications. Dee, K. C., D. A. Pulelo, and R. Bizios. 2002. An Introduction to Tissue Biomaterial Interactions. Hoboken, NJ: Wiley-Liss, Publications. Guidelines for Blood-Materials Interactions. 1985. National Heart, Lung and Blood Institute. Bethesda, MD: U.S. Department of Health and Human Services. Ratner, B., A. Hoffman, F. Schoen, and J. Lemons. 2004. Introduction: Biomaterials science: A multidisciplinary endeavor. In Biomaterials Science: An Introduction to Materials in Medicine, ed. B. Ratner, A. Hoffman, F. Schoen, and J. Lemons, 2nd ed. Boston: Elsevier. Williams, D. F. 1987. Definitions in biomaterials. In Proceedings of a Consensus Conference of the European Society of Biomaterials (1986). Chester: Elsevier. Williams, D. F., J. Black, and P. J. Doherty. 1992. Second consensus conference on definitions in biomaterials. In Biomaterial-Tissue Interfaces, Advances in Biomaterials, ed. P. J. Doherty et al., Vol. 10. Amsterdam: Elsevier. Section I Biology, Biomechanics, Biomaterial Interactions Wound Healing Biology 2 Cutaneous Wound Pathobiology Raison d’etre for Tissue Engineering Lauren K. Macri and Richard A. F. Clark CONTENTS 2.1 2.2 Function and Anatomy of Normal Skin.................................................................................... 7 Biology of Cutaneous Wound Healing...................................................................................... 8 2.2.1 Inflammation.................................................................................................................8 2.2.2 Tissue Formation........................................................................................................... 8 2.2.3 Tissue Remodeling........................................................................................................ 8 2.3 Common Cutaneous Wounds and Abnormal Healing..............................................................9 2.3.1 Burns.............................................................................................................................. 9 2.3.2 Ulcers.............................................................................................................................9 2.3.3 Keloids and Hypertrophic Scars.................................................................................. 10 2.4 The Need for Tissue Engineering............................................................................................ 10 2.5 Tissue Engineering: Healing Cutaneous Wounds................................................................... 11 2.5.1 Tissue Engineering Principle....................................................................................... 11 2.5.2 Approaches to Tissue Engineering.............................................................................. 11 2.5.3 Requirements of the Tissue-Engineered Construct..................................................... 12 2.5.4 Components of the Tissue-Engineered Construct....................................................... 12 2.5.4.1 Cells.............................................................................................................. 12 2.5.4.2 Scaffold......................................................................................................... 13 2.5.4.3 Bioactive Molecules...................................................................................... 14 2.5.5 Fabrication of Tissue-Engineered Constructs............................................................. 15 2.5.5.1 Hydrogels...................................................................................................... 15 2.5.5.2 Fibro-Porous Mats........................................................................................ 15 2.5.5.3 Randomly Integrated Structures................................................................... 16 2.5.5.4 Designed 3D Scaffolds.................................................................................. 16 2.6 Scarring and Cell-Based Therapies......................................................................................... 17 2.7 Conclusion............................................................................................................................... 18 References......................................................................................................................................... 18 2.1 FUNCTION AND ANATOMY OF NORMAL SKIN Skin is the largest organ of the body and is comprised of three layers: (1) epidermis, (2) dermis, and (3) subcutaneous tissue. The epidermis is in direct contact with the external environment and helps maintain homeostasis. It also prevents dehydration and provides the body with protection against toxic 7 8 An Introduction to Biomaterials substances and extreme environmental temperatures. The major cell type found in the epidermis is the keratinocyte, but melanocytes, dendritic cells, adnexal cells, and sensory cells (i.e., Merkel cells) are also located there (Lee 2000). The dermis is separated from the epidermis by the basement membrane and is primarily composed of type I collagen, which is secreted by its primary cellular inhabitants, dermal fibroblasts. Together, collagen and the other less abundant extracellular matrix (ECM) molecules found in the dermis provide the skin with the majority of its mechanical stability (Boyce and Warden 2002). Some other cells active in the dermis are endothelial cells, smooth muscle cells, nerve cells, and immuneresponsive cells. The subcutaneous tissue is primarily made up of fatty tissue and sensory nerve fibers that are capable of responding to vibrations and pressures (Lee 2000). Together, the three layers of the skin provide the body with extraordinary protective, sensory, and wound-healing capabilities (Lee 2000). 2.2 2.2.1 BIOLOGY OF CUTANEOUS WOUND HEALING Inflammation Normal cutaneous wound healing is an extremely dynamic process that progresses through three overlapping stages: (1) inflammation, (2) tissue formation, and (3) tissue remodeling. Immediately after an injury to the skin occurs, blood coagulation and platelet aggregation create a fibrin-rich matrix that helps re-establish homeostasis and serves as a scaffold for cell migration. In addition, the blood clot serves as a reservoir of bioactive molecules secreted by platelets and generated by the coagulation and complement pathways (Singer and Clark 1999). Together these bioactive molecules, specifically growth factors and cytokines, recruit neutrophils and monocytes, which are the major mediators of the inflammatory response (Clark 1996). Neutrophils arrive to the injured site first and are responsible for cleansing the wound of foreign particulate and bacteria. Next, chemoattractants retained by the fibrin clot stimulate monocyte infiltration to the injured site. Once the monocytes adhere to the molecules of the ECM, they differentiate into professional phagocytes, known as macrophages (Ghosh 2007). In addition, monocytes and macrophages secrete a slue of bioactive molecules that play an integral role in transitioning from the inflammatory response to the repair stage of the cutaneous wound-healing process. 2.2.2 Tissue Formation New tissue formation unofficially begins with the re-epithelialization process, which is initiated (along with inflammation) within hours of the injury and is intended to occur rapidly so that dehydration and bacterial infection are prevented. This process is first characterized by the migration of surrounding epithelial cells on the basement membrane, if it exists, or via the dissection of the fibrin/fibronectin clot using proteases (i.e., matrix metalloproteinase-1) (Bugge et al. 1996). Then, 1 or 2 days postinjury, the epidermal cells behind those that have migrated begin to proliferate and complete the re-epithelialization process. The tissue formation stage of wound healing officially begins 4 days after the injury, however, and is mediated by macrophages, fibroblasts, and blood vessels (Hunt 1980). The role of the macrophage is to continue to provide a bioactive molecule-rich environment that will stimulate angiogenesis and fibroblast migration into the wound. The provisional matrix’s structural components (fibrin, fibronectin, and hyaluronic acid) provide an easily penetrable scaffold that facilitates fibroblast infiltration into the wounded area via protease activity (Clark et al. 1982; Toole 1991; Greiling and Clark 1997). Angiogenesis, the formation of new capillary blood vessels, is critical to successful wound healing because blood provides the injured site with oxygen and nutrients vital to cell survival. Gradually, the fibroblast-rich, temporary, provisional matrix is replaced with a longer-term, acellular, collagen-rich scar (Clark et al. 1995; Welch, Odland, and Clark 1990). 2.2.3 Tissue Remodeling The transition from granulation tissue formation to scar tissue occurs during the second and third weeks of wound repair and is the last stage of wound healing. Tissue remodeling is a very dynamic 9 Cutaneous Wound Pathobiology Cell-ECM interactions Cell-ECM-bioactive molecule interactions Cells Extracellular matrix Cell-bioactive molecule interactions Bioactive molecules ECM-bioactive molecule interactions FIGURE 2.1 The “dynamic reciprocity” (Nelson and Bissell 2005, 2006) of the healing process. The three major mediators of the healing process are the cellular inhabitants of the tissue, the extracellular matrix (ECM) molecules present in the wound and its surrounding tissue, and the bioactive molecules necessary to modulate regeneration. The overlapping areas in the schematic represent interactions among the components, resulting in dynamic reciprocity. It is important to take all of these interactions into account to construct a successful tissue-engineered construct (TEC). and interactive process between the tissue’s cellular inhabitants and its matrix, termed dynamic reciprocity (Figure 2.1) (Nelson and Bissell 2005). It is highly dependent on the rates of collagen synthesis and catabolism (Singer and Clark 1999). During this stage of healing, the fibroblasts undergo a phenotypic transformation and become myofibroblast-like cells with large bundles of actin cytoskeletal filaments (Desmouliere and Gabbiani 1996; Welch, Odland, and Clark 1990). This change indicates that compaction and contraction of the wound have ceased (Ghosh 2007). By week 3, the repaired tissue may have 20% of its final strength, and an increase in tensile strength is slow and continuous over the next few weeks (Singer and Clark 1999). Further reorganization of the matrix continues for months to years. At best, the resultant scar tissue can achieve 70% of the tensile strength of normal, intact skin (Levenson et al. 1965). 2.3 2.3.1 COMMON CUTANEOUS WOUNDS AND ABNORMAL HEALING Burns Burns are the most common cause of significant cutaneous tissue loss (Ghosh 2007). In 2007, 500,000 people in the United States sought medical attention for thermal injuries, of which 25,000 were admitted to hospitals with specialized burn care centers. Burns are typically categorized based on the depth of the injury into the following three groups: (1) First-degree burns are those that penetrate into the epidermal layer of the skin and usually manifest as open or closed blisters; (2) second-degree, or partial thickness, burns are those that extend into the dermis of the skin; and (3) third-degree, or full thickness, burns extend through the epidermis and dermis and into the subcutaneous tissue. 2.3.2 Ulcers Skin ulcers affect an even larger number of people than thermal injuries. The primary markers of the presence of an ulcer are (1) infection, (2) inflammation, (3) an extended period of time without healing, and often (4) loss of integrity at the site of the wound. If an ulcer does not heal in a timely fashion, it is classified as a chronic ulcer. Pressure and leg ulcers are the most common types of skin ulcers. Pressure ulcers, also known as bedsores, have been estimated to affect between 1.3 and 3 million people in the United States (2005). They are usually caused by a reduction in blood flow to an area of the body that has remained 10 An Introduction to Biomaterials in a single position for a prolonged period of time. Chronic leg ulcers most commonly develop as a result of insufficient circulation of blood to the leg. Injuries, skin disorders, diabetes, stroke, heart attack, infections, thrombophilia, and tumors can also cause leg ulcers. The two most common types of leg ulcers are arterial and venous leg ulcers (Bergan et al. 2006). Arterial leg ulcers result from a narrowing of the arteries, which is most commonly caused by poor circulation of the blood or diabetes. Venous leg ulcers, the most common type of leg ulcers, are primarily caused by a malfunction of the one-way valves that bridge superficial (located between the skin and the muscles) and deep veins (located between the muscles) (Bergan et al. 2006). This malfunction causes the backflow of blood into the superficial veins, which causes varicose veins. In addition, skin ulcers commonly develop on the feet of 5% of the diabetic population as a result of the patient’s inability to sense and relieve pressure in the injured tissue (Singer and Clark 1999). Abnormal healing in diabetic patients can also be attributed to the following: (1) ischemia, which restricts the supply of oxygen and nutrients to the wound area, (2) infection, (3) malfunctioning macrophages, (4) limited angiogenesis, and (5) a major imbalance in the dynamic reciprocity between fibroblasts and the ECM (i.e., increased protease activity with reduced collagen production) (Fahey et al. 1991; Loots et al. 1998). As a result of the aforementioned inadequacies, the tissue typically spends a prolonged period of time in the inflammation stage of the wound-healing process (Fahey et al. 1991; Loots et al. 1998; Singer and Clark 1999). In the United States, it has been reported that 2 million people are diagnosed with chronic diabetic ulcers each year (Supp and Boyce 2005). The population in the United States is not only increasing year to year, but people are also living longer lives; therefore, it is expected that the number of people suffering from both diabetic and pressure ulcers is also likely to increase. Not only are skin ulcers extremely painful and debilitating, but treatment costs are extremely high; for example, costs associated with pressure ulcers total over $8 billion per year (Supp and Boyce 2005). For venous pressure leg ulcers alone, it has been estimated that it costs the United States approximately $1 billion per year to treat this medical problem (Khan and Davies 2006). The average cost per patient for the duration of his or her treatment typically exceeds $400,000 (Khan and Davies 2006) or about $2,400 per month (Ehrenreich and Ruszezak 2006). This estimation does not include costs endured for psychological issues that may have to be treated as well. 2.3.3 Keloids and Hypertrophic Scars Keloids and hypertrophic scars are two examples of abnormalities that can occur during the wound-healing process. Keloids are different from hypertrophic scars in that keloids are generally restricted to the original site of injury, whereas hypertrophic scars can exceed the boundaries of the original injury and can possibly reduce in size over time (Brown and Bayat 2009). These cutaneous wound-healing defects are due to excess collagen accumulation at the repaired site, which can result from malfunctioning cell activity, that is, migration, proliferation, and matrix production and degradation (Singer and Clark 1999). Both keloids and hypertrophic scars are aesthetically displeasing and can impair function, depending on their location on the body. 2.4 THE NEED FOR TISSUE ENGINEERING The use of skin grafts to heal cutaneous wounds dates back to 3000–2500 BC when it was first documented that Hindus transplanted skin from the buttocks onto the injured site (Herman 2002). Although the surgical techniques used then were primitive, the proof of principle was rather exhilarating. In total, there are three types of skin grafts that have been investigated throughout the years to treat cutaneous wounds: (1) autografts (skin is transplanted from one part of the patient’s body to another), (2) allografts (skin is transplanted from one human to another human), and (3) xenografts (skin is transplanted from a nonhuman species to the human patient). Today, autografting is the Cutaneous Wound Pathobiology 11 standard treatment for most cutaneous wounds and is quite successful for full thickness burns and other acute wounds. Despite its success, autografting is extremely dependent on the availability of donor skin and is limited to patients who have less than 50% of the total surface area of the skin affected by injury. On the other hand, chronic wounds treated with autografts have not had similar success because of the underlying pathologies usually associated with these conditions. Skin grafts do not restore full sensory function (Ward et al. 1989; Ward and Tuckett 1991); they have the potential to fail or be rejected; the operation sites may become infected; scarring may result; and reulceration of the same limb can occur. To a lesser extent, allografts and xenografts are also used to treat cutaneous injuries but can also be rejected; in that case, they must be replaced because of their inability to engraft into the wound site and the potential to mediate the transfer of disease (Eisenbud et al. 2004). In addition, the surgical procedure can take several hours and may have to be completed with multiple, separate operations. The patient must undergo anesthesia and endure pain from both the donor and recipient (ulcer) sites. Furthermore, time must be spent in the hospital for the procedure and recovery, which is both unpleasant and costly. Therefore, tissue engineering offers the tools necessary to create a construct that is capable of stimulating and accelerating the cutaneous wound-healing process with minimal scarring and without the drawbacks of skin grafting. 2.5 TISSUE ENGINEERING: HEALING CUTANEOUS WOUNDS 2.5.1 Tissue Engineering Principle The long-term goal of a tissue-engineered construct (TEC) is to regenerate the tissue to an end result that mimics the biology and function of normal, uninjured skin. The successful regeneration of the skin, or any other tissue or organ in the body, depends on the dynamic reciprocity (Nelson and Bissell 2005, 2006) that exists among the tissue’s native cells, ECM, and bioactive molecules (Figure 2.1). The skin’s cellular inhabitants are the primary mode by which the tissue defect is restored. The molecules of the ECM serve two functions. First, they act as a scaffold for cell attachment and migration during the wound repair process. Second, they bind bioactive molecules, such as growth factors and cytokines, which provide either stimulatory or inhibitory signals to the cells. Because morbidity of the wound site is defined and characterized by failure to restore the anatomy and function of the skin, complete regeneration may not be possible until all the cellular constituents of the skin are addressed by TECs (Boyce and Warden 2002). It is believed that TECs that take into account the aforementioned dynamic reciprocity will offer superior wound-healing repair and regeneration of cutaneous wounds. 2.5.2 Approaches to Tissue Engineering One approach to the challenge of creating a TEC that stimulates healing of cutaneous wounds is to create a product in vitro that is capable of complete integration into the patient’s body (Figure 2.2). The first step to this approach involves taking a biopsy of cells from the patient. In the second step, the cells are cultured and expanded in vitro. The cells are then introduced to a scaffold and/or bioactive molecules in the third step, and the complex is cultured as a single unit in the fourth step. The last step requires that the TEC is implanted into the patient’s wounded area, and the ultimate goal is for perfect engraftment into the wounded site. A second approach to create a TEC for cutaneous wound healing is to engineer a bioresorbable scaffold (with or without bioactive molecules, i.e., growth factors) that can stimulate the patient’s native cells to invade a large defect and heal the wounded area with aesthetically pleasing results (Macri, in press). The overall goal here is to create a TEC that degrades simultaneously with the rate of healing, so that the TEC is completely replaced by the regenerated tissue. 12 An Introduction to Biomaterials Step 1: Take biopsy from patient Step 2: Expand patient’s cells in vitro Step 3: Introduce matrix and/or bioactive molecules Step 5: Implant engineered construct into wounded area Step 4: Culture complex as single unit FIGURE 2.2 A schematic of one approach to tissue engineering of the skin. 2.5.3 Requirements of the Tissue-Engineered Construct The mainstay treatment of cutaneous wounds to those patients who are eligible is the application of split-thickness autologous skin, either as a sheet (Housinger, Hills, and Warden 1994) or expanded by meshing (Tanner, Vandeput, and Olley 1964). The success of the autograft depends on its adherence and integration into the injured site, biocompatibility, ability to restore function and mechanical stability, cost and availability, and tolerance by the immune system (Boyce and Warden 2002). Therefore, regardless of which of the two approaches to tissue engineering is used, the aforementioned requirements are also demanded of TECs. However, some additional criteria should also be met, including that the TEC is biocompatible, degradable at physiologic conditions, FDA approvable, and easily applicable by a medic; has a reasonably long shelf life; facilitates and accelerates wound healing and closure; and produces minimal to no scarring. To successfully accomplish these goals, it is imperative that tissue engineers address the dynamic reciprocity that exists among the cells, the scaffold, and the bioactive molecules involved in the regeneration process. 2.5.4 Components of the Tissue-Engineered Construct 2.5.4.1 Cells The initial goal of most TECs, when attempting to heal a cutaneous wound, is to facilitate wound closure. The restoration of the epidermal layer of the skin is imperative to prevent dehydration of the wounded site and to protect against the invasion of infective agents. This restoration is accomplished by the keratinocytes. Epicel (Genzyme Corporation) is an example of a TEC comprised of autologous keratinocytes. Unfortunately, the success of Epicel has been limited by the 2- to 3-week lag period required to expand out the cells in vitro before a transplantable sheet is produced (Clark, Ghosh, and Tonnesen 2007), by high costs (Rue et al. 1993), and by variable graft acceptance outcomes (Carsin et al. 2000). Even if Epicel was not associated with the aforementioned drawbacks, the regeneration of only the epithelial barrier does not imply that function has been restored to the injured site. For complete regeneration of the skin to occur, the cellular constituents beyond the epidermis must also participate in the healing process. For example, fibroblasts located in the periwound must migrate into the Cutaneous Wound Pathobiology 13 blood clot, degrade it, synthesize new matrix, and organize it into the appropriate infrastructure to facilitate regeneration. Apligraf (Organogenesis Inc.) and OrCel (Forticell Bioscience Inc.) are FDA-approved, organotypic skin substitutes created to mimic the epidermal and dermal layers of the normal skin. The “dermal layer” is predominantly made of bovine type I collagen containing allogeneic dermal fibroblasts. The “epidermal layer” is comprised of allogeneic epidermal keratinocytes. Although both of these products are commercially available, allografts have been shown to be more immunogenic than autografts (Erdag and Morgan 2004), do not enable perfect engraftment into the host’s injured site, and have a relatively short shelf life because of their cellular constituents. Dermagraft (Advanced BioHealing Inc.) is an example of a bilayered TEC that contains viable fibroblasts from a same-species donor. That is, the fibroblasts are from a human donor and are designated as “allogeneic.” The lower layer of Dermagraft is comprised of natural ECM deposited by fibroblasts and the upper layer is comprised of degradable polyglactin (Pham et al. 2007). Unfortunately, it has been difficult to control the quality of the viable fibroblasts residing in the lower layer of the matrix. In addition to the keratinocytes of the epidermis and the fibroblasts of the dermis, there are many other cells types that should be considered when creating a TEC. The incorporated cells can be either differentiated cells from an autologous, allogeneic, or xenogeneic source or undifferentiated cells, ranging from embryonic to adult stem cells. For example, mesenchymal stem cells are known to “home” (i.e., become attracted to) to sites of injury and may therefore play a crucial role in the cutaneous wound-healing process. Also, endothelial and smooth muscle cells are extremely important in that they have the potential to enable rapid vascularization at the injured site. If vasculature is neglected in the healing process, ischemic and nutrient-deprived conditions will result, and will ultimately lead to failure to heal. For aesthetic purposes, melanocytes may be considered, as they have been shown to be culturable and transplantable (Boyce et al. 1993; Kratz et al. 1992; Lerner et al. 1987; Swope et al. 1997). In addition, structural features such as glands and hair follicles can also be transplanted (Jahoda et al. 1996; Steenfos and Jansson 1992). Glands and hair follicles play critical roles in the regulation of body temperature but are yet to be incorporated in present-day TECs. 2.5.4.2 Scaffold In either aforementioned tissue engineering approach, the material from which the scaffold is made is at the crux of the technology. The scaffold can have various purposes: (1) to act as a conduit for cell migration; (2) to encapsulate cells and/or slowly release bioactive molecules, or pharmaceutical agents, into the cellular microenvironment to promote wound repair; and (3) to serve as a protective covering and maintain hydration at the injured site. Scaffolds can be synthesized from three classes of materials: (1) metals, (2) ceramics, and (3) polymers. Metals (e.g., titanium) and ceramics (e.g., aluminum oxide) are not typically used for tissue engineering of the skin because they are not degradable at physiologic conditions. Therefore, TECs for this application are primarily synthesized from biocompatible materials, that is, “materials that elicit minimal toxic response when used in vitro in cell culture protocols and minimal or no unwanted foreign body reaction when implanted in vivo” (Moroni and Elisseeff 2008, p. 44). Biocompatible materials are typically classified into two groups: (1) biomaterials (man-made; e.g., poly[lactic acid], poly[ethylene glycol], and tyrosine-derived polycarbonates) and (2) biopolymers (naturally derived, e.g., collagen, hyaluronic acid, and alginate). Regardless of its origin, the material must exemplify the ability to bond with and integrate into the cutaneous wound defect. The intrinsic chemical backbone or surface properties of the biocompatible material may be modified to enable cell attachment, migration, and differentiation, and ultimately promote integration into the wounded site. If the material itself cannot sustain cell attachment, then cell-binding peptides can be incorporated into the matrix. The most commonly used cell-binding motif is the arginine–glycine–aspartate (RGD) peptide 14 An Introduction to Biomaterials (Shu et al. 2004) to activate β1 integrins (Keselowsky, Collard, and Garcia 2005), however, other peptides may also be beneficial, such as proline–histidine–serine–arginine–asparagine (PHSRN) (Aota, Nomizu, and Yamada 1994). These physicochemical cues must be provided in the optimal spatial pattern to successfully conduct cell migration and induce differentiation into the correct cell lineage. There are a wide variety of options available regarding the structural design of the scaffold, including fibers, sponges, freeze-dried materials, and rapidly prototyped structures (Griffith 2002; Muschler, Nakamoto, and Griffith 2004). Independent of the structural design, the scaffold’s porosity and pore network play an integral role in its ability to contribute to the wound-healing process. Macro- and micropores provide conduits for cell infiltration into the wound and micro- and nanopores allow gas, nutrient, and bioactive molecule exchange. In addition, the structural design has a major impact on the material’s mechanical properties; for example, scaffolds with high percentages of void space tend to be mechanically less rigid. However, the absolute value of the mechanical strength is not as important as creating a scaffold with mechanical properties similar to that of the skin. This will help promote its integration into the wound space by providing a continuous interface between the wound and the peripheral native tissue without compromising nutrient diffusion. Because the materials science industry has excelled at such a rapid pace and has successfully created a multitude of polymers for tissue engineering applications, virtual libraries are now being used to help tissue engineers more accurately choose polymers that best fit their applications (Kohn 2004). These virtual libraries are high throughput screening tools with information that is backed by hard science and computer modeling (Karp and Langer 2007). Integra (Integra Lifesciences Corporation) is an example of a FDA-approved, partially resorbable TEC. The lower layer is made of degradable bovine collagen type I cross-linked with shark-derived chondroitin-6-sulfate that is designed to mimic the dermis and stimulate dermal regeneration. The upper layer is comprised of nondegradable silicone. It is intended to mimic the epidermis by preventing dehydration and serving as a protective barrier. The bilayered TransCyte (Advanced BioHealing Inc.) is similar to Integra except that its lower layer is comprised of natural ECM synthesized and deposited by neonatal human foreskin fibroblasts onto a nylon mesh. Both Integra and TransCyte are advantageous in that they are both acellular products; however, Integra is derived from animals and the nondegradable upper layer of both Integra and TransCyte must be removed and ultimately replaced with an autograft. 2.5.4.3 Bioactive Molecules Bioactive molecules, such as growth factors, cytokines, and DNA, can provide the appropriate conductive cues that will recruit the tissue’s native cells into the wounded site, and inductive cues that will stimulate proliferation and differentiation, which will heal the wound with aesthetically pleasing results. Providing these biochemical cues in the correct spatiotemporal pattern is a major challenge and may not always be necessary for successful cutaneous wound regeneration. The bioactive molecules can be of endogenous origin in which parenchymal and mesenchymal cells in the TEC can secrete bioactive molecules that can facilitate the wound-healing process in both autocrine and paracrine manners. Exogenous sources of bioactive molecules can also be added directly to the TEC if the natural sources are inadequate, inaccessible, or unavailable. For example, REGRANEX Gel (Ortho-McNeil Pharmaceutical, a division of Johnson & Johnson) is used to treat diabetic foot ulcers and is an FDA-approved product that contains platelet-derived growth factor (PDGF) (http://www.regranex.com). It has been used successfully to treat and heal cutaneous wounds in thousands of patients (http://www.regranex.com). Another approach to delivering bioactive molecules to the wound may include tethering to a matrix. For example, epidermal growth factor (EGF) has been shown to promote mesenchymal stem cell spreading and survival (in the presence of Fas ligand) when tethered to a poly(ethylene oxide) backbone compared to soluble EGF (Fan et al. 2007). 15 Cutaneous Wound Pathobiology 2.5.5 Fabrication of Tissue-Engineered Constructs The methods described in this section focus on those used to fabricate scaffolds for the treatment of cutaneous wounds. The structural designs addressed include hydrogels, fibro-porous mats, randomly integrated structures, and designed three-dimensional (3D) scaffolds. 2.5.5.1 Hydrogels Hydrogels are jelly-like materials that are composed of mostly liquid but develop into cross-linked structures via covalent bonds, physical chain entanglements, or association bonds (hydrogen bonds or van der Waals interactions) (Peppas 1987). Hydrogels are commonly used in wound-healing applications because their architectural arrangements are quite comparable to the natural ECM on both the micro- and nanoscales. The fluidity of uncross-linked hydrogel solutions allows infiltration into the wound, which promotes material integration within the tissue and offers the advantage of site-specific, noninvasive delivery via injection with in situ–stimulated polymerization. Hyaluronan is an example of a naturally derived biopolymer that has been successfully used to create a hydrogel TEC. Hyaluronan is a nonsulfated glycosaminoglycan present in both fetal and adult stages of life. Its appearance coincides with cell migration during embryogenesis and morphogenesis (Toole 1991) and with collagen deposition during fetal wound healing (Longaker et al. 1991). In addition, hyaluronan plays a major role in adult wound repair by regulating inflammation (Wisniewski and Vilcek 1997) and stimulating cell activity, such as migration and proliferation (Chen and Abatangelo 1999). As a result of its natural participation in wound healing, it has been successfully used in cutaneous wound-healing TECs. For example, Ghosh et al. (2006) created a biocompatible hydrogel comprised of fibronectin functional domains tethered to a hyaluronan backbone that was shown to recruit and stimulate en masse fibroblast migration in a porcine cutaneous excisional wound model (Ghosh et al. 2006). 2.5.5.2 Fibro-Porous Mats Fibrous scaffolds, referred to as fibro-porous mats, can be comprised of either randomly arranged (Figure 2.3) or specifically oriented (aligned) fibers. Although the ECM molecules of the dermis seem to be randomly arranged, it may be beneficial to explore an aligned matrix that can optimize the three purposes of the scaffold mentioned earlier. The cellular response to the 3D porous architecture of fibro-porous mats was shown to be superior to the more simplistic architectures of hydrogels. Some advantages demonstrated include enhanced cell adhesion, proliferation, and differentiation, 2, 500 x 20.0 kv 10 μm AMRAY #0000* FIGURE 2.3 Scanning electron micrograph of electrospun, fibro-porous mat made of a biocompatible, degradable, tyrosine-derived polycarbonate. 16 An Introduction to Biomaterials possibly because the porous architecture of the fibro-porous mats more closely mimicked the ECM in vivo (Moroni and Elisseeff 2008). Self-assembly and electrospinning are examples of manufacturing processes that have been used to fabricate 3D fibro-porous mats. Self-assembly is the spontaneous formation of nanofibers due to preprogrammed noncovalent bonds, such as hydrogen bonds (Barnes et al. 2007). Self-assembly is unique in that fibers can be synthesized with diameters on the lowest ECM scale (5–8 nm) (Barnes et al. 2007). However, manufacturing using self-assembly is limited to materials that are block polymers or those that possess both hydrophobic and hydrophilic regions such as peptide-amphiphiles, which typically have long hydrocarbon tails (hydrophobic) and hydrophilic heads that are attached to the tails via amide bonds (Paramonov, Jun, and Hartgerink 2006; Ryadnov and Woolfson 2003). Due to the high hydrophobicity of the tails, peptide-amphiphiles tend to form cylindrical micelles less than 1 μm in length (Barnes et al. 2007). Recently, a self-assembled nanofiber scaffold has been shown to accelerate wound re-epithelialization (Schneider, Garlick, and Egles 2008). The nanofibers were fabricated using ionic peptides of 16 amino acids in length that undergo spontaneous fiber formation when exposed to physiological conditions (Schneider, Garlick, and Egles 2008). Using a composite organotypic dermal–epidermal wound-healing model, it was shown that full thickness incisional wounds treated with an EGF-delivering self-assembled nanofiber scaffold had a threefold faster re-epithelialization rate when compared to wounds treated with the scaffold alone at 24 hours, and an even greater difference was shown at 48 hours (Schneider, Garlick, and Egles 2008). Although these results are exciting, manufacturing using self-assembly processing is limited to the bench top at the present time; therefore, this technology is not optimal for commercialization. Electrospinning is a process used to fabricate fibro-porous mats through the use of an electric field. A positively charged polymer jet is attracted to a grounded (or negatively charged) collector. The process produces long, continuous, nonwoven fibers ranging from the nanometer to micrometer scale (Barnes et al. 2007). The electrospun fibers provide a 3D architecture with high surface area that may promote cell attachment. This technique is compatible with a slue of biocompatible materials in either organic or inorganic solvents (Jiang et al. 2004; Li et al. 2002; Matthews et al. 2002; Meechaisue et al. 2006; Yang et al. 2004, 2005; Yoshimoto et al. 2003). For example, Ji et al. (2006a,b) fabricated electrospun, nanofibrous, hyaluronic acid scaffolds that promoted the fibroblast migration and invasion (Ji et al. 2006a,b). Other electrospun scaffolds have sustained human dermal fibroblast attachment, growth, migration, infiltration, and ECM deposition in vitro (Han et al. 2007; Ji et al. 2006b; Kumbar et al. 2008; Zhu et al. 2008). Electrospinning can be used to create randomly arranged fibers or aligned fibers on an industrial-sized scale. 2.5.5.3 Randomly Integrated Structures Phase separation is a commonly used process to fabricate randomly integrated structures, such as porous and/or fibrous scaffolds with randomly interconnected pores. Phase separation involves the thermodynamic separation of a solution into a polymer-rich phase and solvent-rich phase (Barnes et al. 2007). This process enables the tissue engineer to create fibers of varying diameters, ranging from 50 to 500 nm, which is comparable to collagen type I, the predominant ECM molecule found in the dermis. Like self-assembly, phase separation is limited to a few polymers, such as degradable aliphatic polyesters, and is restricted to the bench top (Barnes et al. 2007). The pores are typically created using either salt particles (Cai et al. 2002) or paraffin spheres (Woo, Chen, and Ma 2003), which are later removed via postprocessing. Although the larger sized pores in the scaffold can be controlled through variations in the size of the particles or spheres, one has much less control over any smaller pores created and the wall thickness of these scaffolds. 2.5.5.4 Designed 3D Scaffolds Rapid prototyping processes (RPPs), or solid freeform technologies, are computer-controlled layerby-layer technologies that can be used to create 3D TECs via the precise deposition of material, Cutaneous Wound Pathobiology 17 cells, and/or bioactive molecules. They differ from conventional manufacturing processes in that they are generally additive processes. Although there are many different types of RPPs (reviewed by Hutmacher, Sittinger, and Risbud 2004), this chapter will briefly discuss those that have been used for tissue engineering of the skin: photolithography, 3D inkjet printing, and the bioassembly tool (BAT). Photolithography is a high throughput technique that has been most successful in the semiconductor and electronics industries and has recently been adapted for tissue engineering applications. It is a process that efficiently and precisely (on the micron level) transfers patterns from a photo mask onto a photosensitive substrate. Using this technology, the spatial arrangement of the surface chemistry of the material can be specifically tailored. Hahn et al. (2006) have demonstrated that photolithography can successfully be used to modify the surface of poly(ethylene glycol)diacrylate hydrogel substrates with a pattern of RGDS peptides. Using human dermal fibroblasts, it was shown that only those areas of the substrate patterned with the RGDS peptides were able to support cell attachment and spreading (Hahn et al. 2006). Because of its efficient industrial-sized manufacturing ability and its potential to specifically control the cells’ microenvironment during the wound-healing process, photolithography may be a promising technology for tissue engineering of the skin. Inkjet printing offers the ability to dispense a biological solution onto a layer of powder and construct the layers into a 3D tissue-engineered scaffold. The most common approach has been to first print individual dots of the polymer material onto a substrate lacking cell adhesion properties, and then print cells onto the polymer scaffold (Xu et al. 2006). This two-step procedure is repeated until a 3D scaffold of a specific thickness has been synthesized. Lee et al. (2009) used 3D inkjet printing to create engineered tissue on nonplanar surfaces. Layers of collagen containing either fibroblasts or keratinocytes were printed between multiple layers of collagen hydrogel precursors that were crosslinked by a layer of nebulized aqueous sodium bicarbonate (Lee et al. 2009). Inkjet printing has many advantages in that it is an economic, flexible process with high throughput (Xu et al. 2006). On the other hand, the cells are limited to only two dimensions. 3D inkjet printing is versatile in that it can deposit both biomaterials and biopolymers at room temperature in either aqueous or organic solvents. Some major drawbacks of the technology are that it can be extremely difficult to remove any residual powder in each layer and the resolution of the scaffold’s pores is a direct function of the nozzle size and the properties of the powder. BAT is an RPP similar to inkjet printing but has the ability to dispense solutions of material as either individual dots or as continuous polymer jets (Smith et al. 2004, 2007). The BAT can extrude solution either using pneumatic pens or positive displacement pens and has specialized dispenser tips that allow multiple solutions to be spatially organized within the 3D construct. Because the BAT is controlled by computer-aided design (CAD), aligned fibers with predetermined pores and interconnectivity can be fabricated. This technology has also been used to extrude cells, that is, human fibroblasts, onto a substrate in predetermined 3D patterns (Smith et al. 2004). 2.6 SCARRING AND CELL-BASED THERAPIES In the last phase of the cutaneous wound-healing process (tissue remodeling), scar formation is typically the end result, and its severity can drastically vary from case to case. As described in Section 2.2, the remodeling phase is characterized by the resolution of inflammation, a reduction in vascularization, and the perfect balance between collagen synthesis and degradation (Kloeters, Tandara, and Mustoe 2007). In some cases, collagen deposition occurs at a much faster rate than its degradation, which often leads to hypertrophic scarring, and there are four known contributing factors to this condition. First, genetics may play a role in a patient’s susceptibility to hypertrophic scarring. Second, it has been shown that cutaneous wounds that take longer than 10–12 days to re-epithelialize have a higher incidence of resulting in hypertrophic scars (Mustoe 2004). Third, the areas of the body with tight skin, such as the knees and elbows, often scar because these areas are 18 An Introduction to Biomaterials subjected to high tensile forces that induce fibroblasts to synthesize collagen at a rate that is higher than normal. Lastly, a prolonged inflammatory response can also result in scar formation. Currently there are two strategies used in the clinic to treat or prevent hypertrophic scarring: (1) silicon gel sheeting and (2) steroidal injections. Because most animals do not heal with excessive scarring, it has been difficult to test therapeutics that addresses this problem in a preclinical setting. However, a hypertrophic rabbit ear scarring model has recently been shown to be a reproducible and quantifiable animal model (Kloeters, Tandara, and Mustoe 2007) and has been validated using various therapeutics, such as steroids (Morris et al. 1997), transforming growth factor (TGF-β) blockers (Lu et al. 2005), silicon occlusion (Saulis, Mogford, and Mustoe 2002), and collagen synthesis blockers (Kim et al. 2003; Reid et al. 2006). One promising strategy being pursued to address hypertrophic scarring is the use of adiposederived stem cells (ASCs). Evidence derived from children with progressive osseous heteroplasia, lipomas, liposarcomas, and obese people have all pointed to the existence of stem cells in the fatty tissue (reviewed by Gimble, Katz, and Bunnell [2007]). Liposuction provides from 100 mL to more than 3 L of fatty tissue per procedure, and more than 400,000 surgeries are performed per year in the United States. Because this material is in abundance and often discarded and is believed to contain stem cells, many researchers have begun to investigate the effects of ASCs on wound healing. Using the hypertrophic rabbit ear scarring model, studies have shown that rabbits treated with ASCs had statistically reduced hypertrophic scarring compared to untreated controls (Mustoe et al. 2008). Another approach to prevent hypertrophic scarring is treatment with various cytokines and growth factors. Because TGF-β is a key cytokine involved in dermal scarring, due to its integral role in collagen synthesis, it is not surprising that protein therapies with antiTGF-β antibodies and antisense oligonucleotides to TGF-β1, TGF-β2, and connective tissue growth factor (CTGF; O’Shaughnessy et al. 2007; Sisco et al. 2008) have been shown to reduce hypertrophic scarring. 2.7 CONCLUSION Under normal circumstances, cutaneous wounds heal quickly without adverse responses. However, extensive and/or contaminated wounds, or wounds in the setting of underlying pathobiology heal slowly and sometimes not at all. Failure to heal often necessitates the use of autografts or biological dressings including allogeneic skin substitutes. Autografts can be limited by inadequate harvest sites and/or by increased morbidity secondary to a surgical process resulting in another wound to heal. Biological dressings are a temporary covering that allow more time for the wound site to heal. 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Electrospun fibrous mats with high porosity as potential scaffolds for skin tissue engineering. Biomacromolecules 9:1795–801. 3 Osseous Wound Healing Ayesha Nawab, Mark Wong, Daniel Kwak, Lyndsey Schutte, Aditi Sharma, and Jeffrey O. Hollinger CONTENTS 3.1 Introduction to Bone Anatomy and Development................................................................... 23 3.1.1 Acellular Components of Bone.................................................................................... 23 3.1.2 Bone Biology and Development..................................................................................26 3.1.3 Osteogenic Growth Factors......................................................................................... 27 3.1.3.1 Bone Morphogenetic Proteins...................................................................... 27 3.1.3.2 Platelet-Derived Growth Factors.................................................................. 29 3.2 Types of Osseous Healing....................................................................................................... 30 3.3 Spontaneous Healing............................................................................................................... 31 3.4 Fracture Healing without Surgical Intervention...................................................................... 31 3.5 Fracture Healing Following Surgical Intervention.................................................................. 32 3.6 Primary Fracture Repair.......................................................................................................... 33 3.6.1 Gap Healing................................................................................................................. 33 3.6.2 Contact Healing........................................................................................................... 33 3.7 Secondary Fracture Repair...................................................................................................... 33 3.8 Graft Healing...........................................................................................................................34 3.9 Distraction Osteogenesis.........................................................................................................34 3.10 Promotion of Osseous Healing with Biologics........................................................................34 3.11 Conclusion............................................................................................................................... 36 References......................................................................................................................................... 36 3.1 INTRODUCTION TO BONE ANATOMY AND DEVELOPMENT Bone is a dynamic structural organ comprised of marrow, endosteum, periosteum, nerves, blood vessels, lymphatic channels, and mineralized and organic matrices. Macroscopically, bone is organized into a dense outer layer of cortical bone surrounding a lattice-like cancellous inner core. The hierarchical organization of bone is represented in Figure 3.1. The ability of surgeons to promote the regeneration of bone as a biologically and biomechanically functioning organ is predicated and dependent on their knowledge of the organizational hierarchy of bone as a dynamic composite tissue complex. Consequently, the underlying rationale for this chapter is to introduce the fundamental design composition of bone, emphasizing biology. Bone biomechanics and the highly complex biological dynamics (i.e., modeling–remodeling) are beyond the scope of the chapter; however, the inquisitive student is encouraged to pursue this compelling topic (suggested review modeling–remodeling; Hollinger 2003). 3.1.1 Acellular Components of Bone The composition of bone can be described in three organizational levels according to the size of its components. The smallest level of organization includes the nano and subnanostructures of bone, 23 24 An Introduction to Biomaterials Collagen molecule Cancellous bone Lamella Cortical bone Collagen fiber Collagen fibril Bone crystals Osteon Haversian canal 0.5 μm 10−500 μm Microstructure Macrostructure 1 nm 3−7 μm Nanostructure Submicrostructure Subnanostructure FIGURE 3.1 Hierarchical schematic of the principal components of bone from the subnanostructural to macrostructural scales. (From Rho, J. Y., L. Kuhn-Spearing, and P. Zioupos. 1998. Med Eng Phys 20:92–102. With permission.) composed of inorganic crystal matter and protein. The inorganic material is predominantly composed of platelet-shaped hydroxyapatite (HA; Ca10(PO4)6(OH)2) crystals, commingled with dahllite (Ca10(PO4, CO3)6(OH)2), and substituted with auxiliary ions within its crystal lattice structure (i.e., Na+, Mg2+, Cl–, and F–) (Bonar, Lees, and Mook 1985; Currey 2002). The protein is mainly type I collagen, a triple helix of three polypeptide chains approximately 1000 amino acids in length. The percent composition and organization of hydroxyapatite, collagen, and water are characteristic of the macrostructure of bone, defining the type of bone; for example, the increased percentage of mineral in a variety of bone has been directly correlated to increased mechanical strength (Currey 1988, 1990). It is important to note that although type I collagen comprises approximately 80% of the total protein found in bone (Niyibizi and Eyre 1994), there are over 200 additional noncollagenous proteins of bone (Delmas et al. 1984) responsible for biomineralization and other roles yet to be defined. The organization of hydroxyapatite and collagen defines the larger structural features of osseous tissue. In two dimensions, a “hole” zone exists between the NH2 terminus of one collagen molecule, known as a fibril, and the COOH terminus of the next due to cross-linking between adjacent triple helices; the collection of multiple collagen fibrils is termed the collagenous fiber. In addition to gaps, there are overlap regions of densely packed collagen fibers. Evidence suggests that the threedimensional (3D) conformation (Figure 3.2) of collagen fibrils results in contiguous holes (Hodge and Petruska 1963), forming channels that traverse throughout the fibrils. In addition to hydroxyapatite crystal formation originating between fibril gaps (Glimcher 1984), aggregates of hydroxyapatite are found between the collagen fibrils themselves (Katz and Li 1973). The second hierarchical level of organization describes micro- and submicrostructures, which include the lamellae, trabeculae, and Haversian systems. An image of trabeculae organized by the lines of stress is shown in Figure 3.3. The lamellae are composed of mineralized collagen fibers arranged in either planar or concentric sheets around a canal. These lamellae–canal units are known as Haversian systems or osteons. Haversian systems are the main functional component of cortical bone and are more accurately classified as secondary osteons to differentiate them from primary osteons, which are present in parallel-fibered bone of fibrolamellar (plexiform) bone (Francillon-Vieillot et al. 1990). The acellular components of osteons include the Haversian canals, located in the center of the osteon with lamellar formation developing from this central region outward (i.e., newest lamellae develop closest to the center). Within these canals are blood vessels, lymphatic vessels, and nerves, which travel from lacunae-containing osteocytes through radially oriented channels known as canaliculi. 25 Osseous Wound Healing Collagen fibril 1.23 nm Collagen molecules Overlap Hole zone region 300 nm 40 nm 27 nm Bone mineral crystal 50 × 25 × 3 nm Protein triple helix FIGURE 3.2 Representation of the collagen-mineral nanoscale motif. (From Rho, J. Y., L. Kuhn-Spearing, and P. Zioupos. 1998. Med Eng Phys 20:92–102. With permission.) FIGURE 3.3 Coronal sections from the proximal aspect of the femora. In the normal femur, the trabeculae are aligned along the lines of stress. (From Buckwalter, J. A., M. J. Glimcher, R. R. Cooper, and R. Recker. 1995. J Bone Joint Surg Am 77:1276–89. With permission.) Osteocytes are mature forms of the bone-forming osteoblast cells and are found in lacunae within the lamellae. Though relatively isolated from one another, osteocytes communicate and exchange nutrients through the numerous canaliculi present in the osteon. In addition to these smaller channels, Volkmann’s canals, also housing blood vessels, connect Haversian systems to one another as well as to the periosteum and marrow cavity. In short, osteons and bone in general are highly vascularized structures, and this supports their dynamic metabolic activity; in the first year of life, almost 100% of the skeleton is replaced, and in adults, remodeling proceeds at approximately 10% per year (Wheeless 2010). Figure 3.4 illustrates the organization of macrostructures in a long bone. 26 An Introduction to Biomaterials Epiphyseal line Epiphysis Trabecular bone Cartilage Medullary (marrow) cavity Cortical (compact) bone Trabecular bone Trabeculae Osteoclast Osteoblasts Osteocytes Capillaries in haversian and volkmann’s canals Cortical bone Concentric lamellae Periosteum Capillaries in haversian canals Capillary in volkmann’s canal Interstitial lamellae Osteocyte Circumferential subperiosteal lamellae FIGURE 3.4 Schematic diagram of cortical and trabecular bone. (From Hayes, W. C. 1991. Biomechanics of cortical and trabecular bone. In Basic Orthopaedic Biomechanics, ed. V. C. Mow and W. C. Hayes, 93–142. New York: Raven Press. With permission.) The third and largest hierarchical level includes both types of lamellar bone: trabecular and cortical. Early in osseous healing, woven bone, a less organized tissue is laid down as a foundation. The collagen fibers are haphazardly arranged and account for the anisotropic behavior of this type of bone. Woven bone is also known as immature bone and is found in fracture calluses and as part of the embryologic skeleton. It is subsequently resorbed by osteoclasts and remodeled into organized lamellar bone. 3.1.2 Bone Biology and Development The development of bone in an embryo and in subsequent stages of maturation follows one of two pathways: endochondral or intramembranous ossification. These processes are also recapitulated during osseous healing and discussed in later sections. In general, endochondral ossification occurs in four distinct phases: 1. A region of hyaline cartilage is produced by chondrocytes, which originate from mesenchymal stem cells (MSCs). 2. Chondrocytes rapidly divide by mitosis. 3. Chondrocytes produce and secrete alkaline phosphatase, increasing the concentration of phosphate ions in the environment. 4. Calcification occurs as a result of chondrocytic activity. The calcified matrix discourages nutrient supply to chondrocytes and consequently leads to their death, providing cavities for subsequent occupancy with osteocytes (Poole 1991). Osseous Wound Healing 27 In contrast to endochondral ossification, intramembranous ossification does not occur in a cartilaginous environment and instead involves the differentiation of MSCs into osteoblasts, resulting in the production of bone matrix within a collagen–fiber network. Areas of bone subsequently become organized in response to stress and produce thin sheets and bars of bone known as trabeculae. The trabeculae are further organized into an open-patterned region forming the cancellous portion of bone. Hematopoietic cells present in cancellous bone produce the red marrow, while more primitive cells are found in the fibrous soft-tissue layer covering the outer surfaces of bone known as the periosteum. Lastly, cortical bone is the dense external layer of bone formed by osteoblasts located in the periosteum. During embryonic development, three germ layers are involved in organogenesis: (1) the endoderm, (2) the mesoderm, and (3) the ectoderm. The mesoderm gives rise to osteoprogenitor and chondroprogenitor cells. Though the exact mechanism of embryonic bone development is not completely clear, the effects of hyaluronan (HY) present in the extracellular matrix (ECM) has a large influence over the development, migration, proliferation, shape, and metabolic functions of embryonic stem cells. More specifically, HY (secreted by numerous cell types, including MSCs) has been shown to play a prominent role in chemotaxis by providing a substrate through which MSCs migrate (refer to Chapter 19), while growth factors, particularly the bone morphogenetic proteins (BMPs), have been shown to be essential in healthy embryonic development (e.g., BMP-2 and -4), while others function in a pleiotropic behavior, affecting chemotaxis, mitosis, and differentiation (Reddi 1994). Consequently, endochondral and intramembranous skeletogenesis of the vertebrate embryo is highly dependent on local concentrations of HY, which dictate migration and correlate to the proliferative and differentiating effects of MSCs. In addition, differentiation is also highly associated with cell density/aggregation as well as cell–cell communication (i.e., ectomesenchymal interactions). As discussed in the following sections, there are a plethora of proteins associated with chondrogenesis and osteogenesis, as well as the differentiation of these cell types; some families include the BMPs, fibroblast growth factors (FGFs), platelet-derived growth factors (PDGFs), and vascular endothelial growth factors (VEGFs). 3.1.3 Osteogenic Growth Factors A thorough portrayal of the osseous milieu would not be complete without a discussion of the requisite growth factors (e.g., BMP, FGF, PDGF and VEGF) involved in ossification. The FGF and VEGF families are marked components for inducing neovascularization, angiogenesis, chemotaxis, morphogenesis, and mitogenesis. Despite their prevailing roles in embryogenesis and tissue development, the extensive functionalities of the FGF and VEGF families cannot be discussed in depth here, and the reader is referred to several reviews (Ferrara et al. 1992; Bikfalvi et al. 1997; Ferrara and Davis-Smyth 1997; Seghezzi et al. 1998). However, the following sections include a brief overview of BMP and PDGF, the two key growth factors essential in osteogenesis. 3.1.3.1 Bone Morphogenetic Proteins The remarkable history of the BMPs began when decalcified bone matrix was observed to induce bone healing (Senn 1889) and ectopic osteogenesis (Levander 1934, 1938). However, it was the discovery of the protein mixture (i.e., BMPs) responsible for bone induction that had generated widespread interest in their structure and function (Urist 1965). The following decades yielded profound insight on the heterogenic roles of the BMPs and the identification of their family members of which there are now over 20 identified (Ducy and Karsenty 2000). Because the BMP family is organized by its structural and amino acid sequence homology, it exhibits diverse biological function, some unrelated to bone and cartilage (Table 3.1) (Bessa, Casal, and Reis 2008). In short, the BMPs have direct and indirect effects on morphogen esis, differentiation, mitogenesis, and chemotaxis of MSCs. Furthermore, the developments 28 An Introduction to Biomaterials TABLE 3.1 Bone Morphogenetic Protein (BMP) Members in Humans and Their Main Physiological Roles BMP Nomenclature BMP-2 BMP-2a Cartilage and bone morphogenesis/heart formation BMP-3 BMP-3b BMP-4 Osteogenin GDF-10 BMP-2b Negative regulator of bone morphogenesis Negative regulator of bone morphogenesis Cartilage and bone morphogenesis/kidney formation Limb development/bone morphogenesis BMP-6 Vrg1, Dvr6 BMP-7 OP-1 BMP-8 OP-2 Hypertrophy of cartilage/bone morphogenesis/oestrogen mediation Cartilage and bone morphogenesis/kidney formation Bone morphogenesis/spermatogenesis BMP-9 GDF-2 Bone morphogenesis/development of cholinergic neurons/glucose metabolism BMP-11 GDF-11 Axial skeleton patterning/eye development/ pancreas development/kidney formation BMP-12 CDMP-3, GDF-7 BMP-13 BMP-14 CDMP-2, GDF-6 CDMP-1, GDF-5 Ligament and tendon development/ development of sensory neurons Cartilage development and hypertrophy Chondrogenesis/angiogenesis BMP-8b BMP-10 BMP-15 BMP-16 BMP-17 BMP-18 OP-3 BMP-5 GDF-9b Nodal Lefty Lefty Main Physiological Roles Spermatogenesis Heart morphogenesis Ovary physiology Embryonic patterning Embryonic patterning Embryonic patterning References Wang et al. 1990; Kang et al. 2004; Callis, Cao, and Wang 2005 Hino et al. 2004 Hino et al. 2004 Luyten et al. 1994; Kubler et al. 1998; Oxburgh et al. 2005 Cho et al. 2002; Zuzarte-Luis et al. 2004 Gitelman et al. 1994; Rickard et al. 1998; Kang et al. 2004 Reddi 1998; Kang et al. 2004; Simic and Vukicevic 2005 Ozkaynak et al. 1992; Zhao et al. 1996; Cho et al. 2002 Chen et al. 2003; Kang et al. 2004; Lopez-Coviella et al. 2006 Esquela and Lee 2003; Harmon et al. 2004; Kim et al. 2005; Andersson, Reissmann, and Ibanez 2006 Reddi 2003; Lo, Dormand, and Anderson 2005 Reddi 2003 Yamashita et al. 1997; Reddi 2003; Zeng et al. 2007 Zhao et al. 1996 Chen et al. 2004 Knight and Glister 2006 Celeste and Murray 1999 Celeste and Murray 2000 Celeste and Murray 2000 Source: Bessa P. C., M. Casal, and R. L. Reis. 2008. J Tissue Eng 2:1–13. GDF = growth/differentiation factor; OP = osteogenic protein; CDMP = cartilage-derived morphogenetic protein. of BMP devices (Hollinger et al. 2001; Schmitt et al. 1999; Winn, Uludag, and Hollinger 1999; Hollinger et al. 1996) and the landmark FDA approval of a BMP-2 biomedical device in 2002 (McKay, Peckham, and Badura 2007) testify to the robust osteogenic properties and promising therapeutic application of BMP-2. BMP monomers are synthesized endogenously as a precursor polypeptide with an N-terminal proregion sequence; subsequently, the precursor monomer is cleaved into the mature polypeptide, dimerized into homo- or heterodimers, and secreted. The BMPs conform to a characteristic cysteine knot motif in which the BMP monomer comprises seven cysteines: six are involved in intramolecular disulfide bonding and the seventh is responsible for intermolecular dimerization. Osseous Wound Healing 29 The proregion is known to facilitate secretion to the ECM; however, the noncleaved, propeptide forms of BMPs secreted into the ECM have also been linked to distinct physiological roles, such as synovial rheumatoid arthritis and BMP stabilization (Lories et al. 2007; Degnin et al. 2004; Brown et al. 2005; Gregory et al. 2005). BMPs affect the cell by binding to the serine–threonine kinase receptors and inducing specific intracellular pathways that modulate gene transcription. Of the types I, II, and III receptors for transforming growth factor beta (TGF-β) superfamily members, only types I and II have shown to play significant roles in BMP binding and signaling. The BMP signaling complex is best characterized by the SMAD pathway (involving the human homolog of the mothers against decapentaplegic gene). Two BMP dimers form a tetra complex with transmembrane receptors BMPR-IA, BMPR-IB, and BMPR-II. Following complex formation, BMPR-II phosphorylates BMPR-I, which subsequently phosphorylates the receptor SMADs 1, 5, and 8 (R-SMADs); I-SMAD6 is an inhibitory antagonist of this progression. These phosphorylated R-SMADs then bind to the common mediator SMAD4 (co-SMAD4) and translocate to the nucleus, initiating gene transcription and the ensuing differentiation to osteoblastic phenotype (Celil, Hollinger, and Campbell 2005; Hollinger et al. 2007). In addition to I-SMAD6, several other modulators (i.e., chordin, noggin, cerberus, follistatin, fetuin, gremlin, and sclerostin) inhibit BMP activity by binding to the BMP tetra complex or BMPRs. 3.1.3.2 Platelet-Derived Growth Factors The discovery of the PDGF family was ineludible: the propagation of mesenchymal-type cells (e.g., fibroblasts) in the serum utilized for early cell culturing techniques strongly suggested the presence of growth factors necessary for proliferation. Further investigation led researchers to deduce that the origin of these growth factors was from platelets, and the appropriately termed platelet-derived growth factor family was established (Balk 1971; Antoniades, Scher, and Stiles 1979; Heldin, Westermark, and Wasteson 1979). Years of research following their initial discovery, the PDGF receptors (PDGFRs) were characterized as receptor tyrosine kinases (RTKs), and the RTK pathway served as the archetypical mechanism of action for many other growth factors (e.g., VEGFs, FGFs, epidermal growth factors, hepatocyte growth factors [HGFs], etc.) (Robinson, Wu, and Lin 2000; Manning et al. 2002; Alonso et al. 2004). Additionally, PDGFs have shown to induce chemotaxis and mitogenesis of mesenchymal-derived cells (i.e., fibroblasts, osteoblasts, chondrocytes, and smooth muscle cells) (Hollinger et al. 2008). Similarly, deregulated PDGF expression and/or PDGFR activity has been closely linked with oncogenesis (Yu, Ustach, and Kim 2003; Heldin and Westermark 1999). Since its initial discovery, several isoforms of PDGF, bearing the cysteine knot domain, have been characterized to date (i.e., PDGF-AA, -AB, -BB, -CC, and -DD), all functional as dimers (Heldin, Eriksson, and Östman 2002). These dimers have the capacity to activate dimeric RTKs PDGF-Rα/α, PDGF-Rα/β, or PDGF-Rβ/β, however, only PDGF-BB has shown to bind to all three dimeric receptor types (Figure 3.5). Upon activation, the cytosolic tyrosine kinase domains will undergo autophosphorylation, yielding binding sites for the SH2 domain of signaling molecules, activating downstream pathways of Ras-MAPK, phosphatidyl inositol 3-phosphate (PI3K), and phospholipase Cγ (Heldin, Östman, and Rönnstrand 1998; Rozenkranz and Kazlauskas 1999). One of the pleitrophic effects of PDGF includes its capacity to promote osseous wound healing: first, PDGF induces mitogenesis and chemotaxis of osteogenic cells, as well as neovascularization of the site of osteogenesis; and second, the presence of PDGF has indirect effects on the expression of VEGF, HGF, and interleukin-6 (IL-6), a proinflammatory cytokine. Furthermore, PDGFs can regulate BMP activity by increasing the expression of gremlin and enhancing insulin-like growth factor (IGF) signaling. In turn, PDGF activity is modulated by the inflammatory cytokine IL-1, inhibiting the expression of PDGF-Rα in osteoblastic cells (Hollinger et al. 2008). 30 An Introduction to Biomaterials BB AA FIGURE 3.5 tive ligand. 3.2 CC AB DD PDGF-Rα/α PDGF-Rα/ PDGF-R / PDGF ligand-receptor specificity. Arrows indicate activation of a receptor with its respec- TYPES OF OSSEOUS HEALING Most tissues in the body respond to injury by forming a scar; however, bone has the capacity to heal itself to preinjury strength and function by the regeneration of tissue indistinguishable from the original (Feinberg, Steinberg, and Helman 1997). The sequence of events that occur during bone wound healing are universal regardless of the type and cause of injury and will be described in Section 3.3. Osseous healing is orchestrated by a complex system involving bioactive proteins, physical and chemical factors, and cells (Hollinger and Wong 1996). It begins with hematoma formation in the clotting phase. The exposure of platelets to collagen and thrombin induces activation of these platelets, leading to the formation of a hematoma, or blood clot, at the wound site (Dimitriou, Tsiridis, and Giannoudis 2005). The cessation of bleeding and stabilization of the wound site that results from hematoma formation is termed hemostasis. The hematoma is composed primarily of fibrin and is occasionally referred to as a fibrin clot (Diegelmann and Evans 2004). Activated platelets aggregate and release cytokines and growth factors that initiate subsequent steps in the wound-healing cascade, including the next phase of wound-healing, the inflammatory phase. The two growth factors released by platelets that are of greatest impact in the wound-healing process are PDGF and TGF-β (Doll et al. 2005; Hollinger and Wong 1996). In addition to inducing chemotactic migration of neutrophils, macrophages, and fibroblasts to the wound site, PDGF also stimulates the mitogenesis of fibroblasts (Hollinger et al. 2008). TGF-β attracts macrophages to the wound site and stimulates macrophages to secrete FGF, PDGF, tumor necrosis factor alpha (TNF-α), IL-1, and IL-6 (Diegelmann and Evans 2004). TGF-β also attracts fibroblasts and induces collagen production by these fibroblasts (Hollinger and Wong 1996). Neutrophils migrate to the wound site primarily within the first 24 hours after injury. They are responsible for eliminating particles from the wound site such as microbes, damaged or dead cells, cell debris, and damaged matrix material (Diegelmann and Evans 2004; Feinberg, Steinberg, and Helman 1997). Within 48 hours after injury, monocytes at the wound site become macrophages, which release PDGF and TGF-β. These macrophages serve to clean the wound site by engulfing cell and matrix debris, Osseous Wound Healing 31 engorged neutrophils, and other particulate matter remaining at the wound site (Diegelmann and Evans 2004). As wound healing progresses, the high metabolic activity at the wound site leads to a low pH and reduced oxygen tension, which in turn induces the release of VEGF and other factors such as TGF-β and FGF to initiate angiogenesis. During the entire process of wound healing, BMPs are released from the surrounding pre-existing matrix and are also secreted primarily by MSCs and osteoblasts in order to induce chemotaxis, proliferation, and differentiation of preosteoblasts and MSCs into osteoblasts (Dimitriou, Tsiridis, and Giannoudis 2005). The low oxygen tension at the wound site soon after the fracture is due initially to the destruction of blood vessels, leading to tissue necrosis and low pH (Pietrzak 2008). Osteoclasts at the fracture site contribute to the clearance of necrotic tissue. The process of angiogenesis is required for the efficiency of subsequent steps in the fracture healing cascade as blood vessels are needed for oxygen, nutrient and growth factor transport into the wound site, as well as waste removal from the fracture site. Depending on the conditions present in the site of bone injury, variations in osseous healing occur. Some of the more common types of healing observed during different clinical situations are discussed in Section 3.3. 3.3 SPONTANEOUS HEALING Besides the liver, bone is the only other organ capable of spontaneous regeneration. During this process, bone formation occurs within a segmental defect in a skeletal structure without the assistance of a bone graft. For this to occur, a mix of viable cells must be available in a setting of adequate nutrition, vascularity, and appropriate oxygen content (Chalmer, Grey, and Rush 1975). Periosteum is the cellular fibrous sheet that covers the external surface of the bone, while the inner surface of the periosteum, the cambial layer, acts as a source of new bone-forming cells responsible for the spontaneous regeneration of a skeletal defect. Osteoblasts differentiate from the embryonic fibroblasts contained within the inner layer of periosteum, laying down new bone through intramembranous ossification (Junqueira, Carneiro, and Kelley 1992). Activation of these cells follows a physiological stimulus, usually an injurious event. In order for sufficient bone formation to bridge a skeletal defect, adequate space must be present. In clinical practice, this occurs when the adjacent skeletal segments and soft tissue envelope are prevented from collapsing into the defect, eliminating the necessary space. Spontaneous osteogenesis is most commonly observed in children with highly active periosteal cells (Feinberg, Steinberg, and Helman 1997). However, it has also been seen in older individuals, perhaps as a function of latent osteogenic potential. 3.4 FRACTURE HEALING WITHOUT SURGICAL INTERVENTION Although bone is a living organ with a unique breaking strength comparable to that of medium steel, the ability of osseous tissue to resist nonaxial forces is limited (Feinberg, Steinberg, and Helman 1997). Bone must therefore be capable of repair whenever trauma compromises its integrity. Fracture repair is the process of osseous tissue regeneration within a closely approximated defect following injury. It is a complex series of events that involves the interplay of local and systemic regulatory factors, hormones, cytokines, growth and differentiation factors, cells, and ECM (Dimitriou, Tsiridis, and Giannoudis 2005). Factors that influence this process are the stability of the segments at the fracture site, vascularity, and the availability of osteoprogenitor cells (OPCs) and MSCs (Feinberg, Steinberg, and Helman 1997; Hollinger and Wong 1996). In the absence of surgical intervention, fractures heal through one of two processes: a direct mechanism similar to that associated with intramembranous ossification or through a cartilaginous intermediary, reminiscent of endochondral ossification. Direct or intramembranous ossification involves the development of bone directly from OPCs and MSCs residing in the periosteum as well as endosteum (Dimitriou, Tsiridis, and Giannoudis 2005). The cells that contribute most prominently in this process appear to be located in the cortical bone at the fracture site, the periosteum, 32 An Introduction to Biomaterials Osteoclasts Osteoblasts FIGURE 3.6 Cutting cone. Osteon with bone-resorbing osteoclasts that drill a tunnel into the bone and osteoblasts that lay down new bone (osteoid) to fill the tunnel with a new layer of bone. (From Hollinger, J. O., C. Hart, R. Gruber, and B. Doll. 2007. Protein therapeutics and bone healing. In Tissue Engineering: Applications in Oral and Maxillofacial Surgery and Periodontics. 2nd ed., ed. S. E. Lynch, R. E. Marx, M. Nevins, and L. A. Wisner-Lynch, 3–25. Chicago: Quintessence Publishing Co, Inc. With permission.) and the bone marrow. Preosteocytes and undifferentiated MSCs differentiate into osteoblasts, beginning approximately 24 hours after injury (Dimitriou, Tsiridis, and Giannoudis 2005). By the third postinjury day, osteoblasts produce a disorganized mineralized structure consisting of woven bone called the hard callus (Diegelmann and Evans 2004; Dimitriou, Tsiridis, and Giannoudis 2005). Cutting cones, which are repetitive units consisting of osteoclasts that breakdown matrix, followed by osteoblasts that secrete osteoid, re-establish the osseous continuity between two ends of a fractured bone by burrowing through the hard callus formed during intramembranous ossification (Figure 3.6). In contrast to direct bone healing, secondary healing emulates endochondral ossification. MSCs are induced to differentiate into chondrocytes, or cartilage cells, prior to calcification of the ECM and replacement with bone formed by osteoblasts (Dimitriou, Tsiridis, and Giannoudis 2005). This type of fracture healing involves the formation of a soft callus, consisting mainly of type II collagen and proteoglycans. MSCs are recruited from the periosteum as well as the surrounding soft tissue to first differentiate into chondroblasts from day 7 to day 21 (Dimitriou, Tsiridis, and Giannoudis 2005). Once mechanical stability has been established, the soft callus undergoes mineralization, during which the chondroblasts hypertrophy and are replaced by osteoblasts a rising from the differentiation of MSCs from the periosteum and surrounding tissues (Dimitriou, Tsiridis, and Giannoudis 2005). The soft callus is later replaced by woven bone produced by osteoblasts. After the wound-healing process is complete, remodeling of the newly formed bone begins to re-establish Haversian systems to restore the features of mature or lamellar bone. 3.5 FRACTURE HEALING FOLLOWING SURGICAL INTERVENTION When fractures are treated surgically, healing is affected by the degree of immobility achieved at the fracture site and the amount of space present between the fracture ends. The different clinical conditions under which fractures heal have led to the classification of fracture healing based upon the stability of the fracture segments. Healing by primary intention is observed when reduction at a fracture site occurs without space or mobility in the presence of a rich vascular supply. Healing influenced by mobility or lack of structural continuity proceeds by secondary intention. Osseous Wound Healing 3.6 33 PRIMARY FRACTURE REPAIR Primary healing occurs when rigid fixation immobilizes the bony fragments achieving mechanical stability. Capillaries and osteogenic cells form new bone by proliferating within the medullary zone across the fracture site. Studies by Schenk and Willenegger on cortical bone demonstrated that osseous healing across rigidly fixed fractures differs slightly depending on the presence or absence of space between the fracture ends (Feinberg, Steinberg, and Helman 1997). The two processes have been termed gap healing and contact healing. 3.6.1 Gap Healing Small gaps across a fracture site often persist despite rigid fixation due to deforming forces created by muscle function on bone. Within these spaces, growth of blood vessels from the periosteum and Haversian canals adjacent to the fracture are sources for mesenchymal preosteoblastic cells. These cells and the bone marrow pluripotent precursor cells and endosteal osteoblasts provide the preponderance of bone regenerative cells. Gaps smaller than 0.3 mm are filled with lamellar bone, whereas gaps up to 1 mm are initially filled with woven bone, which matures into lamellar bone. The process takes 6 weeks, and the lamellar bone laid down at the site is initially at right angles to the longitudinal axis of the bone, but later remodels so that the lamellae are parallel to the long axis of the repaired bone. 3.6.2 Contact Healing When there is no gap between the fractured segments of bone, cortical bone heals through the formation of a bone repair unit (BRU) to achieve contact healing (Feinberg, Steinberg, and Helman 1997). Osteoclasts on either side of the fracture line clear a path for vessel ingrowth along an advancing cone. Osteoblasts follow closely behind to lay down bone, and an osteon forms at the rate of 1–2 µm per day. This defined physiological structure responsible for resorption and regeneration is known as a cutting cone. Rigid fixation along the fracture site is required to help maintain stability, while the osteoclasts resorb bone because adequate strength is not achieved until the osteon is formed and has undergone remodeling (Hollinger and Wong 1996). 3.7 SECONDARY FRACTURE REPAIR Secondary healing commonly occurs in fractures that heal spontaneously as well as those treated with nonrigid fixation. The mechanisms involved in secondary fracture repair have already been described and occur in three well-defined stages: (1) the early inflammatory stage, (2) the repair stage, and (3) the remodeling stage. The first 2 weeks after injury comprise the initial inflammatory phase. Injury to blood vessels and subsequent hemorrhage as well as heat generated from the energy necessary to cause a fracture all lead to hypoxia and cell death. Necrosis at the fracture ends causes an inflammatory response, and the extravasation of platelets and red blood cells is responsible for a hematoma formation. The arrival of inflammatory cells, fibroblasts, and endothelial cells under the influence of cytokines, proinflammatory mediators, and physicochemical gradients leads to neovascularization and formation of granulation tissue. The repair phase is characterized by the formation of a callus. A low pH of around 4–5 and low oxygen tension in the early granulation tissue as well as micromovement at the site of immobilized fracture segments promote cartilage formation. The callus forms externally as well as internally between the bone ends. This initial union develops in the first 4–6 weeks and has limited strength so additional stabilization to immobilize the fracture site is required if a fibrous union is to be 34 An Introduction to Biomaterials p revented. Osteoblasts continue their activity so that a soft callus becomes hard, as cartilage is replaced by woven bone if mobility is limited. The remodeling phase occurs over months and involves osteoclasts that resorb and remodel the immature bone into organized lamellar bone. BMPs and other regenerative factors including TGF-β and insulin-dependent growth factor help orchestrate repair through their chemotactic, mitogenic, and differentiating activities (Hollinger and Wong 1996). 3.8 GRAFT HEALING When the space between the segments of discontinuous bone is large and no longer a fracture defect, a hard tissue transfer of autogenous, allogeneic, or xenogeneic bone can be used to provide bony continuity to augment existing reparative activities. Healing at a graft site occurs through the overlapping mechanisms of osteogenesis, osteoinduction, and osteoconduction. Osteogenesis is the process of new bone formation from osteoblastic cells present within a wound or from transplanted cells within the graft. This process is heightened when highly cellular living bone is used to fill the defect. Osteoinduction is the process of bone formation from OPCs into osteoblasts under the influence of inductive proteins such as the BMPs (i.e., BMP-2, -4, and -7). A recipient bed well populated by progenitor cells or a graft with osteoprecursor cells promotes this combination. Osteoconduction occurs when bone is formed by host-derived or transplanted OPCs along a physical matrix/scaffolding in close contact to a bony site. The porosity and interconnectivity of the matrix affects the amount of bone formed by this mechanism (Junqueira, Carneiro, and Kelley 1992). 3.9 DISTRACTION OSTEOGENESIS In situations where osteogenesis is required to augment the quantity of native bone, principles of osseous regeneration can be employed via distraction osteogenesis. Popularized by Ilizarov, distraction osteogenesis is a technique of applying tensile stress across a surgically created bony gap to take advantage of the periosteal- and endosteal-mediated regenerative process. The vector of the distracter determines which direction the augmentation is achieved. Bone formation during distraction osteogenesis has been shown to occur via membranous ossification. Following the surgical osteotomy, an inflammatory response occurs and a hematoma forms. As the distraction progresses, vascular ingrowths occur across the site, MSCs appear, and type I collagen is synthesized across the long axis of the distraction. Mineralization occurs at 2 weeks from the margins of the osteotomy and progresses centrally. Collagen is eventually replaced by bony spicules and the gap closes after distraction is discontinued during a consolidation period. 3.10 PROMOTION OF OSSEOUS HEALING WITH BIOLOGICS When attempts are made to enhance osseous healing, it is advantageous to employ existing physiological mechanisms present within a site of bony injury. The principal biological process to be thwarted is scarring, resulting in the replacement of bone with fibrous soft tissue. Normal bone healing can repair fractures and small gaps, but if the bony defect results in a void greater than a certain dimension, physiological restoration of bony continuity is unlikely. The size of a bone gap that will not heal by bone formation spontaneously is referred to as a critical-sized defect (CSD) (Schmitz and Hollinger 1986). In order for a bone CSD to regenerate bone (regeneration is the restoration of form and function with tissues equivalent to those existing prior to the tissue deficit), a supplemental procedure must be done; that is, at the CSD, a composition must be administered to promote the regeneration of osseous tissue. The composition may consist of autograft, allogeneic bone bank material, xenogeneic material that has been processed at a bone bank to render the material nonimmunogeneic, natural and synthetic polymers, such as collagen and poly(alpha-hydroxy acids), Osseous Wound Healing 35 respectively, cells (e.g., MSCs, adipocytes), biological factors (e.g., rhBMP-2, rhPDGF-BB), and combinations of the aforementioned. Typically, biologicals such as rhBMP-2 and rhPDGF-BB as well as cells, require a scaffold or delivery system. The scaffold or delivery system must provide a biomimetic equivalent to the ECM of osseous tissue. Scaffolds for bone regeneration offer opportunities to direct the wound site through a controlled healing pathway that avoids scarring. Multiple biological and biomechanical requirements need to be met by a scaffold. The scaffold should be a biological and mechanical match to bone. For example, a biodegradable scaffold composed of poly(alpha-hydroxy acids) (polylactic and polyglycolic; see Chapter 15) must resorb in harmony with new bone formation. If the scaffold resorbs too slowly, bone regeneration is thwarted; if resorption is too rapid, the requisite cell attachment cascade will not occur and a pseudarthrosis may occur (nonunion, such as a fibrotic nonunion). Moreover, in terms of biology, the scaffold must fulfill stringent performance criteria at the recipient site as well as systemically. For example, locally and systemically, biocompatibility must be achieved; moreover, chronic inflammation must be avoided (see Chapters 8 and 9). Additional performance criteria for a scaffold, also referred to as structural standards, must address properties such as osteoconductivity, surface format to promote cell attachment (i.e., charge, hydrophobicity, or hydrophilicity), and biomechanical parity to the bone at the anatomical locale being regenerated. The concept emphasized is that the design and development of bone regenerative scaffolds must comply with biological and biomechanical standards mimicking the extracellular matrices (i.e., organic and inorganic) of bone. Therefore, the biologist must define performance standards for the scaffold design, and the standards must be defined in terms of the dynamic quantitative and qualitative properties of bone. Physically, scaffolds must have a pore structure that is interconnected to facilitate cell attachment and cellular migration and thus promote osteoconduction. Mechanically, scaffolds have to withstand both compressive and tensile forces present in the wound site. In addition, if the scaffold is biodegradable, biodegradation products must be noncytotoxic and degradation profiles that match the patient’s ability to regenerate bone. Scaffolds can be composed of many different materials (see biodegradable materials in Chapters 13–24). Autogenous cancellous bone grafts are the gold standard for repairing a bone defect (Bauer and Muschler 2000). These grafts already contain optimized microstructures, mechanical properties, as well as cells and signaling molecules to support bone regeneration. The disadvantage is that they have to be provided in a second surgery (i.e., the donor site), and this procedure exposes patients to additional pain, operating room and anesthesia time, and potentially a higher risk of infection. Along with scaffolds, cells (e.g., MSCs) and proteins (recombinant human growth factors such as rhBMP-2 and rhPDGF-BB) have been used for bone regeneration. The most common cell phenotype used in bone tissue engineering is the bone marrow–derived MSC (BM-MSC). Bone marrow offers a concentrated, extractable source of stem cells, and MSCs are the logical choice because they are the stem cells that differentiate into osteoblasts. A survey of clinical trials using rhBMPs 2 and 7 suggest they are effective in stimulating bone healing (e.g., posterior lumbar spine fusions and recalcitrant tibial fractures, respectively) and may eliminate the need for autogenous bone grafting (Garrison et al. 2007). PDGF is an important growth factor in tissue engineering; it is a mitogen (amplifies mesenchymal-lineage cell quantity), a chemoattractant (attracts cells), as well as an angiogenic factor (promotes blood vessel formation) (Heldin and Westermark 1999; Hollinger et al. 2007; Hollinger et al. 2008). The angiogenic properties of PDGF are particularly important for promoting and sustaining the bone regenerative cascade. Moreover, PDGF has been shown to enhance bone formation (Howes et al. 1988), and this property has been exploited to accelerate fracture healing in a geriatric and osteoporotic preclinical model (Hollinger et al. 2008). In addition to the mitogenic, chemoattractant, and osteopromotive properties of PDGF, it upregulates VEGF (Hollinger et al. 2007; Hollinger et al. 2008). VEGF is crucial for the maturation of the endothelial cells that line blood vessels. 36 3.11 An Introduction to Biomaterials CONCLUSION The intent of this chapter is to introduce the student to a complex biological organ that is an integrated tissue composite of organic and inorganic matrices populated by multiple cell phenotypes and directed in space and time by a lush array of biological modulators. The coauthors of this chapter purposefully focused on biology as road map for the design and development of biomaterial compositions and their structures that may be useful for therapeutic bone regenerative applications. Bone is the second most frequently transplanted tissue after blood and consequently has generated the most attention for biomaterials scientists. 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Section I Biology, Biomechanics, Biomaterial Interactions Cellular Mechanics 4 Cell and Tissue Mechanobiology Wei-hui Guo, Pedro Alvarez-Urena, and Yu-li Wang CONTENTS 4.1 4.2 Introduction............................................................................................................................. 43 Cellular Mechanobiology........................................................................................................44 4.2.1 Cytoskeleton as Intracellular Structural Elements and Force Generators...................44 4.2.2 Mechanical Linkage Mediated by Membrane Adhesive Structures........................... 45 4.2.3 Response of Cultured Cells to Mechanical Signals.....................................................46 4.2.3.1 Responses of Cellular Behavior to Mechanical Signals...............................46 4.2.3.2 Regulations of Cell Growth, Death, and Differentiation by Mechanical Signals.................................................................................. 47 4.2.4 Signaling Mechanism of Mechanotransduction.......................................................... 47 4.3 Tissue Mechanobiology........................................................................................................... 48 4.3.1 Mechanical Signals in Soft Tissue Morphogenesis..................................................... 48 4.3.2 Mechanobiology of Bones........................................................................................... 49 4.4 Mechanobiology and Tissue Engineering............................................................................... 49 4.5 Mechanobiology and Diseases................................................................................................ 50 4.6 Conclusions.............................................................................................................................. 50 References......................................................................................................................................... 50 4.1 INTRODUCTION “A scientist discovers that which exists. An engineer creates that which never was.” (Theodore von Kármán). The unique challenge to a biomedical engineer is not only to create something that never existed but also to make sure that it works seamlessly in something that remains very much to be discovered—the human body. In regenerative medicine, a key task is to understand how cells interact with the surrounding tissues, which may then guide the design of materials that support artificial tissue growth. While previous attention has been focused on chemical factors such as growth factors and cytokines, it is becoming increasingly clear that mechanical interactions play an equally important role in determining cell proliferation, migration, and differentiation. For example, osteocytes in bones receive stress from body weight while endothelial cells of blood vessels receive mechanical stimulations of fluid shear and blood pressure. The active generation of and passive response to mechanical forces now appears to be a common property of adherent cells—cells that use receptors to form physical connections with the surrounding matrix or neighboring cells. In fact, a main function of such adhesion structures appears to be mediating the transmission and detection of mechanical signals, such as mechanical forces and rigidity, to complement chemical signals particularly where diffusion may be impaired by barriers. Proper consideration of mechanical properties of both artificial and tissue materials is likely to increase greatly the success in tissue engineering. 43

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