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Introduction to Biomedical Engineering Apr 29th 2014 Introduction to Tissue Engineering Ming-Long Yeh 葉明龍 1 2 1 Stem cells 2 Morphogenesis, generation of tissue in the embryo 3 Tissue homeostasis 4 Cellular signaling 5 The extracellular matrix as a biologic scaffold for tissue engineering 6 Natural polymers in tissue engineering applications 7 Degradable polymers for tissue engineering 8 Degradation of bioceramics 9 Biocompatibility 10 Cell source 11 Cell culture: harvest, selection, expansion, and differentiation 12 Cell nutrition 13 Cryobiology 14 Scaffold design and fabrication 15 Controlled release strategies in tissue engineering 16 Bioreactors for tissue engineering 17 Tissue engineering for skin transplantation 18 Tissue engineering of cartilage 19 Tissue engineering of bone 20 Tissue engineering of the nervous system 21 Tissue engineering of organ systems 22 Ethical issues in tissue engineering 3 The central tissue engineering paradigm 4 Introduction Tissue Engineering It generally involves the use of materials and cells with the goal of trying to understand tissue function and some day enabling virtually any tissue or organ on the body to be made de novo重新. 5 Introduction A foundation for the understanding and development of the cell-based systems needed for tissue engineering. One of these areas involves the cells themselves; include stem cell biology and cell sources, and various aspects of cell culture including harvesting, selection, expansion, and differentiation. Other important themes involving cells, such as cell nutrition and cryobiology and cellular signaling. 6 Introduction There are a variety of very important materials issues in tissue engineering. Cells adhere to the extracellular matrix material in the body; this matrix has an enormous affect on how the cells behave. However, to try to recreate extracellular matrixes, is a difficult task and therefore various materials have been explored to provide substrates for cell growth in vivo . The physical and chemical properties of these materials are important. These include natural polymers, degradable polymers, and bioceramics. 7 Introduction It also examines the important issue of tissue compatibility and biomaterials compatibility which are critical if these tissues are to be safe and integrate with the body. In addition, scaffold design and fabrication are discussed. Ways of using controlled release from materials is examined; controlled release of different factors (e.g. growth factors to promote vascularization) can provide an important means of controlling and improving tissue function. 8 Introduction Once the scaffold system and the cell system are developed, they have to be put together. This is generally done through a bioreactor where the cells and materials are combined with the right type of media and flow conditions inside the reactor. By combining all of these entities, a new tissue may be created. 9 Introduction Overall, this book provides a very useful guide for those who wish to understand important issues such as cell biology, materials science, and bioreactor design with respect to tissue engineering, providing specific examples of how tissue engineering is accomplished. skin transplantation, cartilage, bone, nervous system and organ systems 10 Introduction In 1997, media all over the world were aroused by a BBC documentary, Tomorrow’s World , showing what is now known as the Vacanti mouse (Cao et al. , 1997). Although the Vacanti experiment is truly exemplary for the discipline of tissue engineering, it is fair to say that the media upheaval was not so much caused by the actual experiment but even more by the spectacular sight of a nude mouse that had apparently grown a human ear on its back. 11 For many, the Island of Dr Moreau *攔截人魔島 (Wells, 1896) had become reality and media hype was born. At first sight one would tend to say that the field has over promised and has a history of not delivering on those promises. This statement would be too simple. The eagerness of the media to report on the advances in the field of tissue engineering is not so much caused by publicity eager scientists; the actual cause is the enormous demand in our society for technologies that are able to repair, or even better regenerate, damaged or worn out tissues and/or organs. 12 Island of Dr Moreau 13 The Mouse and the ear Superhero Science- Limb Regeneration Regenerative Medicine: Re-Growing Body Parts Organ Printing 14 Box 1 The Mouse and the ear: tissue-engineered cartilage in the shape of a human ear A formal formulation of the discipline is traced back to the paper of J. Vacanti and R. Langer in Science (1993). Since then, the number of studies in tissue engineering has grown rapidly. A landmark study published in 1997 in Plastic and Reconstructive Surgery by Y. Cao et al. (1997) attracted the interest of a large audience; thanks also to a BBC service on the subject. In this paper it is shown how to successfully regenerate the cartilagineous part of a 3-year-old child’s ear. The work is useful also from an educational point of view, as most of the ‘ingredients’ to perform a classic tissue engineering experiment are present. 15 The Mouse and the ear Materials Science: First, a plaster mold of the ear of a 3-year-old child was cast from an alginate impression of the ear. Then, a 100 μm thick non-woven mesh of poly(glycolic acid) (PGA) was immersed in a 1% weight/volume solution of poly(lactic acid) (PLA) in methylene chloride for 2 seconds and subsequently shaped into the plaster mold. 16 The Mouse and the ear Biology: calf chondrocytes (cartilage cells) were harvested from the articular surfaces of a calf, isolated by collagenase digestion, filtered, washed in cell medium, labeled with BrdU to control their viability, and seeded onto the polymeric scaffolds (1.5 x108 cells in total). The constructs were cultured in vitro for 1 week in an incubator under physiological conditions (T 37°C; pco2 5%) and then implanted into a subcutaneous pocket on the back of athymic無胸腺 mice. Three groups of scaffolds were considered: (I) scaffolds seeded with cells; (II) scaffolds reinforced with an external stent and seeded with cells; (III) externally stented scaffolds with no cells. 17 The Mouse and the ear Biochemistry: after 12 weeks, the constructs were explanted, sectioned, and histologically stained with specific markers for typical cartilage extra cellular matrix components hematoxylin and eosin for general tissue formation; alcian blue for glycosaminoglycan deposition; Masson’s trichrome for collagen formation). Immunohistochemistry was also performed to confirm that present collagen was specific for cartilage (type II). 18 The Mouse and the ear The results showed extensive cartilage formation in the scaffolds that were seeded with cells, while no cartilage was present in the unseeded scaffolds. Furthermore, scaffolds reinforced with an external stent for the first 4 weeks of implantation maintained the anatomical shape of the ear. In contrast, the other scaffolds lost partially their integrity and appeared of reduced size and distorted shape. From these findings the scientists concluded that cartilage formation is not mature enough in the first 4 weeks to counteract the contraction forces in the healing process. 19 The Mouse and the ear 20 The Mouse and the ear This experiment was surely a success for those years and definitely contributed to boost the interest in the field. If we consider the state of the art nowadays, however, a number of drawbacks still characterize this study, some of which are mentioned here: 21 The Mouse and the ear 1. 2. Skin coverage is missing and is a critical element of any ear reconstruction. Bovine immature (young) chondrocytes were used, while clinical application thereof is of course highly unlikely and these cells are now known to be partially unrepresentative for the use of human cartilage cell sources. Such human sources tend to loose differentiation capacity quite fast and are furthermore frequently characterized by necrosis in the center of scaffolds with a clinically relevant size. 22 The Mouse and the ear 3. An athymic or immunodeficient mouse model is used here. 4. 5. Obviously, this is nothing more than a useful screening model and large animal models are required to test clinical relevance in the presence of a functional immune system. Scaffolds need to provide an adequate mechanical stability to the construct at the time of implantation Implications on the growth rate of the artificial ear compared to the growth rate of a 3-yearold child should be addressed before the final implantation in the patient. 23 Introduction The loss or failure of an organ or tissue is one of the most frequent, devastating, and costly problems in human health care. A new field, tissue engineering, applies the principles of biology and engineering to the development of functional substitutes for damaged tissue. This article discusses the foundations and challenges of this interdisciplinary field and its attempts to provide solutions to tissue creation and repair . Langer and Vacanti (1993) 24 If the need is indeed so high, and the field has so extensively grown over the last decade, then why do we still lack frequent clinical successes? There are two major reasons for this phenomenon. First, bringing a relatively simple medical device from initial idea to a widespread clinical reality frequently takes a minimum of 10 years. As the underlying technology for developing tissue engineering products is less mature, and possibly more complex, it is to be expected that clinical progress in this field will be measured in decades rather than years. 25 Second, tissue engineering is truly a multidisciplinary field where acquired knowledge from individual classical disciplines (e.g. quantum physics, polymer chemistry, molecular biology, anatomy) no longer suffices to make substantial leaps. Individuals active in this field will have to acquire multidisciplinary skills and be willing to look over the borders of their home discipline. 26 A clear example of that is the paragraph on mass transport in the Cell Nutrition chapter ( Figure I.1 ). 27 Figure I.1 A glimpse at mass transport theory. 28 Figure I.2 Diagram sketching the layout of the book (groups biology, technology and evaluation form basic to clinical practice. Fundamental Biology Fundamental Biomaterials Applied Biology Engineering Tissue Engineering 30 Stem Cells Stem Cells (Chapter 1) gives insight into the different aspects of this cell and will show that the stem cell is not a single cell type but, in reality, encompasses different categories of cells ranging from the multipotent embryonic stem cell to the apparently less potent adult stem cell. 31 Two properties: Stem cells The ability to make identical copies of themselves (self renewal) and the ability to form other cell types of the body (differentiation) These properties are also referred to as ‘stemness’. Stem cells may potentially provide an unlimited supply of cells that can form any of the hundreds of specialized cells in the body. It is because of these properties that stem cells are an interesting cell source for tissue engineers. 32 Stem cells Stem cells can be divided into two main groups: embryonic and adult or somatic身體的stem cells. Embryonic stem cells are responsible for embryonic and fetal development and growth. In the human body, adult stem cells are responsible for growth, tissue maintenance and regeneration and repair of diseased or damaged tissue. 33 Stem cells 34 Stem cells 35 Morphogenesis: generation of tissue in the embryo The formation of tissues in the embryo. Although most tissue engineers do not venture into the realms of developmental biology and research with a strong focus on developmental biology is all too scarce in tissue engineering. Obviously most tissue engineering constructs will not be implanted into embryos but into human beings after birth. 36 Morphogenesis: generation of tissue in the embryo Thus important lessons for tissue engineering can be learned from the formation of organs during embryogenesis. (i) the origin of cells that contribute to the formation of a particular organ, (ii) the growth factors and their interrelationship in the formation of an organ, (iii) the mechanisms by which undifferentiated precursor cells are induced to specialize into an organ specific cell type, (iv) the subsequent steps in organ formation and (v) the interaction between cells and their environment consisting of both the extracellular matrix and neighboring cells. 37 Tissue Homeostasis 42 Tissue Homeostasis 3.2 Tissues with no potential of regeneration 3.3 Tissues with slow regeneration time 3.4 Tissues with a high capacity of regeneration Although our body has the capacity to regenerate to a large extent, there are tissues with no or limited capacity of regeneration. For example, the central part of our lens consists of lens lamellas that are embryonic fossils that will not have changed since they were developed during embryonal life. The cell populations of the photo receptor of the retina and the auditory organ of Corti are not regenerated. 43 Tissue Homeostasis 3.3 Tissues with slow regeneration time Our bones are constantly being renewed by an active process which involves the breakdown and rebuilding of new bone matrix by the osteoblasts. This constant renewal, governed by the continuous optimization of the load-bearing role of the bone by a functionally adaptive remodeling activity (which is more active in growing bone), is dominated by high magnitude, high-rate strains presented in an unusual distribution. Adaptation occurs at an organ level, involving changes in the entire bone architecture and bone mass. This process is continuously ongoing and results in a total turnover time of 3 years for the whole bone structure. 44 Tissue Homeostasis 3.3 Tissues with slow regeneration time Cartilage has a similar turnover rate over time, although the individual matrix components are renewed at various rates. The two major extracellular components in articular cartilage, collagen type II and aggrecan, are relatively longlived in the tissue non-enzymatic modifications by reducing sugars, thus ending in accumulation of advanced glycation endproducts. These accumulated endproducts reflect the half life of the components that for collagen is estimated to be 100 years and for aggrecan to be 3,5 years. The cellular turnover in cartilage is probably limited and the localization of stem cells in articular cartilage is so far undetermined. 45 Tissue Homeostasis 3.4 Tissues with a high capacity of regeneration Human regeneration in intestine and epidermis of the skin is characterized by a high turnover rate where cells constantly proliferate and differentiate; a process that is finalized by a discarding of the cells into the intestine, or off the body. The regeneration is maintained by a special resident cell type, the stem cell situated at the basal lamina. These cells persist throughout life their function is to maintain homeostasis, and effect tissue regeneration and repair ( Figure 3.3 ). 46 Tissue Homeostasis 角質層 (stratum corneum) 顆粒層 Stratum Granulosum 為表皮層的一層 stratum spinosum 棘細胞層,棘狀層 47 Cellular signaling Understanding how these cells interact is recognized as pivotal for the success of tissue engineering. Furthermore, in spite of all media attention that befalls stem cells, in reality cells represent only a small part of the dry weight of living tissue. 48 Cellular signaling Cellular signaling: the molecular mechanism by which cells communicate Tissue engineering, in analogy with development and wound healing, is a dynamic process in which the right cell type should be at the right place at the right time in order to establish a normally functioning tissue. Communication between cells and the rest of the body plays a crucial role in the coordination of cell number, position and function. 49 Cellular signaling 50 Cellular signaling A central paradigm can be recognized in most events of cellular signaling, which consists of three distinct steps (see Figure 4.2 ): 1. Signal initiation: an extracellular ligand binds to a receptor on the surface of the cell. Ligand binding changes activity of the receptor, thus generating the signal. 2. Signal transduction: The activated receptor triggers a signal transduction cascade in which intracellular proteins are activated, ultimately leading to activation of a so-called transcription factor in the cell’s nucleus. 3. Gene activation: The transcription factor binds to regulatory sequences in target genes, resulting in gene activation, protein synthesis and changed cellular physiology. 51 Figure 4.2 The paradigm of cell signaling. Although many variations on this theme exist, many signaling events follow the paradigm of signal initiation by an extracellular ligand, followed by signal transduction into the nucleus where the change in gene expression underlies physiological adaptation of the cell as depicted in Figure 4.1. 52 Signal transduction 53 54 The extracellular matrix as a biologic scaffold for tissue engineering All, or at least most, cells interact with an extracellular matrix, which, in contrast to the errant opinion of some engineers and even biologists, presents much more than mechanical support and adds substantially to the biological interactions in our body. As tissue engineering typically combines scaffolds with biologically active components as cells or growth factors we felt that a chapter on the biological equivalent: Extracellular Matrix (Chapter 5) could not be missed. 55 Extracellular Matrix Scaffolds to support the constructive remodeling of injured or missing tissues or organs can be composed of synthetic or naturally occurring materials. Such scaffolds can be degradable or nondegradable, and these scaffolds can be engineered to have specific mechanical and material properties that closely approximate those of the tissue to be replaced. Ultimately, the scaffold should facilitate the attachment, migration, proliferation, differentiation and three-dimensional spatial organization of the cell population required for structural and functional replacement of the target organ or tissue. 56 Extracellular matrix: Definition The extracellular matrix represents the secreted product of resident cells within each tissue and organ and is composed of a mixture of structural and functional proteins arranged in a unique, tissue specific three-dimensional ultrastructure. These molecules, mainly proteins, provide the mechanical strength required for proper function of each tissue and serve as a conduit for information exchange (i.e. signaling) between adjacent cells and between cells and the ECM itself. 57 Extracellular matrix: Definition The ECM is in a state of dynamic reciprocity 互惠(Bissell and Aggeler, 1987) and will change in response to environmental cues such as hypoxia and mechanical loading. In the context of tissue engineering applications therefore, use of the ECM as a scaffold indeed provides structural support, but perhaps more importantly provides a favorable environment for constructive remodeling of tissue and organs. 58 Extracellular matrix: Composition The extracellular matrix represents a mixture of structural and functional molecules organized in a three dimensional architecture that is unique to each tissue. Most of these molecules are well-recognized and they form a complex mixture of proteins, glycosaminoglycans, glycoproteins and small molecules arranged in a unique, tissue specific threedimensional architecture. The logical division of the ECM into structural and functional components is not possible because many of these molecules have both structural and functional roles in health and disease. 59 Extracellular matrix: Composition For example, both collagen and fibronectin, molecules that once were considered to exist purely for their ‘structural’ properties, are now known to have a variety of ‘functional’ moieties with properties ranging from cell adhesion and motility to promotion of or inhibition of angiogenesis. These ‘bimodal’ or multifunctional molecules provide a hint of the diverse occult amino acid sequences that exist within certain parent molecules and which, in themselves, harbor biologic activity. 60 Extracellular matrix: summary 1. Scaffolds composed of extracellular matrix are useful in many tissue engineering applications throughout the body. 2 The ECM is composed of structural and functional molecules arranged in a tissue specific three dimensional structure. 3 The main components of the ECM are collagen, fibronectin, laminin, and glycosaminoglycans. 4 Growth factors exist in the ECM in small quantities but are potent modulators of cell behavior. 5 Following isolation from various tissues, the ECM maintains structural and functional proteins that support the host response to tissue injury. 61 Extracellular matrix 6 These scaffolds promote constructive remodeling of host tissue and elicit various biologic responses including angiogenesis, chemotaxis, and antimicrobial properties. 7 The use of native xenogeneic scaffolds, devoid of cells from the original tissue, results in host accommodation as opposed to scaffold rejection. However, when the ECM is modified by chemical or other cross-linking methods, the biologic response is more consistent with chronic inflammation, tissue destruction, fibrous encapsulation, and scarring. 8 ECM scaffolds are currently used to treat a wide range of tissue injuries, but their full potential will only be realized following additional investigation and clinical application. 62 Scaffolds At this point in the book a shift is made to the more fundamental engineering aspects. Since not all scaffolds and matrices are completely synthetic, a chapter on Natural polymers (Chapter 6) seemed in place as well. Most tissue engineers, actively involved in the design of both synthetic and natural scaffolds or matrices, prefer to have these degrade after implantation. This is with good cause as the prolonged presence of foreign material in the body may induce a variety of unwanted effects such as implant-associated infection or mutagenesis. Two groups of authors discuss the aspects of implant degradation and this is done in two related chapters: Degradable polymers (Chapter 7) and Degradation of Bioceramics (Chapter 8). 63 Natural polymers Natural polymers are derived from renewable resources, namely from plants, animals and microorganisms, and are, therefore, widely distributed in nature. These materials exhibit a large diversity of unique (and in most cases) rather complex structures, and different physiological functions, and may offer a variety of potential applications in the field of tissue engineering due to their various properties, such as pseudoplastic behavior, gelation ability, water binding capacity, biodegradability, among many others. 64 Natural polymers In addition, they possess many functional groups (amino, carboxylic and hydroxyl groups) available for chemical (hydrolysis, oxidation, reduction, esterification, etherification, cross-linking reactions, etc.) modification and/or conjugation with other molecules, which allows an overwhelming variety of products with tailorable chemistries and properties to be obtained. 65 Natural polymers Protein materials may offer an additional advantage as they are able to interact favorably with cells through specific recognition domains present in their structure. On the other hand, the creation of hybrid materials – by means of combining the advantages of different natural polymers – may constitute a useful approach to mimicking the natural environment of the extracellular matrix and to obtaining scaffolding materials with superior mechanical and biological properties. Most popular natural polymers: chitosan, starch, collagen, hyaluronic acid, elastin. 66 Natural polymers An intrinsic characteristic of natural origin polymers is their ability to be degraded by naturally occurring enzymes, which may indicate the greater propensity of these materials to be metabolized by the physiological mechanisms. 67 Natural polymers Another important aspect to consider when using natural polymers, is that they can induce an undesirable immune response due to the presence of impurities and endotoxins (depending on their source), and their properties may differ from batch to batch during large-scale isolation procedures due to the inability to accurately control the processing techniques. 68 Natural polymers Nevertheless, as knowledge about these natural polymers increases, new approaches (including methods for production, purification, controlling material properties and enhancing material biocompatibility) are likely to be developed for designing better scaffolding materials to support the development of more natural and functional tissues. 69 Natural polymers In summary, both natural and synthetic polymers present important characteristics and, therefore, one must recognize that the best biodegradable polymer for biomedical applications might be found by taking steps towards the development of new biomaterials that combine the most favorable properties of synthetic and natural polymers. 70 71 72 73 74 75 76 77 Natural polymers: Summary 1. A wide range of natural origin polymers have frequent been used, and might in future be potential useful in tissue engineering. 2 . Tissue engineering scaffolds comprised of natural derived macromolecules have potential advantages of biocompatibility, cell-controlled degradability, and intrinsic cellular interaction. 3. However, they may exhibit batch variations and, in many cases, exhibit a narrow and limited range of mechanical properties. In many cases, they can also be difficult to process by conventional methods. 78 Natural polymers 4 . In contrast, synthetic polymers can be prepared with precisely controlled structures and functions. However, many synthetic polymers do not degrade as desired in physiological conditions, and the use of toxic chemicals in their synthesis or processing may require extensive purification steps. Many of them are also not suitable for cell adhesion and proliferation. 5 . The combination of natural origin polymer with synthetic polymers and the further development in emerging methodologies such as recombinant protein technologies is expected to lead to outstanding developments towards the development of improved materials to be used in tissue engineering application. 6 . No material alone will satisfy all design parameters in all applications within the tissue engineering field, but a wide range of materials can be tailored for discrete applications. 79 Degradable polymers for tissue engineering A general definition of tissue engineering can be found in The Williams Dictionary for Biomaterials ‘ Tissue engineering is the persuasion of the body to heal itself through the delivery to appropriate sites of molecular signals, cells and supporting structures. ’ Although very short, this definition addresses the three main components of tissue engineering: 1. Molecular signals, such as growth factors, that stimulate the proliferation and differentiation of cells in vitro and/or in vivo, the infiltration by surrounding tissue, 2. Cells, to regenerate the lost or damaged tissue, and 3. Scaffolds and matrices, i.e. porous supporting structures and/or gels that allow cell attachment and tissue ingrowth. 80 Degradable polymers for tissue engineering In addition to this, other important aspects regarding the polymeric material that is to be used are: Biocompatibility is of utmost importance (see Chapter 9) to prevent an adverse tissue reaction of the immune system. It should be possible to tune the rate of degradation of the polymer. For instance, the material should not degrade too quickly in load bearing areas, as it may cause a significant decrease of the mechanical properties and its consequent premature failure. On the other hand, degradation should not be too slow as it may prevent tissue regeneration. 81 Degradable polymers for tissue engineering The resulting degradation products should be non-toxic and, therefore, should not induce an inflammatory reaction. They should dissolve in body fluids and, after transportation via the lymphatic system, the kidneys should be able to excrete them from the body. 82 83 Degradable polymers for tissue engineering 1. Degradable polymers are suitable as scaffold material for tissue engineering as interference with the development and growth of new tissue and unwanted long-term reactions are prevented. 2. Degradable polymers for use in tissue engineering should be biocompatible, and the resulting degradation products should be non-toxic. The time required to complete degradation resorption depends on the intended application and preferably matches the formation of functional tissue. 3. Polymers are long-chain macromolecules formed by covalent coupling of monomers. Copolymerization, i.e. the preparation of polymers from two or more types of monomers, is often employed to tune their properties. 84 Degradable polymers for tissue engineering 4. For a polymeric material to be degradable in the body it is necessary that the main chain of the macromolecule contains labile degradable bonds, such as ester, carbonate, or anhydride bonds. 5. The most important degradable polymers are polyesters. In the presence of water, chain scission occurs by hydrolysis of ester bonds, which lowers the molecular weight. The resulting oligomers dissolve in the surrounding environment or undergo further hydrolysis. 85 Degradable polymers for tissue engineering 6. The soluble degradation products are transported from the implantation site via the lymphatic system to the kidneys which excrete them from the body. 7. Mass loss of the polymeric device, due to removal of the oligomers is referred to as erosion. Distinction is made between surface and bulk erosion processes. 8. Often, in vivo degradation is faster than in vitro degradation. This is due to the tissue response (inflammation, foreign body response) and the presence of enzymes. 86 Degradable polymers for tissue engineering 9. Linear aliphatic polyesters such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and their copolymers (PLGA) have been broadly used in tissue engineering. These polyesters degrade via hydrolysis of the main chain ester bonds. Their degradation products (lactic- and glycolic acid) are part of the Krebs metabolic cycle. 10. More advanced degradable polymers will have an optimal balance between mechanical properties and degradation rate. In addition, functional groups attract cells and/or growth factors required for tissue formation. 87 Cell Sources 1. Adult stem cells are undifferentiated cells present in differentiated tissues with a long term self renewal capacity and a differentiation potential toward the cells phenotypes of the same tissues. 2. The main role of adult stem cells is to maintain tissue homeostasis, and to replace cells lost due to normal tissue turnover, injury or disease. 3. The first adult stem cells to be identified in the 1960s were the hematopoietic stem cells (HSC) isolated from bone marrow. 88 Cell Sources 4. A specific HSC niche exists in the bone marrow where HSCs self-renew and are protected from entering a differentiative or apoptotic fate. Exit of HSC from the ‘niche’ may determine their exposure to a microenvironment promoting their differentiation into a hematopoietic cell progeny. 5. Stratified分層的 epithelia, such as epidermis and corneal epithelium, are organized in different cell layers. Epithelial stem cells are located in the basal layer. Only keratinocytes in the basal layer can divide. Basal keratinocytes that become committed to terminal differentiation, stop dividing, and migrate upward through the different cell layers completing the differentiation process. 89 Cell Sources 6 . Although post-natal neural tissue had long been thought to be unable to regenerate, two primary locations of post-natal neurogenesis, fueled by postnatal neural stem cells, were identified: the subventricular zone of the lateral ventricle wall (SVZ) and the subgranular zone of the hippocampal dentate gryrus (SGZ) in rodents, and the olfactory bulb and hippocampus in humans. 7. In addition to HSC, a second population of stem cells exists in the bone marrow. Originally identified as bone marrow stromal stem cells, these cells were later renamed mesenchymal stem cells (MSC). 90 Cell Sources 8. When cultured in vitro or transplanted in vivo under specific conditions MSC demonstrate a differentiation potential toward several mesoderm derived cell lineages such as osteogenic, adipogenic and chondrogenic. 9. Cells with properties similar to those exhibited by bone marrow-derived MSC exist in other connective tissues, such as adipose, skeletal muscle, etc. 10. Tissue specific stem cells may have the ability to cross lineage specific boundaries, i.e. they are ‘plastic’. True plasticity is defined as the ability of the progeny後代 of a single cell to actually function as another cell type. Some studies have challenged the concept of stem cell transdifferentiation potential by showing that a cell fusion was responsible. 91 Cell Sources 11. In terms of genetic disparity between donor and recipient, grafts can be classified as autografts, isografts, allografts or xenografts. Due to the presence of different cell membrane antigens allogeneic or xenogeneic graft are rejected by the body. 12. Stem cells such as MSC have a direct role in regulating the immune response and can prevent/ reduce the allogeneic or xenogeneic graft rejection. The mechanisms by which stem cells control the immune response are only partially known An Isograft is a graft of tissue between two individuals who are genetically identical 92 Cell Culture: Harvest, Selection, Expansion and Differentiation 1. Autologous or allogenic cells can be utilized alone or in combination with biomaterials to restore lost function of tissue and organs. 2. Cells can be harvested from different locations of the human body and, depending on the tissue to be restored, processed for selection, expansion of cell number or reimplanted directly into the patient. In all cases, care has to be taken to handle the harvested cells in a proper way from the beginning. 3. When a special cell type is needed for implantation, cells can be isolated from the initial harvest by targeting special characteristics of the cells such as size, surface markers, growth behavior and/or functional properties. 93 94 Cells can be harvested by using different methods. (A) Aspiration of bone marrow from iliac crest under local anaesthesia; (B) Harvest of epithelial cells from the bladder by flushing with saline; (C) Dermal core skin biopsy. 95 Empty full thickness defects in the trochlear groove (upper left); tissue implant containing cells and covered with fibrin glue (upper right); defect treated with implant and stapled with resorbable PGA/PDS fixatives (lower left); and treated defect 96 after 12 months (lower right) 97 98 Cell Culture: Harvest, Selection, Expansion and Differentiation 4. The most common way of expanding the initial cell number is by placing the cells in monolayer cultures; although for some cell types such as hematopoetic cells suspension cultures are needed. 5. For clinical TE applications, the selection, expansion and differentiation are made in specially designated laboratories. 6. A potential problem with serial expansion in monolayer is that the cells gradually change their phenotype and that contaminating fast growing cells could become too dominant. 99 Cell Culture: Harvest, Selection, Expansion and Differentiation 7. When the required cell number is achieved, the cells can be differentiated by exposing them to specific feeder cell lines, growth factors and/or to extra cellular matrix proteins. 8. The cells can be combined with synthetic scaffolds, biomaterials, forced into high density cultures or exposed to physical forces to additionally stimulate differentiation. 9. Most of the current clinical cell-based TE therapies include expansion of the cell number in in vitro cultures in special GMP laboratories. Future therapies will be aimed at shortcutting this logistic and relatively expensive procedures. 100 Scaffold Design and Fabrication Although some applications would involve direct injection of cells into the tissues that one intends to regenerate, most researchers active in the field of tissue engineering, and working with cultured cells, strive for implanting combinations of cells and scaffolds. These so-called hybrid constructs will usually have three dimensions and need to give access to both cells and nutrients or have to allow an out flux of active ingredients and waste products. The chapter Scaffold Design and Fabrication (Chapter 14) presents the reader with the different ways to manufacture scaffolds and explains the criteria these scaffolds have to fulfill. 101 Scaffold design and fabrication 102 103 Figure 14.3 a Scanning electron micrographs showing examples of scaffolds produced using various processing techniques. (A) liquid-solid phase separation and freeze-drying using 1,4-dioxane (PEGT/PBT), (B) compression molding and porogen leaching (PEGT/PBT), (C) nonwoven polylactic acid (PLA) mesh with 13 um fiber diameter, (D) 3D fiber deposition (3DF) of PEGT/PBT scaffold with a 175- u m fiber diameter and 0.5104 mm fiber spacing. 105 Scanning electron microscopy images showing chondrocytes attached to 3DF scaffolds containing (A)A homogeneous 1-mm fiber spacing or (B)an anisotropic pore-size gradient. By generating pore-size gradients within RP scaffolds. control over the distribution of cells and amount of ECM components, such as GAG and collagen, could be achieved. (C) Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs. Tissue Eng, 11: 1297– 1311. 106 107 108 109 110 111 112 113 114 Dual spinnerets 115 SFF: Scaffold Free Fabrication 116 117 118 119 Bioreactors for Tissue Engineering Ideally the researcher working on a hybrid construct and following the chapters, as they have been presented so far, now faces one of the main obstacles of tissue engineering: how to initiate cell growth and extracellular matrix formation in three-dimensional constructs of clinically relevant dimensions (usually centimeters versus millimeters in many in vitro and animal studies). These challenges, fundamentals and some solutions are discussed in Bioreactors for Tissue Engineering (Chapter 16). 120 Bioreactors for Tissue Engineering 121 Cell seeding 122 Bioreactor with dynamic compression for cartilage tissue engineering 123 Future automated bioreactor 124 125 Although not complete or covering all of the subjects, for which one could argue would be essential, the editors feel that these first 16 chapters (as well as this chapter), provide a solid basis for students who wish to acquire an understanding for tissue engineering. Much of the international research effort based on tissue engineering is along the lines presented in these chapters. But it is also fair to say that some of the major challenges are still to be found into making tissue engineering a widespread clinical reality. These clinical aspects are treated in the remaining six chapters. 126 17 Tissue engineering for skin transplantation 18 Tissue engineering of cartilage 19 Tissue engineering of bone 20 Tissue engineering of the nervous system 21 Tissue engineering of organ systems 22 Ethical issues in tissue engineering 127 Thanks! 128