Tissue Engineering

Front Cover
Tissue Engineering
Tissue EngineeringThird EditionEditorsJan de BoerClemens A. van BlitterswijkAssistant EditorsJorge Alfredo UquillasNusrat Malik
Contents
Contributors
Preface
1 - An introduction to tissue engineering; the topic and the book
1.1 Learning objectives
1.2 What inspired you to pick up this book?
1.3 What is tissue engineering about?
1.4 Tissue engineering's origin and progression over time
1.5 Tissue engineering's limitations and promises
1.6 The future of tissue engineering
1.7 Tissue engineering and you
1.8 How to use this book? A guide for students and teachers
1.9 How to use the chapters?
1.10 References
2 - Stem cells
2.1 Learning objectives
2.2 Introduction
2.3 What defines a stem cell? Self-renewal, proliferation, and differentiation
2.4 Self-renewal
2.5 Stem cell proliferation
2.6 Stem cell differentiation
2.7 Stem cell quiescence and activation
2.8 Cell death is normal—apoptosis, autophagy, necrosis, and necroptosis
2.9 Characterization of stem cells—protein expression
2.10 Characterization of stem cells—RNA analysis by RT-PCR, microarray, and RNA-sequencing
2.11 Characterization of stem cells—cell differentiation
2.12 Stem cell signaling—the Wnt and β-catenin pathway
2.13 Hematopoietic stem cells
2.14 Mesenchymal stem cells
2.14.1 MSC modulation of the immune system
2.14.2 Epithelial stem cells—skin and intestine
2.15 Skin stem cells
2.16 Lgr5+ stem cells of the intestine
2.17 Central nervous system stem cells
2.18 Induced pluripotent stem cells—iPS cells
2.18.1 Deextinction of the northern white rhino by iPS cells
2.19 Natural pluripotent and embryonic stem cells
2.19.1 Differentiation of pluripotent stem cells
2.19.2 Application of pluripotent stem cells
2.20 Organoids, exosomes, and extracts from stem cells
2.21 Stem cell mechanobiology: stretch and strain
2.22 Future perspective
2.23 The dark side: cancer stem cells
2.24 Recommended literature
2.25 Assessment of your knowledge
2.26 Glossary
2.27 Further reading
3 - Tissue formation during embryogenesis
3.1 Learning objectives
3.2 Introduction
3.2.1 Organ formation during embryogenesis
3.2.2 The formation of the three germ layers during gastrulation
3.2.3 Establishment of the body plan by morphogen signaling
3.2.4 Neural crest cells
3.3 Cardiac development
3.3.1 Geometrical changes transform a single beating heart tube into a four-chambered heart
3.3.2 Cardiomyocytes
3.3.3 Future perspective
3.4 Blood vessel development
3.4.1 Vasculogenesis and angiogenesis
3.4.2 Blood pressure drives specification of vessels in arteria or veins
3.4.3 Vessel wall stabilization by smooth muscle cells and pericytes
3.4.4 Recruitment of mural cells is mediated by PDGF signaling
3.4.5 Future perspective
3.5 Development of peripheral nerve tissue
3.5.1 Development of the schwann cell lineage
3.5.2 Myelinating and nonmyelinating nerve fibers
3.5.3 Structure of the peripheral nerve sheath
3.5.4 Future perspective
3.6 Embryonic skin development
3.6.1 Interfollicular epidermis
3.6.2 Follicular epidermis
3.6.3 Dermis
3.6.4 Tissue engineering of embryonic and newborn skin
3.6.5 Cell–cell interactions and growth factors
3.6.6 Future perspective
3.7 Bone development
3.7.1 Skeletal precursor cells
3.7.2 Endochondral ossification
3.7.3 Intramembranous ossification and osteoblast differentiation
3.7.4 Osteoclast differentiation
3.7.5 Tissue engineering of bone
3.7.6 Tissue engineering of articular cartilage
3.7.7 Future perspective
3.8 Recommended literature
3.9 Assessment of your knowledge
3.10 Glossary
4 - Cellular signaling
4.1 Learning objectives
4.2 Paradigm of cellular signaling
4.3 Signal initiation
4.4 Signal transduction
4.4.1 G-protein–coupled receptor–mediated signaling
4.4.2 Receptor tyrosine kinase signaling
4.4.3 TGF-β superfamily signaling
4.4.4 Wnt signaling
4.4.5 Rho kinase signaling
4.4.6 NF-κB signaling
4.4.7 Vitamin D signaling
4.5 Gene activation
4.6 Variations on a theme
4.7 Future perspective
4.8 Recommended literature
4.9 Assessment of your knowledge
4.10 Glossary
4.11 References
5 - Extracellular matrix as a bioscaffold for tissue engineering
5.1 Learning objectives
5.2 Introduction
5.3 Native extracellular matrix
5.3.1 ECM composition
Collagen
Fibronectin
Laminin
Glycosaminoglycans
Growth factors
5.4 ECM scaffold preparation
5.5 Constructive tissue remodeling
5.5.1 Default mammalian wound healing versus constructive remodeling
5.5.2 Mechanisms behind ECM-mediated constructive tissue remodeling
Scaffold degradation
Endogenous cell therapy by ECM bioscaffolds
Modulation of the host immune response by ECM bioscaffolds
Macrophage heterogeneity
ECM bioscaffolds promote a constructive macrophage phenotype
Antimicrobial properties of ECM bioscaffolds
5.6 Clinical translation of ECM bioscaffolds
5.6.1 Skeletal muscle reconstruction
5.6.2 Esophageal mucosa reconstruction
5.7 Commercially available scaffolds composed of ECM
5.8 Future perspective
5.9 Recommended literature
5.10 Assessment of your knowledge
5.11 Glossary
5.12 References
6 - Synthetic biomaterials
6.1 Learning objectives
6.2 Introduction
6.2.1 Why are biomaterials important?
6.2.2 Synthetic biomaterials and their features
6.3 Biomaterials and synthetic chemistry: a molecular view
6.3.1 Atoms, molecules, and interactions
6.3.2 Classes of materials
Polymers: naturals, synthetics, and hybrids
Inorganics: ceramics and glasses
Composites
6.3.3 Synthetic transformations
6.4 The extracellular matrix: a chemical view
6.4.1 What are we trying to mimic?
6.4.2 The ECM is nothing but polymers and composites
6.5 Rational design
6.5.1 Degradation
6.5.2 Mechanical properties
6.5.3 Stimuli response
6.5.4 Bioactivity
6.5.5 Biomimicry
6.6 Future developments
6.6.1 Spatiotemporal complexity
6.6.2 Biohybrid approaches
6.6.3 Rational design
6.6.4 Precision
6.7 Case study: vascularization
6.7.1 How to create a synthetic system for vascularization
6.7.2 Designing a material system
Clinical/industrial solution
Academic solution
Testing
6.8 Recommended literature
6.8 Recommended literature
6.9 Assessment of your knowledge
6.10 Glossary
6.11 References
7 - Degradation of biomaterials
7.1 Learning objectives
7.2 Introduction
7.3 Bioceramics and glasses
7.3.1 Properties of bioceramics and glasses that influence degradation
7.3.2 Degradation mechanisms of bioceramics
Physicochemical degradation of bioceramics
Cellular degradation of bioceramics
7.4 Biodegradable polymers
7.4.1 Introduction
7.4.2 Mechanisms of polymer degradation and erosion
7.4.3 Bulk erosion
Overview
Surface erosion
Degradation kinetics
7.4.4 Factors that influence degradation
7.4.5 Material composition
7.4.6 Bulk eroding polymers
7.4.7 Surface-eroding polymers
7.4.8 Molecular weight
7.4.9 Crystallinity
7.4.10 Glass transition temperature
7.4.11 Architecture
7.4.12 Processing
7.4.13 In vivo degradation
Conditions at implantation site
Inflammatory response
Size and shape
7.4.14 In vitro testing and characterization
7.4.15 In vivo testing and characterization
7.5 Biodegradable metals
7.5.1 Principles of metal corrosion
Corrosion in the in vivo environment
Localized corrosion effects
7.5.2 Magnesium-based implants
Magnesium corrosion
Controlling magnesium degradation rates
Mg-based tissue scaffolds: designing for function and enhanced properties
7.6 Future perspective
7.7 Recommended literature
7.8 Assessment of your knowledge
7.9 Glossary
7.10 References
8 - Cell–material interactions
8.1 Learning objectives
8.2 Introduction
8.2.1 Cell–material/cell–extracellular matrix interactions
8.2.2 Integrins
8.2.3 Integrin-mediated adhesion structures
8.2.4 Mechanotransduction
8.2.5 Cell adherence to synthetic materials
8.3 Surface chemistry
8.3.1 Hydrophobicity and hydrophilicity
8.3.2 Presentation of chemical groups
8.3.3 Patterning using surface chemistry
8.3.4 Ligand spacing
8.3.5 Dynamic chemistry
8.4 Material mechanics (stiffness)
8.4.1 Cell behaviors and matrix mechanics
8.4.2 Mimicking tissue stiffness in vitro
8.4.3 Stem cell differentiation and substrate mechanics
8.5 Topography
8.5.1 Cell guidance by micro- and nanostructures
8.5.2 Nanotopography and stem cell differentiation
8.6 Future perspective
8.7 Recommended literature
8.8 Assessment of your knowledge
8.9 Glossary
8.10 References
9 - Biomaterials discovery: experimental and computational approaches
9.1 Learning objectives
9.2 Introduction
9.3 The challenges of biomaterials discovery
9.4 Approaches to materials discovery
9.5 Experimental high throughput materials discovery
9.5.1 The supporting substrate and coating
9.5.2 Materials libraries
9.5.2.1 High throughput screening systems
9.5.2.2 Combinatorial polymer libraries
9.5.2.3 Genetic methods for materials design
9.5.2.4 design of experiments
9.5.2.5 Synergistic effects
9.5.3 Biological assays
9.6 Computational materials discovery
9.6.1 Problems and opportunities raised by the size of chemical space
9.6.2 Introduction to computational modeling
9.6.3 Ontologies
9.6.4 Computational modeling of structure–property relationships
9.6.4.1 Descriptors
9.6.4.2 Feature selection
9.6.4.3 Statistical and machine learning models
9.6.4.4 Model validation and assessment of predictivity
9.6.5 Illustrative example: correlating measured material properties with cellular attachment
9.6.6 Evolving materials computationally
9.6.7 White box modeling
9.7 Future perspective
9.8 Recommended literature
9.9 Assessment of your knowledge
9.10 Glossary
10 - Microfabrication technology in tissue engineering
10.1 Learning objectives
10.2 Introduction
10.2.1 Background
10.2.2 Microfabrication meets tissue engineering
10.3 Microfabrication techniques in tissue engineering
10.3.1 Photolithography
10.3.2 Soft lithography
10.3.3 Microfluidic fabrication of microtissues
Microfluidic fabrication of point-shaped microtissues
Microfluidic fabrication of line-shaped microtissues
Microfluidic fabrication of plane-shaped microtissues
10.3.4 Microtissue assembly and application perspectives
Self-assembly of microtissues
Textile techniques for the assembly of line-shaped microtissues
Templated molding of microtissues
10.4 Future perspective
10.5 Recommended literature
10.6 Assessment of your knowledge
10.7 Glossary
10.8 References
11 - Scaffold design and fabrication
11.1 Learning objectives
11.2 Introduction
11.3 Scaffold design
11.3.1 Morphology
11.3.2 Porosity
11.3.3 Interconnectivity
11.3.4 Pore characterization
11.3.5 General scholium to scaffold design
11.4 Classical scaffold fabrication techniques
11.4.1 Porogen leaching
11.4.2 Phase separation
11.4.3 Ice templating
11.4.4 Micromolding
11.4.5 Gas foaming
11.4.6 Classical nonwoven textiles
11.4.7 Knitted and braided textiles
11.5 Electrospinning
11.5.1 Electrospinning principles
11.5.1.1 Collection systems
11.5.1.2 Electric field variations
11.5.1.3 Spinneret configurations
11.5.2 Cell/electrospun scaffold interactions
11.5.3 Melt electrospinning
11.6 Additive manufacturing
11.6.1 Direct writing and extrusion of polymers
11.6.2 Inkjet/powder systems (3D printing)
11.6.3 Stereolithography
11.6.4 Digital light processing
11.6.5 Digital light synthesis
11.6.6 Two-photon polymerization
11.6.7 Selective laser sintering
11.6.8 Melt electrowriting
11.7 Hybrid fabrication
11.8 Clinical translation of scaffold guided tissue engineering
11.9 Future perspective
11.10 Recommended literature
11.11 Assessment of your knowledge
11.12 Glossary
11.13 References
12 - Controlled release strategies in tissue engineering
12.1 Learning objectives
12.2 Introduction
12.2.1 Bioactive molecules of interest in tissue engineering
12.2.2 Modes of controlled release
12.3 Physical mixtures of bioactive factors within matrices
12.4 Bioactive factors entrapped within gel matrices
12.5 Bioactive factors entrapped within hydrophobic scaffolds or microparticles
12.6 Bioactive factors bound to affinity sites within matrices
12.7 Bioactive factors covalently bound to matrices
12.8 Matrices used for immunomodulation
12.9 Recommended literature
12.10 Assessment of your knowledge
12.11 Glossary
12.12 References
13 - Bioreactors: enabling technologies for research and manufacturing
13.1 Learning objectives
13.2 Introduction
13.3 Basic requirements
13.4 Mimicking physiological culture conditions
13.4.1 Mass transport
13.4.2 Physical stimuli
13.5 Bioreactors for cell expansion and cell-based products
13.5.1 Cell-based products
13.5.2 Bioreactor types
13.5.2.1 Mixing bioreactors
13.5.2.2 Perfusion bioreactors
13.5.3 Scale-up versus scale-out
13.6 Bioreactors for tissue engineering
13.6.1 Cell seeding
13.6.2 Differentiation
13.7 Future perspective
13.8 Recommended literature
13.9 Assessment of your knowledge
13.10 Glossary
13.11 References
14 - Strategies to promote vascularization, survival, and functionality of engineered tissues
14.1 Learning objectives
14.2 Introduction
14.3 Strategies to improve vascular ingrowth into TE constructs
14.3.1 Modification of scaffolds
Chemical composition
Physical properties
Biological properties—decellularized matrices
14.3.2 Fabrication of predefined hollow channel networks—new technologies
3D printing
Microfluidics
Sound wave technology
14.4 Strategies to improve vascular ingrowth into TE constructs—biological features
14.4.1 Incorporation of growth factors
14.4.2 Incorporation of vascular or proangiogenic cells
14.4.3 Incorporation of microvascular fragments
14.5 Strategies to promote neo-vascularization
14.5.1 In vitro prematuration
14.5.2 Strategies to improve cell survival
14.5.3 Strategies to promote inosculation
14.5.4 In situ prevascularization
14.6 In vivo models
14.6.1 Assessments of the vascularity within constructs—in vivo models
14.6.2 Assessment of graft vascularization and functionality of engineered tissues—in vivo imaging
14.6.3 Assessment of graft vascularization and functionality of engineered tissues—ex vivo analysis
14.7 Translation into clinics
14.8 Recommended literature
14.9 Assessment of your knowledge
14.10 Glossary
15 - Skin tissue engineering and keratinocyte stem cell therapy
15.1 Learning objectives
15.2 Introduction
15.2.1 Burn wounds
15.2.2 Chronic wounds
15.3 Structure and function of the epidermis
15.3.1 Keratins
15.4 Structure and function of the dermis
15.5 Epidermal and hair follicle stem cells of the skin
15.5.1 Keratinocyte stem cells
15.6 In vitro keratinocyte culture
15.6.1 Cultured keratinocyte sheet grafts for the treatment of burns
15.7 Cultured three-dimensional skin models
15.8 Immunogenicity with allogeneic and biosynthetic materials
15.9 Development of in vivo somatic keratinocyte stem cell grafting
15.9.1 Evolution of epidermal replacement
15.9.2 Evolution of dermal replacement
15.10 Poor keratinocyte “take”
15.11 Skin tissue engineering
15.11.1 Acellular dermal matrix
Integra dermal regeneration template
AlloDerm
Matriderm
Matriderm
Matriderm
Matriderm
Biobrane
Spincare
15.11.2 Dermal matrix with fibroblasts
Dermagraft
15.11.3 Skin cell suspensions (applied as sprays or injections)
Recell and Keraheal
15.11.4 Dermal matrix with keratinocytes
Kaloderm and Holoderm
Full-thickness skin equivalents
Apligraf
DenovoSkin
15.12 The use of adult stem cells in tissue-engineered skin
15.12.1 Induced pluripotent stem cells
15.12.2 Mesenchymal stem cells and adipose-derived stromal cells
15.13 Future perspective
15.14 Recommended literature
15.14 Recommended literature
15.15 Assessment of your knowledge
15.16 Glossary
15.17 References
16 - Cartilage and bone regeneration
16.1 Learning objectives
16.2 Introduction: cartilage
16.3 Cellular structures and matrix composition of hyaline cartilage
16.4 Collagen
16.5 Proteoglycans
16.6 The chondrocyte
16.7 Stem cells in cartilage and proliferation of chondrocytes
16.8 Pathophysiology of cartilage lesion development
16.9 Artificial induction of cartilage repair
16.10 Rationale for cell implantation
16.11 Cartilage specimens for implantation
16.12 Cell seeding density
16.13 What type of chondrogenic cells is ideal for cartilage engineering?
16.14 Allogeneic versus autologous cells
16.15 Articular chondrocytes versus other cells
16.16 Embryonic stem cells andinduced pluripotent stem cells
16.17 Xenograft cells
16.18 Direct isolation of tissue
16.19 Scaffolds in cartilage tissue engineering
16.20 Bioreactors in cartilage tissue engineering
16.21 Growth factors that stimulate chondrogenesis
16.22 Future developments in cartilage biology
16.23 Introduction: bone—basic bone biology: structure, function, and cells
16.24 Intramembranous and endochondral bone formation
16.25 Fracture repair
16.26 Critical size defect
16.27 Skeletal stem cells
16.27.1 Immunomodulatory properties
16.28 Expansion and differentiation
16.29 Growth factors for bone repair
16.29.1 Scaffolds for bone regeneration
16.30 Scaffold biocompatibility
16.31 The function of the vasculature in skeletal regeneration
16.32 Animal models in bone tissue engineering
16.33 Clinical experience in bone tissue engineering
16.34 Future perspectives for bone regeneration
16.35 Assessment of your knowledge
16.36 Glossary
16.37 References
16.38 Further reading
17 - Tissue engineering of the nervous system
17.1 Learning objectives
17.2 Introduction
17.3 Peripheral nerve
17.3.1 Peripheral nerve anatomy
17.3.2 Peripheral nerve injury
17.3.3 Autologous nerve grafts (autograft)
17.3.4 Use of nerve guides (tubes) in the lesioned PNS
17.3.5 Critical gap length
17.3.6 Nerve guides as supports for regeneration strategies
Matrices
Oriented matrices
Scaffolds
Acellular allografts
Schwann cell grafts
Neurotrophic factors
17.3.7 Biofabricated nerve guides
17.3.8 Bioprinting for the PNS
17.3.9 PNS summary
17.4 CNS: spinal cord
17.4.1 Summary of anatomy and injury response
The tissue engineering challenge to overcome spinal cord scarring
17.4.2 SCI models
Contusion animal model
Hemisection model
Full transection models
Intrathecal delivery
Gender and age
Species selection for SCI models
17.4.3 Cell transplantation
Schwann cells
Olfactory ensheathing glia
Stem cells
Genetically modified cells
17.4.4 Nanomedicine to treat SCI
17.4.5 Matrices and scaffolds
Relevant peptide sequences
Matrices
Scaffolds
17.4.6 Nerve guides in the CNS
17.4.7 Summary
17.5 CNS: brain
17.5.1 Trauma and stroke
17.5.2 Neurodegenerative diseases
17.5.3 Drug delivery to the brain
Brain targeted therapies
17.5.4 Bioprinting for the brain
17.6 CNS: optic nerve
17.6.1 Regenerative therapies
17.7 CNS: retina
17.7.1 Diseases of the retina
17.7.2 Cell transplantation
17.8 Future perspective
17.9 Recommended literature
17.10 Assessment of your knowledge
17.11 Glossary
17.12 References
18 - Principles of cardiovascular tissue engineering
18.1 Learning objectives
18.2 Introduction
18.3 Heart structure, disease, and regeneration
18.3.1 The myocardium
18.3.2 Myocardial infarction and heart failure
18.3.3 Cardiac ECM—function and pathological changes after MI
18.3.4 Endogenous myocardial regeneration
18.3.5 Potential therapeutic targets and strategies to induce myocardial regeneration
18.4 Cell sources for cardiovascular tissue engineering and regeneration
18.5 Biomaterials—polymers, scaffolds, and basic design criteria
18.6 Biomaterials as vehicles for stem cells or bioactive molecule delivery after MI
18.6.1 Stem cell delivery
18.6.2 Delivery of bioactive molecules
18.7 Bioengineering of cardiac patches, in vitro
18.7.1 Strategies for in vitro cardiac patch engineering
18.7.2 Biomimetic scaffolds and integration of cell–matrix interactions
18.7.3 Perfusion bioreactors and stimulation patterns
18.8 Vascularization of cardiac patches
18.8.1 Prevascularization in vitro
18.8.2 Prevascularization in vivo
18.9 Three-dimensional bioprinting of vascularized tissues and components of heart
18.9.1 3D bioprinting strategies for generation of a vascular network
Direct printing of cellular microfluidic channels
Channel networks fabrication methods based on sacrificial technology
18.9.2 3D bioprinting of heart-like structure
18.10 Challenges for clinical application
18.11 Future perspective
18.12 Recommended literature
18.13 Assessment of your knowledge
18.14 Glossary
18.15 References
19 - Tissue engineering of organ systems
19.1 Learning objectives
19.2 Introduction
19.3 Urogenital tissue engineering
19.3.1 Bladder
19.3.2 Urethra
19.3.3 Kidney
19.4 Reproductive organs
19.4.1 Uterus
19.4.2 Vagina
19.4.3 Ovaries
19.5 Liver tissue engineering
19.6 Gastrointestinal tissue engineering
19.6.1 Natural biomaterials for intestinal repair
19.6.2 Combining biomaterials with cells for intestinal repair
19.7 Pancreas tissue engineering
19.7.1 Creating new β cells for cell therapy in type I diabetes
19.8 Lung tissue engineering
19.9 Future perspective
19.10 Recommend literature
19.11 Assessment of your knowledge
19.12 Glossary
19.13 References
20 - Product and process design: scalable and sustainable tissue-engineered product manufacturing
20.1 Learning objectives
20.2 Introduction
20.2.1 ATMPs—definitions and current manufacturing status
20.2.2 Current challenges of TEP manufacturing
20.3 Regulatory aspects of TEP manufacturing
20.3.1 Legal framework for TEPs
20.3.2 TEP product quality
20.3.3 Regulation of TEP manufacturing
20.4 The TEP manufacturing process
20.4.1 Unit operations
20.4.2 Process monitoring
20.4.3 Process scalability and cost
20.5 Manufacturing process development: quality by design
20.5.1 Design-of-experiments
20.6 Smart manufacturing driven by digital twins
20.6.1 In silico models in digital twins
20.7 Future perspective
20.8 Recommended literature
20.9 Assessment of your knowledge
20.10 Glossary
20.11 References
21 - Clinical translation
21.1 Learning objectives
21.2 Introduction
21.2.1 Historical perspective
21.2.2 Framework for clinical development of conventional medicinal products
21.3 Clinical translation of tissue-engineered products
21.3.1 Medical device regulation
21.3.2 Advanced therapy medicinal products
21.3.3 ATMP preclinical phase
21.3.4 ATMP clinical phase
21.4 Typical challenges for tissue engineering encountered in the clinical phase
21.4.1 Exploratory trial
21.4.2 Dosing
21.4.3 Defining the comparator
21.4.4 Randomization
21.4.5 Blinding a trial
21.4.6 Standardization of patient care and follow-up
21.4.7 Outcome measures
21.5 Implementation of a clinical trial
21.5.1 Protocol
21.5.2 Investigator brochure
21.5.3 Investigational medicinal product dossier and investigational new drug application
21.5.4 Informed consent
21.5.5 Case report form and database
21.5.6 Institutional Review Board/Independent Ethics Committee and Competent Authority
21.5.7 Monitoring, audits, and inspections
21.5.8 Sponsor
21.6 Special points to consider
21.6.1 Define the patient
21.6.2 Manufacturing challenges and up-scaling
21.6.3 Exploratory trials in the academic environment
21.6.4 Hospital exemption (EU only)
21.6.5 Voluntary harmonization procedure (EU only)
21.6.6 Combination products
21.7 Future perspective
21.8 Recommended literature
21.8.1 Recommended websites
21.9 Assessment of your knowledge
21.10 Glossary
21.11 References
Index
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
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