CHAPTER | Tissue Organization “The human body is organized hierarchically (Figure 2.1) and has about ten major organ systems. ‘These organ systems carry out major physiological functions, such fs respiration, digestion, and mechanical motion. (See Table 2.1.) Each organ is ‘Comprised of many repeating functional units, which typically include a number of tiesues: epithelial, connective, nerve, and muscle. The circulatory system is made uup of blood vessels, which deliver a continual flow of blood to each functional unit. In this chapter, we introduce the two major constituents of tissues, extracellular matrix (ECM) and cells; familiarize the reader with the major tissue types (epithelial, ‘connective, muscle, and nerve); and present the characteristic length scales at which, tissue organization is observed. 2.1 | Tissue Components ‘The word “tissue” is derived from a French word meaning “to weave.” The basic components of the tissue “fabric” are the cells and the extracellular matrix (ECM). ‘A tissue is therefore a collection of cells and ECM that perform a given furtction. For example, an epithelial lining is a tissue, whereas the collection consisting of epithelial lining, supportive connective tissue structure, nerves, and blood vessels form an organ.” The cells continually produce and degrade the ECM to form the basic geometsic structure of tissues and to provide many essential tissue properties. 2.1.1 Extracellular matrix ‘A substantial part of the volume of tissues is made up of an interconnected network fof macromolecules referred to as extracellular matrix. ECM is composed of a large umber of different proteins and polysaccharides. A partial list of ECM components is given in Figure 2.2 along with a visual depiction of the varied structures observed in differing ECM constituents. ECM is synthesized, secreted, oriented, and modified24 Tissue Components 21 = ° t cat ~ aia ° 3 c = 6 664 ca ii 1 } \ difecttaion es pita sc Newe Ma major 7 + t 1 aaa oan — * tT aber of rae stunt “ahlar sant “xe (ex. nepon) Nephoon basic Organ (ECM). (eg, kidney) tnction, Organ system (eg, urinary system) perties, ‘Total organism (human being) Figure 2.1 Representation of the hierarchical organization in the human body. Modified from (369) aetwork fa large ponents observed. nodified22. Chapter? Tissue Organization TABLE 2. System Organs Circulatory “Heart, blood vessels, and blood (some classifications also include lymphatic vessels and Iymph in this system) “Transport of blood throughout the body's tissues Respiratory ‘Nose, pharynx, larynx, trachea, bronchi, and Jangs “Exchange of carbon dioxide and oxygens regulation of hydrogen-ion concentration Digestive Mouth, pharynx, esophagus, stomach, intestines, salivary glands, pancreas, lives, and gallbladder Digestion and absorption of organic nutrients, salts, and water “Urinary Kidneys, ureters bladdes, and urethra ‘Regulation of plasma composition through controlled excretion of salts, water, and organic ~ Musculoskeletal Cartilage, bone, ligaments, tendons, joints, and skeletal muscle Support, protection, and movement of the body production of blood cells Spleen, thymus, and other lymphoid tissues Defense against foreign invaders; return of extracellular fiuid to blood; formation of white blood cells ‘Nezvous ‘Brain, spinal cord, peripheral nerves and ganglia, and special sense organs [Regulation and coordination of many activities in the body; detection of changes inthe internal and external environments; states of consciousness; learning; cognition Endocrine “All glands secreting hormones: Pancreas, testes, ‘ovaries, hypothalamus, kidneys, pituitary, thyroid, parathyroid, adrenal, intestinal, thymus, heart, and pineal ‘Regulation and coordination of many activities in the body Reproductive Tntegumentary Male: Testes, penis, and associated ducts and slands; Female: Ovaries, uterine tubes, uterus, vagina, and mammary glands Skin Production of spermy transfer of sperm to female; Production of eggs; provision of a nutritive ‘environment for the developing embryo and fetus; nutrition ofthe infant Protection against injury and dehydration; defense against foreign invaders; regulation of temperature by the cellular components of tissues, In turn, ECM can exert control over many callular fate processes through binding to a class of receptors known as integrins. For example, cell migration rates vary with ECM density. Similarly, cell adhesion, spreading, cell shape, and apoptosis also vary with ECM density.eral ogeins, ‘Though detailed ECM composition varies dramatically throughout the body, some general patterns are common to all tissues. In particular, the basement mem brane (basal lamina), a thin collection of ECM components found at the base of Integrin apt api csp apt spt asp expt exp expo exp aves aves aves evs 0p up? aps Extracellular Matrix In Cellular Interactions Ligands Collagen (IV, VD, laminin Collagen (HIV, VD, laminin Collagen 1) laminin, Sbronectin,entactin,epiligrin ibronectingyy, VCAM 1, thrombospondin ibronectn,thrombospondin Laminin ibronectin ICAM 1, CAM-2,1CAM-3 ICAM 1 iC3b, fbronogen, Fetor X denaturated protein Fibronogen, iC3b, denaturated protein Vitronectin, fibrinogen, Bbronectin, thrombonspondin Vitronectin Fibronectin Laminin ibronectingr, VCAM~1, MASCAM- ‘Fbronogen, fibronectin, vitronectin, vWF ibronogen, fibronectin vitronectin, WWE collagen (IV), entactin @) 2.1. Tissue Components 23 Figure 2.2 ‘The extracellular matric (ECM). (A) ECM interacts with cell surface through integrin and non-integrin ‘membrane-bound receptors. ECM can also bind other ECM molecules as well as soluble growth factors. Extracellular matrix is produced by cells and degraded by a clase of enzymes called matrix ‘etalloproteinases (MMP), (B) Integrins are adhesion molecules with the potential to regulate cell-ECM interactions. Adapted from [261]24 Chapter 2 Tissue Organization [[eompenest Fenaion ______iteeien_| a Trahan ay Tawar Seta aod ‘evi Kaa — SS a aT petieatce atten = an a a a a a St ee TT aa at TS aaa tense Features of ECM ea + Lange (MW ~ 10-105) Se Malina! + galt fstons Sd + Ramainatiachs to “leon ‘Calariace prone mediating cll Ubiqutouy atte cells after isolation satcae jettles * Change Cee a eee to neha = Se ——— a) © Figure 2.3 (A) Key Features of ECM and its major components. (B) A prototypic ECM molecule, flbronecti, has Yenious call plis ECM-binding domains. (C) Characteristics of ECM molecules. epithelial tissues, is typically composed of collagen TV, laminin, and heparansulfate proteoglycan. However, connective tissue ECM is much more prevalent and is typi- {ally composed of collagen I, fibronectin, elastin, and glycosaminoglycans. ECM molecules have a number of important characteristics. Figure 2.3 depicts a typical multimodal ECM molecule, flbronectin. Some important characteristics of ECM components are as follows:sulfate stypi- lepicts tics of « They are very large molecules (molecular weight: 105106) and therefore have very low diffusivities ‘They are multimodal in that they often have cell-binding domains as well as domains to bind other ECM molecules. « They can regulate cell fates, including differentiation, apoptosis, and migration. «They can change conformation under mechanical load, thereby exposing active binding sites. « Many of the critical binding domains that have been identified can be mimicked by small peptides. + These molecules often remain bound to isolated mammalian cells, occupying a subpopulation of integrin recepto: These features are critical ro understand for tissue engineering applications. In partic: ular, ECM or small peptides are often utilized to modulate cell adhesion, migration, and phenotype. A quantitative description of some cell-ECM interactions as they impact key cellular fate processes will be presented in Chapter 6. 24.2 Cells There are over 200 different fully mature cell types in the human body. Table 2.2 lists some examples of these; however, there are much fewer basic categories of cells. Here we will describe two main categories of cell types: epithelial and mesenchymal. Epithelial cells Epithelial cells grow in contiguous two-dimensional sheets. In ep- ithelial sheets, the cells have a cuboidal geometric arrangement. The characteristic morphology of individual cells tends to be independent of the large-scale sheet of which they are members. They are tightly connected with their neighbors and thus cannot migrate individually. They are polar, and each surface of the sheet has dif- ferent characteristics. The basal side (the “inside”) is in contact with basal lamina that is comprised of ECM molecules. Mesenchymal cells are usually located on the other side of the basal lamina, On the superior surface (the “outside”) isa vesicle or a tubule. ‘The epithelia are defining features of the carly embryo, and the mesenchymal cells appear as “fillers” in the spaces between epithelial sheets. Through interactions between the epithelia and the mesenchyme, the embryo develops the diverse structures that form the many tissues and organs. Figure 2.4 depicts a characteristic epithelial cell as compared to a mesenchymal cell and details the characteristic features of tissues composed of these cells Mesenchymal cells The term “mesenchyme” has been used to describe cells that exist alone or as small, loosely connected cells. These cells tend to have a bipolar shape. They can migrate and move about individually. Fibroblasts are the most commonly encountered mesenchymal cells, and they grow readily into confluent layers on two-dimensional surfaces i vitro. ‘These cels display individual properties, such as migration, but also show large-scale patterns when grown on a flat, two- dimensional surface. Their growth is typically contact-inhibited, which means they 2A. Tisue Components. 25,26 Chapter 2. Tissve Organization ‘TABLE 2.2 ies Absorptive cells (e.g, intestine) Ciliated cells (e.g, trachea) Secretory cells (e-» paneth, B-cell, hepatocyte) Connective tissue Fibroblasts Osteoblasts (bone) ‘Adipose calls (fat storage) Chondrocytes (cartilage) Endothelial cells (blood vessels) Newvoustssue = SSSOSOSCS*~S~S~S* Neurons Glial cells Schwann cells Oligodendrocytes Bool eee ythrocytes (red blood cells) “Megakaryocytes (platelet precursors) Lymphocytes (immune system) ‘Monocytes/Macrophages ‘Neutrophils, eosinophils, basophils ‘Muscle — Skeletal Cardiac Smooth Germ cells Sperm Bee Sensory exis Hair cells Rod cellslcone eels Olfacto stop growing when the cell membrane of one cell touches that of another cell. As will be discussed in Chapter 5, populations of mesenchymal cells have the potential to differentiate into specialized cells such as osteoblasts, chondrocytes, and fibroblasts. In addition, we will see that fibroblasts are significant participants in dynamic tissue processes such as morphogenesis and wound healing..swill fal to olasts, tissue 7, Free surface —_ ‘ailing edge Leading lamellipodium Epithelium Mesenchyme ‘Characteristic Epithelia Mesenchyme Linked in contiguous sheets + Polar with outer free surface, apical Golgi apposition + Juxtalomenal ight junctions + Lateral gap junctions and desmosomes + (ora very few) Basal surface on extracellular matrix Bipolar and stellate shape Migratory ‘Vimentin, typeI collagen, fibronectin (can produce fibroblasts with type I collagen) (Cytokeratin, laminin type IV collagen + aeergrcce 2.2 | Tissue Types ‘There are several major tissue types found in organs including epithelial, connective, muscle, nerve, and glandular. ‘These major tissue types provide covering, support, movement, control, and production, respectively. We will briefly describe the major tissue types and their distinguishing characte 2.2.1 Epithelial tissues Epithelial tissues are typically characterized by the number of epithelial sheets that they contain and the shape of the cells contained in these sheets. Single layers of cells are“ simple” compared with multilayered epithelia, which are “stratified.” Epithelial 22 Tissue Types 27 Figure 2.4 ‘Characteristics of epithelial and mesenchymal cell types Adapted from [98].28 Chapter? Tssve Organization ) 6 © Figure 2.5 Different geometric arrangements of epithelial tissues: (A) squamous; (B) cuboidal; (C) pseudostratiied and ciliated; (D) columnar; and () scratified © tissues are further described by cell shape: squamous (“fried-ege” shaped), cuboidal, columnar, or pseudostratified. (See Figure 2.5.) “Pseudostratified” refers to simple epithelial layers that may appear stratified due to the varied location of nuclei rela~ tive to their basement membrane. Multiple cell layers can also be described by cell shape such as stratified squamous (esophagus), or transitional (bladder), where cells transition between cuboidal and columnar depending on the state of the organ. 2.2.2 Connective tissue Connective tissues offer mechanical support (bones and ligaments), store much of the body's fluid (fat and blood), aid in the immune response, and are often important in tissue repaiz. Though diverse in form and function, all connective tissue origi- nates from a common source—the mesoderm (or middle layer) of the embryo. These include cells that are relatively fixed such as fibroblasts and adipocytes, wandering, cells such as macrophages and plasma cells, and noncellular components. Noncellu- lar components are either fibrous such as collagen I or amorphous such as hyaluronic acid, 2.2.3 Other tissue types Other tissue types, such as muscular, nervous, and glandular, will be presented only briefly here. The reader is referred to a standard anatomy and physiology text for 1 comprehensive description of tissue organization in humans. Most of the move- ‘ments in the body are caused by fibers in muscle tissues that contract forcefully upon shortening. The body is composed of three different types of muscle tissue: skele~ tal, cardiac, and smooth. Skeletal muscle pulls on bones to cause movements and is composed of striated, multinucleated myofibers. Cardiac muscle is part of the heart and serves to:propel blood through the blood vessels. Compared with skeletal ‘muscle fibers, cardiac muscle fibers are striated; however, they have one nucleus, anduboidal, > simple slei rela- A by cell vere cells gan, much of ‘portant ue origi- 0. These andering Foncellu- raluronic ited only y text for he move- ully upon xe: skele- ents and art of the hh skeletal sleus, and they branch and join at junctions called intercalated discs, which are composed of gap junctions for propagation of the action potential. Smooth muscle fibers, unlike fardiac and skeletal muscle fibers, lack striations (hence the name) and occur pri- marily in the walls of hollow organs (ie., intestine) to squeeze substances through these organs by alternate contraction and relaxation. In general, organ structure is specialized for a particular function in three ways: cellular structure, tissue struc- ture due to multicellular organization, and interfacing with the rest of the body. As described earlier, the subcellular and multicellular organization varies inthe three dif- ferent types of muscle (ie., striations, no striations). Different organization of muscle tissue is therefore necessary for slow, small-magnitude contractions of the gut; fast, coordinated depolarization of heart muscle; and the coordinated large contraction force needed in skeletal muscle. ‘Nervous organs such as the brain, spinal cord, and nerves regulate and control body functions via their main component, the nervous tissue, which consists of neu- rons and supporting cells. Neurons generate and conduct electrical impulses called action potentials. Electrical impulses are received at a neuron’s cell body (soma) via branching extensions called dendrites, while a single extension called an axon transmits the electrical impulses to other neurons’ dendrites. Supporting cells of the nervous tissue such as astrocytes and Schwann cells are not believed to participate in electrical conduction, but they play important roles in nourishing, insulating and protecting their neuron neighbors. Glandular tissue is composed of polarized epithelial cells that make and secrete protein products from precursors in their microenvironment. Most of the secreted product is made in the rough endoplasmic reticulum (ER) and packaged into secre tory vesicles in the Golgi apparatus for subsequent exocytosis. Therefore, glandular cells usually have well-developed rough ER, a large Golgi apparatus, and secretory vesicles in the cytoplasm of their apical domains. Glands are classified as exocrine ‘or endocrine. Exocrine glands, which include sweat glands of the skin and the liver (secretes bile), secrete their products onto body surfaces (skin) or into body cavities (digestive tube). Exocrine glands can be unicellular or multicellular, with the latter having ducts to carry their products to epithelial surfaces. Endocrine glands, on the other hand, secrete their hormonal products directly into the bloodstream. Each target organ of the hormone reacts in some characteristic way. For instance, the pancreas releases enzymes to digest a meal upon stimulation by a hormone secreted by endocrine cells in the intestine. Having discussed the different tissue types in che body, we next describe how organs are composed of repeating functional subunits. 2.3 | Functional Subunits Cells and tissues are organized into functional subunits containing many different cell types. ‘These subunits perform basic tissue and organ functions. Examples of these subunits include the alveoli in the lung and the nephron in the kidney; other examples are shown in Figure 2.6. Separating the functional subunits into their individual cell cohorts leads to the loss of tissue-specific function, but one can study cell-specific properties with such purified preparations. A fundamental observation that follows from the preceding discussion is that tissue function is a property of 23° Functional Subunits 2930 Chapter 2 Tissue Organization Figure 2.6 Levels of organization of an ‘organ system with relative length scales. Organ System Organ cell-cell interactions, mediated by the ECM, soluble signals, and direct contact, The size of the functional subunits is on the order of 100 microns, whereas the size scale of a cell is 10 microns. Each organ is then comprised of tens to hundreds of millions of functional subunits. The sizing of organs represents an evolutionary challenge that js also faced by tissue engineering in scaling up the function of reconstituted tissues ex vivo, 2.4 | Problem Decomposi ‘The tissue engineer faces fundamental challenges in the implementation of cell-based therapies. These are as follows: (see Figure 2.7). 1. the proper reconstitution of the microenvironment for the development of basic tissue function and properties; 2, the scale-up to generate numerically enough properly functioning microenviron- ‘ments to be clinically meaningful; 3. the automation of system operation at a clinically meaningful scales and 4, the implementation of the automated device in a clinical setting with all the cell handling and preservation procedures that are required for the implementation of cell-based therapies. “The primary focus of this text is on the first two issues, although some of the challenges faced with the latter two are discussed briefly in Part IV. ‘The first two issues are further illustrated in Figure 2.8 from the point of view of every cel in the body. This diagram demonstrates the “spheres” of influence and the relevant length scales around a cell at which these become dominant. At the centerThe scale lions ethat ssues based basic ecell- tation of the iew of nd the center Mesenchiym Progenitor cell al accessory cell 1. 100 um 4 Tissue 7 Hematopoietic accessory cell Microenvironment 2 Wem Bioreactor 3. 10-200 cm. ‘Automated Cell- Culture System 4 140m Cell-Manufacturing Facility of this diagram is a cell chat communicates with the rest of the body through its local microenvironment. The cell microenvironment is characterized by neighboring cells, ECM, cyto/chemokine and hormone trafficking, cell geometry, dynamics of respiration, and the provision of nutrients and removal of metabolic byproducts ‘The microenvironment is thus very complex. To achieve proper reconstitution, cone must accurately mimic these dynamic, chemical, and geometric variables. The microcirculation connects all the microenvironments in every tissue to their larger whole-body environment. With few exceptions, essentially all metabolically active calls in the body are located within a few hundred microns from a capillary. The 24 Problem Decomposition 31 Figure 27 ‘The four principal size scales in tissue engineering and cellular therapies. ‘Adapted from [275]Chapter? Tasue Organization Figure 2.8 ‘cell and its communication with other body pars. Cells interact with neighboring cells, ECM, and soluble factors and ace inluenced by local ‘geometry and mechanical seimoli. By interacting with the microcirculation, cells then interact with the major homeostatic mechanisms of the body. Modified from (227) A cell and its communication ‘with other body parts <> 110mm (receptor-ligand interaction) 10-20 um (cell) ‘100-200 rm (functional subunit) capillaries provide a pecfusion environment that connects every cell 0 a source of oxygen and sink for carbon dioxide (the lungs), a source of nutrients (the small in- testine), the clearance of waste products (the kidney), and so forth. The engineering of these functions ex vivo is the domain of bioreactor design. Such culture devices ‘must appropriately simulate and provide respiratory, gastrointestinal, and renal func- tions. Further, these cel-culture devices have to respect the need for the formation of microenvironments and thus must have perfusion characteristics that allow for uni- formity down to the 100-~um length scale. These are stringent design requirements. ‘We will see in Chapter 16 that techniques for tailoring biomaterials on this length scale are now readily available.2.6 Further Reading 33 2.5 | Summary 4 Tissues are composed of cells and ECM; cells are primarily epithelial or mes- enchymal. Epithelial tissues are described by cell shape and the number of cell layers. Connective tissues are described by ECM structure, Organs are com- posed of both epithelial and connective tissues. « Tissues have fundamental subunits that are comprised of several cell types, each of a developmentally different origin. If separated, tissue function is lost. The dimension of such tissue subunits is about 100 microns. Human organs are comprised of tens to hundreds of millions of such subunits, ‘The challenges that face the tissue engineer in implementing cellular therapies can be broken down into four major categories: the design of the microenvi- ronment based on in vivo tissue function; the scale-up to a large number of similar, if not identical, subunits, connecting a large number of subunits with each others process automation; and the preservation and manufacturing of a clinically meaningful number of cel functions. 2.6 | FURTHER READING Martini, EHL, Fundamentals of Anatomy and Physiology, Marieb, EN., Human Anatomy and Physiology, 4" ed, 4® ed. Uppeer Saddle River, NJ: Prentice Hall, 2001. Menlo Park, CA: Addison Wesley Longman, 1998. Vandes, A.J, |. Sherman, and D'S. Luciano, Hionan Phys- ology 6" ed., NY: McGraw Fill, 1994,