Approaches to Tissue Engineering

The first part of this book has proposed that tissue engineering is a modern realization of a practice with ancient origins. Tissue engineering is different because technologies that are now available permit generation of synthetic materials that mimic biological materials as well as clinically useful quantities of biological components (such as proteins and cells). These technologies have emerged from rapid advances in the biological sciences and engineering over the past few decades. Since tissue engineering is new, however, few examples of successful tissue engineering are available. The reader, upon recognizing this early stage of development, might presume that the prospects for a compelling chapter on “Approaches to Tissue Engineering” are bleak. Instead, I am convinced that this is the most exciting of times to write such a chapter, because the precedents are not yet assembled and the field has not yet been reduced to systematic divisions. But there are many challenges. The challenge begins with organization of information. Written reviews of tissue engineering to date adopt different organizational structures. For example, an early influential review was organized around replacement strategies for different organ or tissue systems. A similar, although more encyclopedic, approach was used in the first two editions of an edited textbook. This is a sensible arrangement, given that tissue engineering is an interdisciplinary area of study that has emerged in response to rather specific clinical needs, such as the shortage of donor livers and the paucity of grafts for skin. But it is a difficult arrangement for the teacher and student, as it does not require reconciliation between approaches used to solve different problems. For example, although regeneration of skin and liver differs in many essential ways, there are important areas of intersection. As a consequence, an organ or tissue-based approach does not easily allow for assimilation of new knowledge that is acquired by successes made on particular problems. What is tissue engineering and how can the basic principles, which are developed in Part 2 of this book, be integrated into a strategy for the engineering of replacement tissues? The previous three chapters describe important, but focused, elements of tissue engineering practice: cell delivery, agent delivery, and cell interactions with synthetic materials.

Cell Delivery and Recirculation

Perhaps the simplest realization of tissue engineering involves the direct administration of a suspension of engineered cells—cells that have been isolated, characterized, manipulated, and amplified outside of the body. One can imagine engineering diverse and useful properties into the injected cells: functional enzymes, secretion of drugs, resistance to immune recognition, and growth control. We are most familiar with methods for manipulating the cell internal chemistry by introduction or removal of genes; for example, the first gene therapy experiments involved cells that were engineered to produce a deficient enzyme, adenine deaminase (see Chapter 2). But genes also encode systems that enable cell movement, cell mechanics, and cell adhesion. Conceivably, these systems can be modified to direct the interactions of an administered cell with its new host. For example, cell adhesion signals could be introduced to provide tissue targeting, cytoskeleton-associated proteins could be added to alter viscosity and deformability (in order to prolong circulation time), and motor proteins could be added to facilitate cell migration. Ideally, cell fate would also be engineered, so that the cell would move to the appropriate location in the body, no matter how it was administered; for example, transfused liver cells would circulate in the blood and, eventually, crawl into the liver parenchyma. Cells find their place in developing organisms by a variety of chemotactic and adhesive signals, but can these same signaling mechanisms be engaged to target cells administered to an adult organism? We have already considered the critical role of cell movement in development in Chapter 3. In this chapter, the utility of cell trafficking in tissue engineering is approached by first considering the normal role of cell recirculation and trafficking within the adult organism. Most cells can be easily introduced into the body by intravenous injection or infusion. This procedure is particularly appropriate for cells that function within the circulation; for example, red blood cells (RBCs) and lymphocytes. The first blood transfusions into humans were performed by Jean-Baptiste Denis, a French physician, in 1667. This early appearance of transfusion is startling, since the circulatory system was described by William Harvey only a few decades earlier, in 1628.

Cell Adhesion

The external surface of the cell consists of a phospholipid bilayer which carries a carbohydrate-rich coat called the glycocalyx; ionizable groups within the glycocalyx, such as sialic acid (N-acetyl neuraminate), contribute a net negative charge to the cell surface. Many of the carbohydrates that form the glycocalyx are bound to membrane-associated proteins. Each of these components— phospholipid bilayer, carbohydrate-rich coat, membrane-associated protein—has distinct physicochemical characteristics and is abundant. Plasma membranes contain ∼50% protein, ∼45% lipid, and ∼5% carbohydrate by weight. Therefore, each component influences cell interactions with the external environment in important ways. Cells can become attached to surfaces. The surface of interest may be geometrically complex (for example, the surface of another cell, a virus, a fiber, or an irregular object), but this chapter will focus on adhesion between a cell and a planar surface. The consequences of cell–cell adhesion are considered further in Chapter 8 (Cell Aggregation and Tissue Equivalents) and Chapter 9 (Tissue Barriers to Molecular and Cellular Transport). The consequences of cell–substrate adhesion are considered further in Chapter 7 (Cell Migration) and Chapter 12 (Cell Interactions with Polymers). Since the growth and function of many tissue-derived cells required attachment and spreading on a solid substrate, the events surrounding cell adhesion are fundamentally important. In addition, the strength of cell adhesion is an important determinant of the rate of cell migration, the kinetics of cell–cell aggregation, and the magnitude of tissue barriers to cell and molecule transport. Cell adhesion is therefore a major consideration in the development of methods and materials for cell delivery, tissue engineering, and tissue regeneration. The most stable and versatile mechanism for cell adhesion involves the specific association of cell surface glycoproteins, called receptors, and complementary molecules in the extracellular space, called ligands. Ligands may exist freely in the extracellular space, they may be associated with the extracellular matrix, or they may be attached to the surface of another cell. Cell–cell adhesion can occur by homophilic binding of identical receptors on different cells, by heterophilic binding of a receptor to a ligand expressed on the surface of a different cell, or by association of two receptors with an intermediate linker. Cell–matrix adhesion usually occurs by heterophilic binding of a receptor to a ligand attached to an insoluble element of the extracellular matrix.

Cell and Tissue Mechanics

Mechanics is the branch of physics that is concerned with the action of forces on matter. Tissue engineers can encounter mechanics in various settings. Often, the mechanical properties of replacement biological materials must replicate the normal tissue: for example, there is limited use for a tissue-engineered bone that cannot support the load encountered by its natural counterpart. In addition, the mechanical properties of cells and cell–cell adhesions can determine the architecture of a tissue during development. This phenomenon can sometimes be exploited, since the final form of engineered tissues depends on the forces encountered during assembly and maturation. Finally, the mechanics of individual cells—and the molecular interactions that restrain cells—are important determinants of cell growth, movement, and function within an organism. This chapter introduces the basic elements of mechanics applied to biological systems. Some examples of biomechanical principles that appear to be important for tissue engineering are also provided. For further reading, comprehensive treatments of various aspects of biomechanics are also available. Consider an elongated object—for example, a segment of a biological tissue or a synthetic biomaterial—that is fixed at one end and suddenly exposed to a constant applied load. The material will change or deform in response to the load. For some materials, the deformation is instantaneous and, under conditions of low loading, deformation varies linearly with the magnitude of the applied force: . . . σ[≡F/A]= Eε (5-1) . . . where σ is the applied stress and ε is the resulting strain. This relationship is called Hooke’s law, after the British physicist Robert Hooke, and it describes the behavior of many elastic materials, such as springs, which deform linearly upon loading and recover their original shape upon removal of the load. The Young’s modulus or tensile elastic modulus, E, is a property of the material; some typical values are provided in Table 5.1. Not all elastic materials obey Hooke’s law (for example, rubber does not); some materials will recover their original shape, but strain is not linearly related to stress. Fortunately, many interesting materials do follow Equation 5-1, particularly if the deformations are small.

Elements of Tissue Development

This book began with a reflection on the miracle of development, wherein a single cell transforms into a human. The transformation from fertilized egg to adult results from a complex tapestry of events, which scientists are only beginning to dissect and unravel. Certain processes occur frequently during development; that is, the tapestry is woven from threads of elemental colors and textures. A central assumption of subsequent chapters is that key concepts underlying tissue regeneration first appear during fetal development. The elements of developmental biology are presented in this chapter; more complete descriptions are available in any of several excellent textbooks. The relevance of developmental processes in the study of tissue engineering is detailed in subsequent chapters. One of the most intimidating aspects of developmental biology is the vocabulary; therefore, important words are indicated in small capitals on first occurrence and collected in a glossary at the end of the chapter. Developmental biology is an ancient science. One of the central concepts in developmental biology, EPIGENESIS, came from Aristotle in the fourth century B.C. Epigenesis is a continuous, stepwise process in which a simple initial structure becomes complex. Through much of history between Aristotle and the present, epigenesis was not widely accepted as operating in development; many scientists, particularly during the 17th and 18th centuries, were preformationists who believed that the structure of animals was preformed at conception. To the preformationist, the embryo begins as a small replica of an individual which changes only in size during the course of development. Preformationists differed as to whether the preformed individual resided in the ovum or the sperm, but they agreed that all of the attributes of an adult were present from the outset of development. Epigenesis is now well established and many of the steps underlying epigenesis are understood. Human development is part of a larger cyclic process; fertilized eggs develop into newborns who grow to adults and produce new eggs and sperm. This chapter will introduce some of the mechanisms underlying human development from egg to newborn.

The State-of-the-Art in Tissue Exchange

It is an impressive spectacle. Multicellular organisms—from fruitflies to humans—emerge from a single cell through a coordinated sequence of cell division, movement, and specialization. Many of the fundamental mechanisms of animal development are known: differentiated cells arise from less specialized precursor or stem cells, cells organize into functional units by migration and selective adhesion, and cell-secreted growth factors stimulate growth or differentiation in other cells. Despite extensive progress in acquiring basic knowledge, however, therapeutic opportunities for patients with tissue loss due to trauma or disease remain extremely limited. Degeneration within the nervous system can reduce the quality and length of life for individuals with Parkinson’s disease. Inadequate healing can cause various problems, including liver failure after hepatitis infections, as well as chronic pain from venous leg ulcers and severe infections in burn victims. The symphony of development is difficult to conduct in adults. Tissue or whole-organ transplantation is one of the few options currently available for patients with many common ailments including excessive skin loss and artery occlusion. During the past century, many of the obstacles to transplantation were cleared: immunosuppressive drugs and advanced surgical techniques make liver, heart, kidney, blood vessel, and other major organ transplantations a daily reality. But transplantation technology has encountered another severe limitation. The number of patients requiring a transplant far exceeds the available supply of donor tissues. New technology is needed to reduce this deficit. Some advances will come from individuals trained to synthesize basic scientific discoveries (for example, in developmental biology) with modern bioengineering principles. Tissue engineering grew from the challenge presented by tissue shortage. Tissue engineers are working to develop new approaches for encouraging tissue growth and repair; these approaches are founded on basic science of organ development and wound healing. A few pioneering efforts are already being tested in patients; these include engineered skin equivalents for wound repair, transplanted cells that are isolated from the immune system by encapsulation in polymer membranes for treatment of diabetes, and chondrocyte implantation for repair of articular cartilage defects.

Delivery of Molecular Agents in Tissue Engineering

The previous chapter provided some examples of tissue engineering, in which cells that were isolated and engineered outside of the body are introduced into a patient by direct injection of a cell suspension, typically into the circulatory system. But the field of tissue engineering also points to treatments that are conceptually different from variations on cell transfusion technology; tissue engineering promises the regrowth of adult tissue structure through application of engineered cells and synthetic materials. In support of this broad claim, the field of tissue engineering can point to some initial successes. For example, synthetic materials are now available that accelerate healing of burns and skin ulcers. In addition, in vitro cell culture methods now allow the amplification of a patient’s own cells for cartilage repair or bone marrow transplantation. But major obstacles to the widespread application of tissue engineering remain. Tissue engineers have not yet learned how to reproduce complex tissue architectures, such as vascular networks, which are essential for the normal function of many tissues. In fact, the tissue engineering concepts that have been demonstrated in the laboratory to date involve arrangements of cells and materials into precursor tissues (or neotissues) that develop according to natural processes that are already present within the cells or the materials at the time of implantation. These methods may be suitable for production of some tissues in which either the structure is relatively homogeneous (such as cartilage, in which a tissue structure can reform after the implantation of chondrocytes into a tissue defect) or the structure develops naturally (such as in some tissue-engineered skin, in which the stratified epithelium develops naturally by culturing at an air–liquid interface). The engineering of many tissue structures—such as the branching architectures found in many tissues or the intricate network architecture of the nervous system—will probably require methods for introducing and changing molecular signals during the process of neo-tissue development. For example, it is well known that chemical gradients of factors known as morphogens induce the formation of structures during development; some of the attributes of morphogens were introduced in Chapter 3.

Objecives of Tissue Engineering

Tissue exchange is an ancient art, but tissue engineering is a new concept. The new thinking about tissue engineering is supported by technologies that were developed during the twentieth century, including advanced cell culture, gene transfer, and materials synthesis. Tissue engineering arose from a diverse group of historical precedents that included pharmacology, surgery, and materials science; each historical line of inquiry engaged different motivations and diverse tools. Therefore, as a substitute for a single definition, this chapter observes tissue engineering from several different angles and attempts to illustrate the field by practical example. The field of tissue engineering can be subdivided in various ways; usually it is organized by organ system, as in hepatic tissue engineering or bone tissue engineering, which are concerned with engineering replacements for liver and bone function, respectively. A coarse subdivision can also be made according to the general objective; most tissue engineering strategies involve replacement of a tissue’s metabolic function, structural function, or both. Here, several overlapping views of tissue engineering are presented: tissue engineering as a logical extension of contemporary medical and surgical therapies; tissue engineering as a method for controlling the normal healing response of tissues; tissue engineering as an effort to repopulate the cellular component of tissues without replacement of the whole organ; tissue engineering as a variety of controlled drug delivery; and tissue engineering as a new method for developing models of human physiology. Metabolism is a coordinated ensemble of chemical transformations that are individually regulated by the action of enzymes. Many metabolic disorders are caused by the defective production of a single enzyme. It is sometimes possible to identify, produce, and use enzymes to reconstitute missing elements of metabolism. For example, the enzyme adenosine deaminase (ADA) is involved in the degradation of purine nucleosides; individuals who lack the gene for ADA cannot produce the enzyme in their bodies. As a result, high concentrations of certain purine nucleoside metabolites accumulate within cells; toxicity due to these metabolites is particularly harmful to B and T lymphocytes.

Cell Migration

Cell migration is crucial to the life of unicellular and multicellular organisms. Unicellular organisms migrate to find food and avoid predators; this migration can occur by swimming through a fluid, which is achieved by flagellar or ciliary beating (exemplified by E. coli or Paramecium, respectively), or crawling along a surface (as in amoebae). In multicellular organisms, migration of a particular cell population is often a component of complex multicellular behaviors including tumor invasion and metastasis, embryogenesis, angiogenesis, and immune responses. In both cases, the speed and pattern of migration are determined by the nature of the cell and by chemicals (both soluble and surface-bound) in the environment. Since cell migration is critical in the formation or regeneration of tissues, a clearer understanding of the dynamics of cell migration would greatly enhance our ability to design materials and processes for tissue engineering. Cell migration is a fundamental mechanism for forming of structures within developing embryos. Accordingly, the migration of cells during embryogenesis is under exquisite control; development of tissue structure and cell migration are interdependent. Chapter 3 discussed limb regeneration in salamanders, a process in which positional gradients of cell adhesion influence cell migration and, ultimately, tissue structure. Similarly, the rate and migration of myogenic cells from the somitic mesoderm is influenced by the presence of local signals in the form of diffusible factors and extracellular matrix composition. These local signals are also produced by cells, with the result that cells throughout the developing limb (which are present in a particular arrangement or tissue structure) control and coordinate the migration of myogenic cells by secretion of activating factors and expression or organization of extracellular matrix molecules. A better understanding of the mechanisms underlying cell migration in the embryo, and the strategies that nature uses to control migration, will almost certainly provide inspiration for tissue engineering. Cell migration underlies many important physiological functions in adults. For example, the immune system, with its widely dispersed ensemble of B and T cells, relies heavily on the coordinated migration of individual cells to patrol the body and to provide opportunities for cell–cell interaction.

Cell Growth and Differentiation

The expansion in size of a region of tissue, often called growth, is critical to embryonic development and tissue repair. Growth of a tissue most often occurs by an increase in cell number. In fact, sequential cell division—and a resulting increase in total cell number—is the most important change of early development. As development proceeds, however, the rate of increase in cell number slows but the overall size of the organism continues to increase steadily. Growth throughout life can occur by a variety of mechanisms in addition to increased cell number; for example, increases in cell volume or extracellular volume also produce growth. The overall growth of an organ or tissue can involve multiple mechanisms. For example, in the nervous system, neurons increase in size, but not number, as a juvenile grows to adulthood. By contrast, glial cells within the nervous system divide and proliferate throughout life. Overall, however, cell proliferation (which occurs by the process of sequential cell division) is the most important feature of tissue growth. Growth is only one of the changes that occurs with development. As a child grows to adulthood, her increase in size is probably less astonishing than her overall change in behavior and ability. Underlying this overall change are dramatic alterations in function and operation of individual cells; this observation is related to the discussion in Chapter 3, in which the processes of cellular differentiation and specialization were introduced. The child develops by reference to a fixed instructional program, the genome, which somehow encodes all of the molecular signals that lead to increases in size, changes in shape, and inexorable dynamics of aging. But the child is also influenced by her environment and the opportunities for change that her environment presents. One child becomes a doctor and another a cellist; the factors and forces that nudge each down her path are not programmed by the genes alone. Similarly, differentiation of a cell is influenced by its genetic composition and the environment that surrounds it. This chapter begins with a discussion of mechanisms and kinetics of cell division. Later parts of the chapter consider some of the factors that influence cell differentiation. The relationship of cell growth during development of a normal organism and cell growth in culture is introduced in the final sections.