Biomaterials: A Systems Approach to Engineering Concepts

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Chapter 1

Cell Biology

Abstract

The goal of this chapter is to present the reader with a working knowledge of cell types and distinctions, compositional make up of cells, and descriptions of activities that cells perform. The rationale for understanding cell anatomy and physiology is due to the fact that disease can arise from cell dysfunction, and any synthetic intervention leads to cells that ultimately encounter these synthetic, hybrid, and transplanted materials. It is important to create relevant classifications of specialized cell types and to distinguish between functional physiology and pathology or disease. Ultimately bioengineered solutions using materials require some haptic interface with biological tissue and by colocation, cells as well. There are multiple complete books that are dedicated to this one topic. Those are terrific references for this chapter. There are great image resources on the web that can supplement the bulk of the text. The presentation of this chapter coupled with Chapter 2, Cell Expression: Proteins and Their Characterization, tied to proteins and amino acids should help engineers reading this book appreciate the more detailed nuances of normal cell physiology, and allow readers to possess a deeper language linked with cell biology in the context of biomaterials used to replace tissue and organ function, and to seamlessly interface with viable tissues.

Keywords

Cell nomenclature; classifications; cell function; mitosis; motility; communication; trafficking; apoptosis; nucleus; mitochondria

Learning Objectives

By reading this chapter, the reader should be able to

1. Recognize the various naming conventions for cells including functions, tissues from which they are derived, morphological features, and what they ultimately produce as proteins.

2. Describe features and subunits of typical eukaryotic cells which include mitochondria, the nucleus, the cytosol, the lipid membrane, and other structural features that are tied to the cytoskeleton and to the glycocalyx.

3. Understand functions of cells, which include cell division, protein expression, extracellular sensing and communicating within the environment, assembly into larger tissue constructs, and cell death, whether by apoptosis, cell rupture, or some other mechanism.

4. Recognize cell types contained within blood, tissues, and organs.

1.1 Introduction

The genesis of living matter is regulated and organized by cellular production, encoded by different genetic sequences by cells in the presence of local tissues and fluids. There are a number of different classifications for cells in terms of their shape, function, location, and origin and these differences are noted in terms of their activities. The goal here is not necessarily to create a stand-alone cell biology textbook within this treatise. Other books are both more comprehensive and objectively, just better. There are some exemplary images and micrographs to represent detailed structural features of different cell types, organelles, and ensembles thereof. The goal here is to classify cells based on their attributes, functions, and influences sufficiently for bioengineers, and other interested scientists and engineers who have a working knowledge that is useful. A second goal is for readers to have sufficient background so that they can effectively communicate with collaborating life scientists using a more common language. Here the focus is also to identify both morphological features and chemical characteristics of normal eukaryotic cells, followed by a broader description of cellular physiology including the mechanics of cellular function.

1.2 Cell Composition and Make-Up

Normal eukaryotic cells are highly organized and regulated structures and are shown schematically in Fig. 1.1. Eukaryotic (mammalian) cells are distinguished by a lipid bilayer that separates them from their environment. They contain a series of organelles (machinery) to drive cellular function. One such organelle is the nucleus that houses the genetic encoding (DNA, chromatin) and is contained by a separate lipid membrane isolating the DNA from the other organelles contained by the larger cell membrane. Examples of organelle machinery include the mitochondria that convert stored chemical energy channeled through adenosine triphosphate (ATP) into useable energy for cellular function, the endoplasmic reticulum (ER) that is involved in protein synthesis, and the Golgi apparatus that is involved in sorting and protein separations. Each distinct region is partitioned as a separate entity and identified as a different spatial location of the cell through microscopy. The cytosol makes up the fluid fractions of the cell outside of these functional domains with the proteins shuttled from one domain to the next as part of the production cycle. Also contained with the cell are filamentous proteins that compose the cytoskeleton. As a result, the cell has a mechanical structure with mechanical properties such as stiffness and recovery due to the equilibrium structure of the cytoskeleton. Pushing on a cell takes force to sustain the compression and cell retraction results upon unloading from the perturbed state. Contained both inside and outside of the cell and bound to the lipid bilayer are the so-called receptor molecules. Receptor proteins protrude either into the ECM or into the cell and are used to probe the external environment, communicate with neighboring cells, and respond through what is called receptor-binding links to trigger specific physiologic consequences.

Figure 1.1 Schematic of the average eukaryotic cell showing mitochondria and other organelles contained within the cell membrane.

1.2.1 The Nucleus

The nucleus of a cell is also bound by an internal lipid bilayer that separates the genetic encoding sequences (transcription) from the rest of the cytoplasm, as shown in Fig. 1.2. The nucleus is often the largest and most easily identifiable organelle in a cell. Functions within the nucleus include controlling gene expression and mediating the replication of DNA during the cell cycle. Mutations in cellular DNA that lead to protein defects and the failure to control DNA replication usually trigger either apoptotic cell death during the normal cellular function or the formation of a cancer cell that grows in an uncontrolled fashion into a larger tumor mass.

Figure 1.2 Structure of the nucleus in 2D. Note that the chromatin, uncoiled DNA occupies the space within the nuclear envelope. The nucleolus is found somewhere near the center of the nucleus. Pores are often found in the nuclear membrane that allow for trafficking of proteins between the nucleus and the cytoplasm.

1.2.2 The Endoplasmic Reticulum, ER

The ER houses a network of cisternae (sac-like structures) fixtured and shaped by the cytoskeleton, as shown in Fig. 1.3. A lipid bilayer isolates the cisternal space (or lumen) from the fluid region of the cell identified as the cytosol. The ER is involved in protein synthesis as the ribosomes are contained within a morphologically distinct region called the rough ER. The specific role of each ER is cell dependent, as not all cells produce proteins that are commonly transported (exocytosed) out of the cell. The ER ultimately sorts proteins that are conveyed out of the ER usually through a series of vesicles, lipid bilayers surrounding the secreted proteins. Ultimately, proteins are internally digested, internally sequestered, routed to the cell membrane, or exocytosed outside of the cell.

Figure 1.3 Schematic of endoplasmic reticulum (ER) in 2D: The rough ER possesses ribosomes on its solid surfaces and newly formed proteins are conveyed in the interstices of the ER to the Golgi apparatus. The gaps between solid surfaces of the ER are on the order of 10s to 100s of nanometers.

Protein folding occurs in the ER that is regulated by amino acid sequencing, the local charge, the presence of chaperone molecules to control local charge and ionic strength to drive the appropriate conformational shape. Defects linked with protein synthesis (primary amino acid structure) and folding (tertiary and quaternary structure) are often conveyed for digestion, while correct shapes are expressed appropriately. Inappropriately folded or amyloid protein structures that are expressed are the current focus of a range of age-related diseases called amyloid diseases including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and a host of tissue amyloidosis related diseases that are often encountered in the last hours of life [1].

1.2.3 Mitochondria

Mitochondria drive energy conversion in cells due to their production of ATP, used as a primary cellular source of chemical energy, as shown conceptually in Fig. 1.4. Individual cells can have multiple mitochondria which are also involved in a range of other cellular functions associated with the cell cycle, cell signaling, and apoptosis.

Figure 1.4 Conceptual picture of mitochondria and function. Carbohydrates and fatty acids are ultimately converted to adenosine triphosphate, ATP, through a process called oxidative phosphorylation. The source material is drawn from the cytosol (1) and undergoes a conversion first to acetyl coenzyme A (2) and then followed by a series of oxidative complexes (3) ultimately leading to ATP conversion in (4).

1.2.4 The Golgi Apparatus

The Golgi apparatus is an organelle downstream from the ER. Vesicles containing proteins are fused to the input region of the Golgi that causes the vesicle to break down and helps to convey the now liberated proteins into the entering lumen of the Golgi, as shown in Figs. 1.5 and 1.6. Once separated from the vesicles, many proteins are grafted with hydrophilic phosphates (a process called phosphorylation) and carboxylic acids (glycosylation). Specific glycosylation or phosphorylation steps are kinds of labeling exercises that facilitate protein separations and to redirect proteins destined for different fates. There is often a small change in the molecular weight of the protein as a result of the grafting, which are discussed in Chapter 2, Cell Expression: Proteins and Their Characterization.

Figure 1.5 Proteins leaving the trans Golgi complex are either marked for internal use and consumption, recycled within the Golgi or conveyed in vesicles that facilitate transport into the cytosol destined for secretion as either growth factors, antibodies, cytokines, or some other expressed secretion.

Figure 1.6 A schematic of the structural fiber make up within a moving cell. The microtubules shown in blue (grey in print version), actin and myosin filaments that interact to form intermediate filaments (shown in black), and other structural elements like lamellapodia that are stretched as the cell membrane also undergoes displacement or spreading.

Other proteins synthesized in the ER can be decorated with glycosaminoglycans (GAGs) synthesized in the Golgi to produce proteoglycans and other polysaccharides. It is these labeling events that increase the selectivity of protein synthesis for a desired function. It is amazing to consider how well assemblies of these small systemic functions effectively work in concert to produce proteins that can organize into connective tissue, and how under homeostasis, there is a balance of inputs and outputs that regulate normal function.

1.2.5 Cell Structure

The cytoskeleton is essentially composed of a range of actin and myosin filaments, microtubules, and other tubules of intermediate dimensions that are expressed to reinforce the cell membrane structure, regulate cell movement (motility) and intracellular membrane protein transport by the extension and contraction of these fibers. An example schematic of the fibrous interior of a cell is shown in Fig. 1.6. The interaction between actin and myosin fibrils allows a cell to alter its shape including the formation of microvilli, lamellipodia, and filopodia; form contractile bundles; and enhance cell mitosis by restructuring the two cell surfaces from the one larger one. A great reference on cell structure is found in Chapter 11 of Ref. [2]. It is also the displacement of these fibers that enhances extracellular signaling and modulations in cell receptor interactions can also induce structural rearrangements of the cytoskeleton that can regulate cell gene activity and function as well.

1.2.6 The Membrane Structure: Phospholipids

The lipid membrane is composed of phospholipids, derived from glycerol. Two fatty acid substitutions are made on glycerol to form hydrophobic tails and to the center ol function is attached a hydrophilic phosphate group, as shown in Fig. 1.7. In solution, the tails and heads have sufficient mobility to reorganize (self-assemble) into lipid bilayers or vesicles in which the hydrophilic groups are pointed outward from the cell membrane, and the hydrophobic tails intermix among themselves, as shown in Fig. 1.8 to form the cytoskeleton [2]. The inner side is also hydrophilic by default. This resulting lipid bilayer structure is both stable and a relatively effective permeation barrier with a dimension on the order of 5–10 nm in thickness. The lipid membranes (nuclear and cellular) are organized such that the environments within the nucleus and within the cell are effectively isolated from the environments outside of the membrane. As a result, different chemistry can occur within the cell that would not be as well controlled as it is within each contained membrane region. While the membranes are effective barriers, they have their limits as stretching the membrane too far can cause the membrane to rupture, and proteins and ions contained in the cell can be released outside of the cell through various channels and pores. Also contained within the lipid bilayer are molecules that decorate the surface of the cell membrane either on the inside or outside of the cell which are used for communication. The proteins that protrude through the lipid layer are called receptors.

Figure 1.7 An example of a section of the phospholipid cell and nuclear membrane boundary. Lipid-rich legs extend from a hydrophilic phosphate function leading to an amphiphilic structure. Ensembles of these amphiphiles self-organize yielding a membrane barrier that can create hydrophilic environment both inside and outside of the cell.

Figure 1.8 Pictures of model receptors: In this case, a transmembane protein extends from the lipid bilayer presenting a tertiary/quaternary structure that surveys the local environment. Molecules or proteins that interact with the protein can allow this cell to sense its external environment without allowing transport across the membrane. Receptors can traverse the entire membrane or not, depending on their molecular weight.

1.2.7 Receptors

Some proteins synthesized by the cell are sequestered and expressed on its surface. These bound molecules have phospholipid linkages that keep fragments of the proteins jutting out into the cytoplasm. An example is shown in Fig. 1.8. These bound protein molecules are available to sample the chemical environment, adjacent cells and their receptors, viruses, etc. Examples of receptors on cells include integrins, selectins, and cadherins, which are classes of transmembrane proteins that are situated into the lipid bilayer. Chemical reactions that ensue between ligand molecules in the external environment and the bound proteins are called receptor–ligand binding events, and receptor binding regulates cellular function, particularly for the so-called trans-membrane proteins which contain extracellular and intracellular protein linkages in addition to the lipid bilayer. Receptor binding is often considered as kind of a lock and key effect; in other words, if the right ligands are present in the environment and available to initiate receptor binding, this can set off a cascade of conformational changes in the protein that can alter protein structure within the cell, and can change cell function and protein production [3]. The specificity of receptor binding is regulated often by the conformation of the extracellular membrane protein, and some receptors are more selective than others. Some examples of typical receptor/ligand linkages and the cell types to which they are found are included in Table 1.1.

Table 1.1

Examples of receptors, ligands, and cells where these receptors are commonly found.

As an example integrins are composed of two fragments, the so-called α and β fragments, that are coupled so that one integrin (α6β5) targets one set of ligands. Other integrins composed of different combination of fragments are selective to some other series of ligands. It is possible to conserve one fragment (e.g., α6) but still have a different ligand affinity due to the different β fragments. It is these decorations on the cell surfaces that regulate cellular communication and sensing with the extracellular environment. One can consider antibody–antigen interactions also as mediated by receptor binding.

The types of receptors include those like transmembrane receptors, channel-like receptors that create pores in the membrane, enzyme-based receptors activated by enzymes, G-coupled protein receptors where a signal activation outside the cell creates a signaling response within the cell, and intracellular receptors that upon direct interaction with a chemical cue, triggers a response. The action of the receptor binding to various chemistries is shown in Fig. 1.9.

Figure 1.9 Receptor binding examples showing receptors activated through channel-like linkages (top left), through enzyme linkages, through G-protein coupling (bottom left), and through intracellular signaling (bottom right).

Receptor binding is at the core of modern biotechnology as both drug discovery and tissue engineering are based on creating a more comprehensive understanding of cellular response ex vivo that can be translated into in vivo therapies without abusing surrounding cells not targeted for a response. Complications in regulating cell receptor binding relate to how fast therapies take hold and how permanent these induced changes remain. In other words, the answer to the question of how large of a dose is needed and for how long to induce cell apoptosis in a particular cancer cell requires an adequate understanding of the uptake rate and metabolism of a chemotherapeutic drug. Targeting receptors that interact with a fast kinetics of association might lead to therapies that require less persistence, thereby reducing the dose required to treat an oncology patient.

Receptor–ligand binding forms the basis of cell adhesion both between the same type receptor on different cells called homotypic binding and different receptors on different cells called heterotypic binding. Receptor binding can also be mediated by soluble catalysts that facilitate binding through an intermediate species. Calcium and zinc ions are potent intermediate facilitators of binding through receptors called cadherins. Ligand binding can also occur with receptors on surfaces of the extracellular matrix or other foreign bodies. A single cell expresses many different cell adhesion molecules, targeted for creating multiple binding efficiency.

Erythrocytes (red blood cells, RBCs) are decorated primarily with selectins and possess relatively weak interactions with other cell types. Erythrocytes exposed to shear often tumble where they bind and decouple to vascular wall surfaces with significant regularity. It suggests that a sufficient residence time with cells expressing selectins may be needed to form a stronger interaction. Clearly the presence of the selectins slows those interacting cells as they encounter the luminal wall of the cardiovascular network. Leukocytes (white blood cells) often express more integrins, and with a variety of α and β fragments, these cells can target a variety of foreign bodies that are presenting other ligands. Cadherins are transmembrane receptors that usually undergo heterotypic binding with another protein receptor called catenin, mediated by the presence of calcium ions.

How are these receptors manifested though and how fast are these extracellular connections made? There are models that describe the kinetics of binding and association, and they are likely influenced by temperature, pH, and ionic concentration [3]. Some receptors are only capable of linking with one type of ligand yielding significant selectivity while others have several possible ligands that can bind lowering selectivity. Ligand binding can trigger intracellular conformational rearrangements that can alter protein expression or cell function. Functionally, ligand binding and its impact on cytoskeletal reorganization as receptors toggle between a bound and unbound state is the mechanism by which a cell can react to external chemistry by altering its physiology and expression.

So the average eukaryotic cell possesses organelles, a membrane, a cytoskeleton, receptors, etc. Cells range on order of approximately 10 µm in diameter, with erythrocytes being slightly smaller and platelets much smaller than average. A lot more time is required for characterizing cellular response since there are clear differences in what specific cells are tasked to perform. Better ways are needed of distinguishing between different cells, which are collected from different locations, have different shapes and morphologies, and produce different proteins. The focus here is to classify cells extracted from different structures which should help to organize the terminology in terms of cellular production and morphology, key considerations that engineers and scientists might be more familiar with.

1.3 Cell Classifications

1.3.1 Stem Cells

There is much interest and focus on regenerative medicine applied to identifying, isolating, and ultimately characterizing the response of less-differentiated cells that maintain more regenerative capacity. Stem cells are identified both in terms of their renewal potential and their differentiation potential. A third discriminator for stem cells is the capacity to repopulate damaged, diseased, or replicated tissues as functional cells following some sort of transplantation procedure, whether by autografting (from the same person) or allografting (between different people).

Stem cells have been discovered in large numbers of different tissues and not all stem cells are the same. Within an adult population’s bone marrow, both hematopoietic and mesenchymal stem cells can be isolated. These stem cells are separable by different labeling and isolation strategies, and they can transform into different terminal cell types. Hematopoietic stem cells, also known as universal blood stem cells, are essentially capable of producing all cell types resolved within blood. Other stem cells derived from marrow, called mesenchymal cells, can be directed into connective tissue, neurons, and myocytes, when coerced under the most appropriate culture conditions. It is also possible to extract stem cells from a growing embryo to form the so-called embryonic stem cells that have significant potential to be directed into a variety of terminal cell types.

Stem cells are graded in terms of the degree of differentiation potential available when isolated. As a result, stem cells are like trains on rails, as sufficiently differentiated cells are not capable of redirecting beyond one pathway might be considered as cells on a single track with one destination in mind or unipotent. These cells display an asymmetrical mitosis pathway, transforming to yield two daughter cells, a new stem cell and a second cell, committed to differentiate along one pathway yielding a cell with specific shape and functions.

Other, less-differentiated cells are like nodes with multiple pathways and destinations called cell lineages possible, depending on how the local chemistry (signaling molecules, pH, reactive oxygen species, etc.) and receptor binding affects cell differentiation. Depending on the possible pathways available, these cells might be defined as a specific number such as tri-potent (3), or an undetermined number such as pluripotent and multi-potent cells. Stem cells identified from blood remain the best-characterized at this stage, as hematopoietic stem cells isolated from red marrow express specific receptors such as CD34+ and are negative for other receptors such as CD10, CD14, CD15, CD16, CD19, and CD20. An example differentiation tree showing the evolution of a variety of cell types from the human universal stem cell is shown in Fig. 1.10. Other stem cells in different culturing conditions will lead to different terminal cell