CHAPTER Tissue Dynamics All tissues are dynamic and have their own characteristic replacement and pro- duction rates of cells. (See Table 3.1.) However, the time scales of celular processes that determine tissue dynamics are relatively long, and one tends to overlook their importance. The bone marrow is the most proliferative tissue in the body, followed TABLE 3.1 Tissue or eel production Species ‘Characteristic time processes (days) Enythropoiesis nat 28 Myelopoiesis rat 14 Hematopoiesis ‘human 25 Small intestinal epithelium ‘human 46 rt rey Epidermis ‘buman 7-100 Corneal epithelium ‘human 7 Lymphatic cells rat (chymus) 7 rat (spleen) 1s Epithelial cells sat (vagina) 39 ‘human (cervix) 37 Spermatogonia human 4 Renal interstitial cells mouse 165 Hepatic cells eat 400-5003.2. Homeostaisn Highly ProliicTiswes 35 by the lining of the small intestine, followed by the epidermis. The replacement rate of the cell numbers in these three tissues is on the order of a few days. Other tissues are much slower in their cell replacement rates. For instance, the cellular content of the liver replaces in about one year. In the last chapter, we described the basic components of tissues, but here we will describe the different dynamic states of tissues. We then relate these states to the dynamics of the processes that the constituent cells undergo. 3.1 | Dynamic States of Tissue: Overall there are three dynamic states of tissue: Tissue homeostasis: the normal steady-state function of tissue. Some tissues produce cells (bone marrow, skin) as their main function, while others produce a secreted product (glands). Some tissues primarily carry out mass-transfer oper- ations (lungs, kidneys) while others are biochemical “refineries” (liver). Tissues adapt to the physiological need. For example, exercise will lead to hypertrophy of muscle, Tissue repair: wounded tissue displays a healing process that is relevant to tis- sue engineering. A biopsied piece of tissue is expected to initially display a healing-type response after being placed in culture, and the integration proce- dure following grafting of tissue is expected to display components of this type of response as well. Tissue formation: the formation of tissue involves developmental biology and morphogenesis. The way tissues form is of key concern in cellular therapies, and the younger the cell source, the more organogenic potential it possesses. Understanding stem-cell biology is of particular importance, since stem cells can divide and differentiate into a large number of mature progeny, while some daughter cells maintain their undifferentiated state. ‘We will now briefly describe the first two of these dynamic states, while the third will be discussed in Chapter 4. 3.2 | Homeostasis in Highly Prolific Tissues Many tissues produce cells at a high rate. Prolific tissues have been found to have stem cells that are the source of all the basic types of differentiated cells in that tissue, These tissues display a highly organized pattern of stem-cell replication and differentiation to mature progeny. 3.2.1 Bone marrow The highly dynamic nature of bone-marrow function was discussed in Chapter 1, (Figuce 1.6). The bone marrow produces as many cells as it is comprised of ap- Proximately every two to three days, representing the body's most dynamic tissue.36 Chapter 3 Tusue Dynamics Figure 3.1 Vill in the small intestine. (A) Rows of villi of epithelial intestinal cells {the diameter of a villi is about 80 microns.) (B) A schematic showing the villi and the crypt indicating the ritotic state of the cells in various locations, Modified from {7} 3.2.2. Villi in the small intestine The lining of the small intestine is comprised of villi, shown schematically in Fig- ture 3.1, that absorb nutrients. The intestinal epithelial cell layer is highly dynamic. Is cellular content turns over approximately every five days (Table 3.1) and is thus the body’s second most prolific tissue. Between the villi are tube-shaped epithelial infoldings. An infolding is known as aa cryptus. All intestinal epithelial-cell production takes place in the crypt. Towards the bottom of the crypt is a ring of slowly dividing tissue-specific stem cells. The number of stem cells per crypt is about 20. After division, the daughter cell moves Villu (no cell vision) a” Cross section of villus Epithelial cells Caypt Loose — connective tissue Nondividing. differentiated cells Direction lRapidly dividing of call cals (cycle ‘movement time = 1h) Stowiy dividing stem call (yee thevaties \S Nondividing Gifferentiated cells3.2. Homeostasis in Highly Profi sues up the crypt where it becomes a rapidly cycling progenitor cell, with a cycling time on the order of 12 h. These cells have relatives in many tissues and are often referred +o as “transit amplifying” cells. The cells then move up the erypt and differentiate. Once they leave the crypt, they are mature and enter the base of the villi. Then they move, over a period of about five days, from the base of villus to the top, where they dic and slough off. ‘The villi thus represent a tube or a cylinder with a slowly moving layer of mature intestinal epithelial cells that emerge from the crypt and fall off once they reach the outer edge of the villi, During this passage, the cells carry out their organ specific fanction as mature parenchymal cells. They function in the absorption and digestion of nutrients that come from the lumen of the gut. 3.2.3 Skin ‘Skin has two principal cell layers separated by a basal lamina (Figure 3.2). Collagen Vil is an important component of this basal lamina. A connective tissue layer, the dermis, is under the basal lamina and is comprised primarily of fibroblasts. On the outside of the basal lamina is the epidermis, which is a cell layer comprised of differentiating keratinocytes, with the least differentiated ones located at the basal lamina. Thin skin has a squamous columnar organization (each column about 30 zm in diameter). The cells located at the basal lamina replicate. Since the area ofthe skin is constant, cells must leave the replicative cell layer that is on the basal lamina. These Squame about to flake off from surface Karatinized squames Granular |} cell layer Prickle cell layer Basal cell ayer Basal Jamina Connective tissue of Basal cell Basal celldividing dermis passing into prickle cell layer Figure 3.2 ‘The cellular arrangement and differentiation in skin shown in cross-section, schematically. The cellular arrangement in the epidermis and the differentiation stages that the cells undergo are indicated. Redrawn from [7]38 Chapter3 Tissue Dynamics cells then cease to divide and begin to undergo the differentiation that divides the epidermis into well defined cell layers of increased differentiation. AAs the cells leave the basal lamina, they undergo differentiation to prickle cells, then to granular cells, which develop into keratinized squamous cells that eventually flake off. Thus, only the cells on the basal lamina are cycling, while cells in the outer layer are differenti- ating. It is believed that approximately one in 10 to 12 basal cells is an epidermal “progenitor” cell that is responsible for the cell production in the squamous column above it. The net proliferative rate of skin varies with the region of the body. The turnover of skin is on the order of a few weeks, which generates a large amount of flakes over time. It has been estimated that about half of dust found in houses originates from Jhuman skin. Skin is the body’s third most prolific tissue. 3.3_| Tissue Rep: ‘When tissue is injured, a healing response is induced that is comprised of a coor- inated series of cellular events that vary with age. Fetal wound healing proceeds rapidly and leads to the restoration of scarless tissue, Healing of postnatal tissue is slower and often leads to scarring, which generally permits satisfactory tissue restora- tion, although not always fully restoring normal tissue structure, Some pathological states resemble wound healing gone awry. A variety of fibrotic diseases involve sim- ilar processes to tissue repair and subsequent scarring. Wound healing is further discussed in Chapter 18. 3.3.1 Sequence of events that underlie wound healing ‘Thecomplex set of events that contribute to wound healing are schematically depicted in Figure 3.3. Immediately following injury, control of bleeding starts with the rapid adhesion of circulating platelets to the site of damage. Within seconds, the platelets are activated, secrete contents of their storage granules, spread, and recruit more platelets to the thrombus that has started to develop. Within minutes of injury, the extent of hemorrhaging is contained through the constriction of surrounding blood vessels. (See Figure 3.3A.) “The next phase of the wound-healing process involves the release of agents from the platelets atthe injured site that cause vasodilatation and increased permeability of neighboring blood vessels. The clotting, cascade is initiated, resulting in the cleavage of fibrinogen by thrombin to forma fibrin plug. The fibrin plug along with fibronectin, holds the tissue together and forms a provisional matrix. This matrix plays a role in the early recruitment of inflammatory cells and later in the migration of fibroblasts and other accessory cells. (See Figure 3.3B.) Epidermal cells also begin to proliferate and migrate across the wound, Inflammatory cells now migrate into the injured site. Neutrophils arrive early oon the scene migrating from circulating blood. As the neutrophils degranulate and die, the abundance of macrophages at the site increases. All tissues have resident macrophages, and their number at the injury site is enhanced by macrophages mi: ‘grating from circulation. ‘They act in concert with the neutrophils to phagocytose3.3. Tissue Repair ‘Quantity (azbitrary units) 2 Time (days) Figure 3.3 Wound healing in adult skin, (A) hemostasis of the wound occurs; (B) thrombosis (clotting) forms 2 fibrin plug; (C) inflammation (migration of white blood cells); (D) formation of granulation tissue with new blood vessels, cxlfular debris, combat any invading microorganisms, and provide the source of chemoattractants and mitogens. "These factors induce the migration of endothelial calls and fibroblasts to the wound site and stimulate their subsequent proliferation, IF the infiltration of macrophages into the wound site is prevented, the healing pro- ‘ess is severely impaired. In the epidermal layer, the basal layer has reformed. (See Figure 3.3.) At this time, so-called granulation tissue has formed in the dermis. It is com- prised of a dense population of fibroblasts, macrophages, and developing vasculature that is embedded in a matrix that is comprised mainly of fibronectin, collagen, and hyaluronic acid. ‘The invading fibroblasts begin to produce collagen, mostly type 1 and Ill. The collagen increases the tensile strength of the wound. Myofibroblasts ‘contract at this time, shrinking the size of the wound by pulling the wound margins together. At this point, the epidermal layer has stratified. (Sec Figure 3.3D.) Subsequently, the matrix undergoes remodeling, which involves the coordinated synthesis and degradation of eonnective-tissue protein. Remodeling leads to a change inthe composition of the matrix with time. For instance, collagen type Ill is abundant carly on, but gives way to collagen type I with time. The balance of these processes40 Chapter 3 Tesue Dynamics Figure 3.4 Cartilage repair: time scales and tissue events. Note the similarity of cell-fate processes to wound healing in the skin, From R. Sab, ucsb. Cellsignaling, Cel differentiation, Matrix adtesion, migration prelferation apoptosis remodeling _ nl Failure Second Hour ond Hour Day Week Month Year determines scar formation. Although the wound appears healed atthis time, chemical and structural changes continue to occur within the wound site. The final step of the ‘wound-healing process is the resolution of the scar. The formation and degradation of matrix components returns to its normal state, a process that may take many months. Finally, the composition of the matrix and the spatial location of the cells returns close to the original state. {A similar course of events can also be seen inthe healing of cartilage in Figure 3.4 Understanding the wound healing process is important to the tissue engineer, since the placement of reconstituted tissues in vivo induces responses similar to the wound- healing process. 3.3.2. Engineering wound healing ‘Wound healing can be modified with tissue-engincering strategies. One example utilized a porous template designed to be an analog of the extracellular matrix in the dermis of the skin [382, 383]. This template was composed of collagen T and chondroitin-6-sulfates and was fabricated with a range of pore sites and degradation rates. These templates were investigated for their ability to delay wound contracture and promote dermal regeneration in a classic wound-healing model, square full- thickness wounds (removal of dermis and epidermis). The time required for the wound area to decrease by 50% was measured. Optimal pore size for delay of ‘wound contracture was found to be between 20 and 120 jam, which correlates with a typ time of 27 days compared with 7 days. It is thought that the delay in wound contracture related to improved healing correlates with fibroblast migration through the template. Normally, part of wound contraction occurs after fibroblasts have spanned the provisional matrix in the wound bed and changed to a myofibroblast phenotype. Delay of fibroblast migration allows time for wound healing before ‘wound contraction and scar formation. At very small pore sizes, fibroblasts cannot penetrate the analog, whereas at large pore sizes, the surface area available for cell adhesion (per unit volume) de- clines. (See Figure 3.5A.) Furthermore, the degradation rate of the template was found to be optimal when it was tuned to approximate the normal wound-healingmical of the lation ceells woh. “ound- ample tix in Land dation -acture re full for the elay of es with wound hrough ts have sroblast before eas at me) de- ate was ‘healing — — 5 4 Sab 4 a 3 y 1 Em 210 4 oy 1 4 011101001000 Tem ‘Average pore diameter, an « co sn Ungraited control 7 L Li 125 5 i050 25 100 250 Degradation rate ®) rate (Figure 3.5B). This criterion can be described mathematically by the following equation, which simply states that the template degradation rate, t, is on the same order of magnitude as the normal wound healing rate, t, unit = oni GB.) Here, 4 is approximately three weeks for skin and six weeks for peripheral nerve. ‘The template degradation rate for skin was approximately two weeks, satisfying the criterion just described [382]. Another classic example of the wound healing response is found in the formation of “tissue analogs” by cell-contracted extracellular matrix gels. This phenomenon 33° Tisue Repair 41 Figure 3.5, Effect of porous template ‘on wound healing. (A) Role of pore size on optimal delay in wound contracture. {B) Role of scaffold degradation time on delay fof wound contracture, From [383].42 Chaptec3 Tissue Dynamics Figure 3.6 ‘Contraction of cellladen ‘matrices. (A) Cellladen collagen scaffolds contract to create tissue equivalents (B) D is the diameter of scaffold, ¢ is time. Increase in all concentration increases contraction. The effect of cells on matrix ‘contraction is inhibited by matrix metalloproteinase inhibitors. “ D| exploits the cell-based contraction of extracellular matrix to form tissuelike con- structs [28]. This process is utilized to form a dermal equivalent in a commercial tissue-engincered skin product. The process and kinetics of contraction are shown in Figure 3.6. Note the dependence on cell density: An increase in the number of cells increases contraction. Conversely, increased ECM concentration reduces the degree and speed of contraction, as do inhibitors of matrixproteases (TIMPS). The ability of cells to modify the ECM environment, seen in wound healing it tivo, is therefore utilized to engineer tissue in vitro. 3.3.3 Fetal wound healing Fetal wound healing occurs rapidly, efficiently, and perfectly without scar formation. ‘Adult wound healing, on the other hand, is imperfect and results in fibrosis and scar formation with poor reconstitution of epidermal and dermal tissues at the healed ‘wound site. (See Table 3.2.) Even though the processes of re-epithelialization and connective-tisste contraction are common to embryos and adults, the means by which they occur is fundamentally different. In the embryo, a gap in the epidermis is closed by contraction of a rapidly assembled actin purse-string, while in the adult, epithelial cells crawl over the exposed substratum to close the defect. The exposed connective tissue in the adult wound contains specialized myofibroblasts that contract to bring the two edges of the wound together. In the embryo, however, the same type of result is achieved by standard embryonic fibroblasts exerting similar tractional forces. “Another key difference between fetal and adult wound healing is chat in the adult, there is an extensive inflammatory response, but in the embryo, such inflammation is practically nonexistent. Besides having a minimal inflammatory response, fetal wounds contain collagen matrix that is of a “basketweave” form compared with the bundles of collagen in the adult wound. In addition, the composition of the overall ECM between fetal and adult wounds is fundamentally different (ie. fibronectin in adule versus collagen II in fetal wounds). The fetal wounds also have low tension compared with adult wounds. Due to the advantages of minimal scar formationke con- mercial sown in of cells e degree zaling it mation. and scar ae healed ition and by which, sis closed epithelial onnective to bring eofresult roes. theadult, ammation nse, fetal with the the overall ‘onectin in ow tension formation TABLE 3.2 ‘Adult wound healing Fetal slow Rapid Imperfect ficient, minimal scarring fibroblasts, myofibroblasts fibroblasts epidermal migration epidermal (actin purse-string) High inflammation Minimal inflammation High epithelial proliferation Low epithelial penetration Results in bundled collagen “Basketweave” collagen matrix [ECM (fibronectin, enasin) ECM (Collagen Ill, hyaluronic acid) High tension. Low tension and rapid wound healing, detailed insights into the processes of fetal wound healing can provide us with strategies to improve adult wound healing, as well as to better integrate TEMPs into the body with minimal fibrosis and scar formation. Tissue Dynamics as Interacting 3.4 | Cellular-Fate Processes Underlying the dynamic states of tissue function are complex processes that involve interplay among many different cell types. ‘The cells communicate and coordinate their efforts through the principal cellular fate processes. (Sce Figure 3.7.) The cellularfate processes that underlie the dynamic states of tissue function fall into the following categor 1. cell replication, an increase in cell number; . cell differentiation, changes in gene expression and the acquisition of a particular function; cell motility, the motion of a cell into a particular niche or location; 4. cell apoptosis, programmed cell death; and cell adbesion, the physical binding of a cell to its immediate environment, that may be a neighboring cell, extracellular matrix, or an artificial surface. ‘The biology and dynamics of these processes are discussed in detail in Chap- ter 6. The mechanisms by which they are regulated and coordinated are discussed in ‘Chapter 7. ‘The homeostatic function of many tissues involves cell production, as previously outlined. Morphogenesis, or tissue formation, involves the growth and formation 34 Tissue Dynamics as InteractingCelllorFate Processes 4344 Chapter 3 Tse Dynamics Figure 3.7 Tissue dynamics. The three dynamic states of tissues and the underlying, cellular-fate processes. Tissue Dynamics ‘+ Tissue function (homeostasis) ‘+ Tissue repair (wound healing) ‘+ ‘Tissue formation (morphogenesis and developmental biology) Cell differentiation Cal death Cell motion Celladhesion of tissue from immature tissue-specific stem cells. These tissue-specific stem cells are formed in early embryogenesis. Our knowledge about the early embryonic events is ‘growing. The basic events that lead to the formation of the basic body plan and the commitment to various organs and tissue formation can in many cases be traced in detail. The subsequent chapter is devoted to this issue. 3.5 | Summary ‘Tissue dynamics can be divided into three categories: tissue homeostasis, tissue repair, and tissue formation. ‘In tissue homeostasis, cell production and replacement rates range from days (bone marrow) to years (lives). « Tissue repair occurs in phases. Early in the process (days), there is a coordina- tion of cell proliferation, adhesion, and migration. Remodeling of the wound occurs later (Weeks to years) as a result of differentiation (e.g,, fibroblasts to myofibroblasts) and secretion of ECM and proteases. ‘¢ Wound healing can be modified by the use of biomaterials, Degradation rate, pore size, cell seeding density, and the presence of protease inhibitors can affect the healing process.lis are onts is adthe ced in tissue n days ordina- wound asts to on rate, naffect « Fetal wound healing, like adult wound healing, requires both wound contraction and cell migration. However, fetal wound healing is more rapid and results in ‘minimal scar formation before the third trimester of development. «Tissue dynamics result from a coordination of cell adhesion, migration, differ- entiation, replication, and apoptosis. «« Pathological states can result if the underlying cellular fate processes are not properly coordinated. 3.6 | FURTHER READING Alberts, etal. Molecular Biology of the Cell, Garland Pub- Kumar, V., Cotran, R. S., Robbins, $. L., Robbins Basic 3.6 Further Reading 45 Pathology, Saundars, Philadelphia, 2003,