Integrated processes responsible for soft tissue healing

Vol. 82 No. 5

November 1996

II REVIEW ARTICLE

Integrated processes responsible for soft tissue healing Mark E. K. Wong, DDS, a Jeffrey O. Hol!inger, DDS, PhD, b and Gerald J. Pinero, PhD, c Houston, Tex. UNIVERSITYOF TEXAS HEALTHSCIENCECENTER AT HOUSTONAND OREGONHEALTHSCIENCES UNIVERSITY Wounded soft tissues undergo repair through a complex series of interrelated events that involve both physical and chemical activities. These processes are currently undergoing extensive investigation as efforts are directed toward achieving augmented and accelerated healing. Early wound-healing research focused on expanding traditional histologic descriptions of tissue healing by attempting to characterize the environment and biologic mediators responsible for healing. These initial studies successfully identified a number of agents and physiochemical factors present in healing wounds, but their precise roles and importance remain largely unknown. This review article summarizes the current literature on soft tissue healing. An effort has been made to correlate the activities of the major growth factors and cytokines with the individual reparative processes including the inflammatory response, hemostasis, fibroplasia, angiogenesis, and remodeling. Explanations and characteristics of growth factor function as well as brief descriptions of several major factors and their spectrum of activity are also provided. (Oral Surg Oral Meal Oral Pathol Oral Radiol Endod 1996;82:475-92)

Tissues of the body may be Separated into two broad categories: hard and soft tissue. The physiologic responses for repairing hard and soft tissue after injury. will be presented in a two-part series on wound healing. Mechanisms involved in soft tissue healing are discussed in this article, and hard tissue repair will be described in a second article. The soft tissues of the body include skin, muscles, nerves, tendons, and ligaments. This article will focus primarily on epidermal repair. Interestingly, many of the cells and mediators as well as the different stages of soft tissue repair are nearly identical with those of bone healing.

Formerly, healing of soft tissue wounds was characterized with histologic descriptions of fibroplasia, collagen synthesis, and neovasculaturization. However, recent advances in cell biology have provided more accurate descriptions of the mechanisms, cell types, and chemical agents that control and direct these activities. Included in this group of chemical modulators are various growth factors secreted by cells that participate in the healing process. Even though they have not been completely identified, enough is known to assign this collection of biologically active proteins a prominent role in the events that comprise soft tissue healing.

aAssistant Professorand Chief, Divisionof Oral and Maxillofacial Surgery, LBJ General Hospital, University of Texas Health Science Center at Houston. bprofessor of Surgery, Cell, and Developmental Biology, School of Medicine, Department of Surgery, Oregon Health Sciences University. CProfessorof Anatomy,Department of Basic Sciences, University of Texas Dental Branch, University of Texas Health Science Center at Houston. Received for publication Dec. 8, 1995; returned for revision Feb. 13, 1996; accepted for publication June 5, 1996. Copyright 9 1996 by Mosby-Year Book, Inc. 1079-2104/96/$5.00 + 0 7/12/75606

O R G A N I Z A T I O N OF EVENTS IN TISSUE HEALING Soft tissue injury is a necessary corollary to most forms of surgery and provides the stimulus to begin the healing process. Although bone is capable of healing by the regeneration of tissue indistinguishable from its previous form, soft tissue healing is characterized by repair in which the damaged area is replaced by scar tissue. The events that comprise epidermal healing may be broadly categorized into four phases: wounding, in475

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Fig. 1. A, Skin 24 hours after injury. Wound site is filled with blood clot (BC) and there is disruption of surface epithelium (E). At margins of wound area, accumulation of inflammatory cells signals development of healing module. B, Oral mucosa 1 hour after injury. Within 1 hour of incisional injury, margination and diapedesis of leucocytes occurs. Leucocytes (arrows) pass through endothelium andl toward injury site under influence of chemotactic factors. C, Skin within 24 hours of injury. Neutrophils (N) and macrophages (M) accumulate at periphery of wound. (Original magnification for all x200.) flammation, proliferation, and remodeling (Fig. 1). Tissue injury initiates the inflammatory phase through the activation of several cascades including the coagulation cascade, the tissue complement system, and kinin cascade. A cascade is a physiologic mechanism composed of interrelated processes in which antecedent occurrences provoke a heightened response. The different inflammatory cascades alter the local environment, produce vasodilation of adjacent vessels, decrease the pH level and oxygen tension, and increase local lactate concentrations. As a result of these activities, a specifically ordered progression of cells enter the wounded area and begin to replicate. This signals the start of the proliferation phase. Inflammatory cells and macrophages populate the early cellular response, and endothelial cells appear later during healing (Fig. 1). The different cell types present after epidermal wounding include fibroblasts, endothelial cells, and epithelial cells. The remodeling phase begins as the

biologic products of these cells undergo transformation into the soft tissue components responsible for the formation of scar tissue (Fig. 2). The specificity of cell types during the different stages of healing suggest that a chemoattractantreceptor-specific mechanism is responsible, whereas the production of increasing numbers of cells appears to be under the control of growth stimulating agents or mitogens. Many of the chemoattractant and mitogenic activities expressed are mediated through growth factors produced and released by cells recruited by the inflammatory phase. In addition, the microenvironment of the wound is also of considerable importance in determining the cell types present and their respective number. The events that comprise the different stages of wound healing therefore include: the initial tissue response to injury; an inflammatory reaction and activation of the complement system; a vascular response with the implementation of a coagulation cascade; the

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Fig. 2. A, Skin 2 days after injury. Epidermis (E) begins to undercut desiccated dermis (D) and blood clot. (Original magnification x200.) B, Skin 12 days after injury. Wound tract indicated by arrows. New fibroblasts appear within wound 2 days after injury and start to replicate under mitogenic effects of growth factors. Their population reaches maximum number by 12 days by which time they have secreted significant amount of new collagen. Note increased number of fibroblasts in wounded area. creation of a novel microenvironment within the wound; the appearance of different cell types under the influence of various chemoattractants and chemical gradients; the replication of cells as a result of mitogens; and the elaboration of a new vascular system within a connective tissue matrix produced by the cellular infiltrate. Whereas simplification of the complex events of healing into separate categories may facilitate understanding of the phenomenon, in reality there is an amalgamation of the different reactions, both spatially and temporally. This is especially true when different parts of the same wound are considered.

PHASES I AND I1: W O U N D I N G AND INFLAMMATION Inflammatory reaction and activation of the complement system Injury to epithelium and the underlying dermis results in bleeding, cell activation, and initiation of the coagulation, complement, and kinin cascades. 1 The inflammatory reaction that occurs immediately after wounding includes a series of defensive events. The local manifestations were first described by the Roman physician Celsus as the four "cardinal signs of inflammation" (rubor, calor, dolor, and tumor) and may be observed in the soft tissues around a recently injured site. Systemic effects of inflammation, including the development of pyrexia, leukocytosis, and elevation of the erythrocyte sedimentation rate are also evident and m a y be present in the postinjury patient. The extent of the systemic response is proportional to the degree of soft tissue wounding. A significant component of the body's defense

system is the development of tissue exudate. The main functions of a cell-rich tissue exudate are to provide cells capable of producing the components mad biologic mediators necessary for the directed reconstruction of damaged tissue while diluting microbial toxins and removing contaminants present in the wound. The mechanism of exudate formation involves a number of different physiologic responses to injury. Tissue damage results in the liberation of a histamine-like substance. In combination with a local axon reflex, perfusion of the wound increases through overall vasodilation of the remaining capillaries and arterioles. Local vasodilation is also promoted by b i ologically active products of the complement and kinin cascades. The complement cascade involves 20 or more proteins that circulate throughout the blood in an inactive form. After tissue injury, activation of the complement cascade produces a variety of proteins with activities essential to healing. For instance, Csa is chemotactic for neutrophils and macrophages that facilitates healing by phagocytosing debris and contaminant microbials. These cells also express various growth factors that modulate healing. C3a and C4a stimulate mast cells and basophils to release histamine. This causes further vasodilation and exudate formation. The kinin cascade is responsible for the transformation of the inactive enzyme kallikrein, which is present in both blood and tissue, to its active form, bradykinin. Bradykinin also contributes to the production of tissue exudate through the promotion of vasodilation and increased vessel wall permeability. 2 In addition to bradykinin and other activated components of the kinin (e.g., kallidin and leukotaxine) and complement cascades, the dilated vessels are tar-

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CELLULAR COMPONENTS OF THE HEALING MODULE

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geted by an assortment of chemical and enzymatic agents that further increase the permeability of endothelial walls to plasma proteins, leukocytes, and red blood cells. These agents include globulin permeability factors, serotonin (from injured endothelium, platelets, mast cells), Hageman factor, prostaglandins, and cyclic AMP. Diapedesis of leukocytes into the tissue bed is enhanced by the presence of chemotactic factors released into the interstitium as a result of tissue injury. Despite the benefits of an inflammatory response, potentially insidious sequelae associated with excessive exudate formation, such as vessel occlusion or impingement on neighboring vital structures, may also occur. Attenuation of this component of the inflammatory response may be achieved through gentle surgical techniques and, although controversial, the administration of cell membrane stabilizers in the form of intra- and postoperative steroids. Hemostatic reaction to injury Soft tissue injury damages not only the epithelial and connective tissue elements but also blood vessels in the area. Although major vessels may be secured with ligatures, capillaries and small caliber arterioles and venules rely on three mechanisms for the prevention of further blood loss: vessel and vessel wall

retraction, a platelet reaction, and activation of the coagulation cascade. The vascular reaction to trauma differs according to the size of the vessel involved. Larger vessels constrict under the influence of their innervation, whereas smaller vessels are stimulated by vasoactive substances that contract myofilaments in their endothelial walls. These substances include serotonin and the catecholamines that are released from platelets and serum during injury. Small vessels are also subject to collapse as a result of raised extravascular pressures produced by leakage of fluid from vessels in a state of increased permeability. As the intravascular volume is reduced, increased blood viscosity helps reduce blood flow to a traumatized region. The association between platelets and hemostasis has long been apparent. As early as 1882, Hayem noted that a deficiency of platelets resulted in persistent bleeding. 3 During the platelet reaction, the individual cells coalesce irreversibly through the separate processes of adhesion, cohesion, and fusion, and when combined with fibrin, produces a mechanically stable platelet plug that occludes the vessel end. Contemporary research has revealed that platelets not only participate in hemostasis but also contain an array of pharmacologically active substances involved in inflammation and the propagation of repair. Some

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of the substances found in platelets are enzymes, such as adenosine triphosphotase (ATP' ase), which have a fundamental role in the generation of energy required for healing. The vasoactive amines serotonin, adrenaline, noradrenaline, and histamine are also found in platelets and constitute potent chemical mediators of the inflammatory reaction. Growth factors released from degranulating platelet a granules, such as platelet-derived growth factor (PDGF), promote healing by attracting fibroblasts, vascular smooth muscle, endothelial cells, and monocytes. This carefully sequenced series of cellular events has been referred to as the healing module (Fig. 3). The third component of the hemostatic mechanism is the coagulation cascade that begins with tissue injury and concludes with clot formation. Between these two events, a number of interrelated step-wise reactions are responsible for the conversion of fibrin from its precursor fibrinogen. Production of fibrin involves cleavage of the larger inactive fibrinogen molecule by the enzyme thrombin and involves two pathways termed the intrinsic and extrinsic routes. Traumatized tissue releases a complex referred to as tissue thromboplastin that activates the extrinsic pathway. After vessel injury, the intrinsic pathway is activated by interaction between platelets and the Hageman factor (Factor XII). Both routes lead to the formation of prothrombin activator that converts the inactive prothrombin into thrombin.

PHASE II1: PROLIFERATION As discussed earlier, activated products of the complement system attract macrophages to the wound. Growth factors secreted by macrophages in turn stimulate migration of fibroblasts and epithelial and endothelial cells. 4 Fibroblast proliferation and secretion of different types of collagen in combination with the development of new blood vessels (angiogenesis) from endothelial cells leads to the formation of granulation tissue.

Fibroplasia and angiogenesis Once fibroblasts and endothelial ceils enter a wounded area conducive to their activities, the foundations for tissue healing are laid. As with other events in tissue repair, an interdependence is created between the separate processes of fibrous tissue formation and development of a new vascular supply. The production of a connective tissue matrix relies on nutrients from an intact vasculature; in return, blood vessels require support and protection from the matrix to remain patent and for extension of the vascular system. The connective tissue in healing wounds is composed primarily of Types I and III collagen, 5 cells, vessels, and a matrix that contains glycoproteins and

proteoglyCans. Collagen is secreted by fibroblasts that have been attracted to the area and stimulated to multiply by growth factors such as PDGF, TGF-[3, FGF, EGF, and IGF-1 and tissue factors such as fibronectin. The formation of collagen involves four steps (Fig. 4): (1) the intracellular formation of a protocollagen chain; (2) conversion of protocollagen to procollagen c~ chains that undergo winding into a super helix moiety; (3) secretion of procollagen from the fibroblast into the matrix followed by assembly into collagen fibrils; (4) organization of collagen fibrils into collagen fibers with the classic one quarter stagger arrangement. Each of these steps use enzymatic reactions and various cofactors to control their progress. Collagen formation begins with the activation of the enzymes prolyl and lysyl hydroxylase. These enzymes catalyze the hydroxylation of half the proline and lysine present in protocollagen to form procollagen. 6 This reaction requires a minimum oxygen tension of 20 rnm Hg combined with the cofactor activity of ascorbate and ferrous ions. 7 It has been recently noted that lactate may play a role in the activation of the enzyme prolyl hydroxylase, a feature that again underscores the importance of the microenvironment within a healing wound. Once the procollagen is secreted from fibroblasts, cross-linking and polymerization occurs under the influence of lysyl oxidase with copper used as a cofactor. The completed collagen fiber interacts with other matrix components such as proteoglycans to form the necessary support for new blood vessels and cells. Proteoglycans are significant in their ability to influence both the architecture and the ultimate strength of the repair. 6 A collagenous matrix facilitates angiogenesis in several ways. The structural protection afforded to new and friable vessels provides the necessary time needed for anastomotic connections to form. Additional matrix components secreted by fibroblasts termed attachment factors perpetuate endothelial ingrowth by promoting attachment of these cells to basement membrane collagen. 8 Laminin is a glycoprotein that serves this function. Angiogenesis occurs in response to the hypoxic state created by tissue damage as well as to factors released from cells during injury. The primary responsibility for initiating the formation of new vessels lies with the platelet reaction to injury. Activated platelets attract macrophages to a wound through the chemotactic actions of PDGF and basic FGF.bFGF in turn is a potent chemotactic and mitogenic agent for endothelial cells. TGF-[3 is also found in platelets and exerts an indirect effect on angiogenesis by promoting the release of tumor necrosis factor-a and interleukin-1 from macrophages. 9 More recent investigations indicate that angiogen-

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phenotype; negative factors must be decreased. Thrombospondin was the first negative angiogenic factor to be implicated in tumor angiogenesis, A second negative angiostatic factor, angiostatin, is a pro-

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tein with 98% homology to an internal fragment of plasminogen. 12 These and possibly other negative angiogenic factors normally serve to maintain blood vessels in a state of homeostasis. A hypothesis developed to explain angiogenesis in tumor growth 12 may be applicable to wound healing as well. This hypothesis proposes that vessel formation occurs with the upregulation of angiogenic stimulators (e.g., FGF, vascular endothelial growth facor) and downregulation of circulating angiogenic inhibitors (e.g., thrombospondin, angiostatin). Limits to the degree of angiogenesis are imposed by the relatively short half-lives of angiogenic stimulators (which also suggest local activity) and the much longer half-lives of the different angiogenic inhibitors. Given the importance of blood supply in wound healing, there is great interest in the potential therapeutic application of natural and synthetic molecules that act as positive and negative regulators of angiogenesis. New vessel formation begins with the activation of hydrolytic enzymes such as collagenase, stromolysin, and plasminogen by bFGE These enzymes act on vessel walls dissolving their basement membranes and freeing endothelial cells. 13 The individual cells then migrate toward a wounded area under the influence of a chemotactic gradient. On arrival at hypoxic sites within a wound, the cells form lumens and fuse into loops to allow a continual flow of blood to feeder vessels. 14 Mesenchymal cells surrounding new vessels are important from several viewpoints. Some become intimately associated with the vasculature and are known as pericytes. These cells play a role in the inhibition of adjacent endothelial cell growth, thereby regulating angiogenesis. ~5 Others have been implicated as targets for differentiation into osteoblasts under the influence of bone morphogenic proteins, thereby promoting osteogenesis. Additional negative feedback mechanisms for the control o f angiogenesis include a reduction of platelet-associated factor release and the recapture of bFGF by endothelial cellsJ 4 Finally, as oxygen tensions rise through the establishment of new vessels, angiogeneic factor release from cells like macrophages decreasesJ 4 Fig. 5 summarizes the process of angiogenesis. Several types of collagen have been immunolocalized within healing epithelial wounds. Types I and III predominate, but small amounts of types IV and VII collagen can also be found. 5 Type I collagen is the primary constituent of connective tissue; type III is found in blood vessels and skin, type IV in basal lamina, and type VII in skin. The different collagens in combination with glycosaminoglycans, hyaluronans, and assorted attachment factors (e.g., fibronectin, the integrins) are

components of the extracellular matrix (ECM). The role of the ECM in wound healing includes the attachment and direction of cell migration as well as the polarity and orientation of cells. Other characteristics of the proliferation stage include an elevated water content, an increased number of vessels, and epithelialization of the wound surfaces from epithelial cells migrating centripetally. PHASE IV: REMODELING The fourth phase of soft tissue repair involves a conversion of the initial healing tissue to scar tissue. A decrease in water content of the reparative tissue and a replacement of collagen isotypes primarily with types I and III occurs during this stage. The vascularity of the tissue also decreases to normal levels. The increase in types I and III collagen and other aspects of the remodeling process are responsible for wound contraction and Scar formation. A significant feature of wound healing relates to the difference between a clinically " h e a l e d " wound with a regenerated epithelial surface and the restoration of mechanical strength to the repaired tissue. Despite the presence of an intact epithelium at 3 to 4 weeks after the injury, the tensile strength of the wound has been measured at approximately 25 % of its normal value. Several months later, only 70% to 80% of the strength may be restored. 6 The reason for this lies partly in the orientation of the collagen fibers in the healing portion of the wound. For several months after injury, the collagen fibers in fibrous scar tissue tend to remain more vertical than the usual horizontal alignment of fibers in normal epithelium (Fig. 6). Furthermore, the turnover rate of collagen in a newly healed scar is very high, and factors that adversely affect synthesis will allow lysis to predominate. Failure to appreciate these features of healing and provide continued biologic and physical support for a wound may result in dehiscence or a widened scar. The interrelated processes that begin with tissue injury and conclude with tissue repair have been divided into three phases (inflammation, proliferation, and remodeling) to facilitate understanding. The primary components of each phase and their approximate period of activity is summarized in Fig. 7.

SPECIAL MECHANISMS RESPONSIBLE FOR W O U N D HEALING Creation of a suitable wound-healing environment Tissue injury along with the resultant inflammatory, complement, and hemostatic reactions produces a unique environment within wounds that promote repair. Specific physical and chemical characteristics of this environment appear to play important roles

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determining cell types, regulating cell function, and supporting cellular metabolism. Within minutes of injury, the wound environment becomes hypoxic, acidic, hypercarbic, and contains high levels of lactate. These characteristics are not homogenous, and concentration gradients are present in different areas of a wound. Oxygen gradients, for

example, are established with the lowest tensions expressed at the cut edge and the highest toward adjacent inviolated tissue. 16, 17 This gradient is produced by the varying diffusion of oxygen as distances from a peripheral vascular supply increase. Contributing to the central hypoxic environment is the cellular infiltrate produced by the inflammatory reaction that

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Fig. 6. A, Skin 12 days after injury; wound is clinically healed. Surface epithelium has been regenerated, and wound tract (arrows) is difficult to visualize in histologic section. B, Same section as Fig 6A viewed under polarized light. Dark wound tract (arrows) demonstrates disorganized and consequently weaker new collagen. consumes oxygen as a result of normal metabolic activities. TM The elevated lactate content of wounded areas was previously felt to be a consequence of cells undergoing anaerobic glycolysis. However, current investigations suggest that lactate is produced largely through aerobic glycolysis by the significant number of cells in the infiltrate. 19 Detailed analysis of the different amino acid, lipid, a n d inorganic substances required to support cell proliferation have also been studied. Whereas one source of energy for wound healing is provided by glycolysis, another mechanism involves the oxidation of glutamine to pyruvate in cell mitochondria. In addition to meeting the energy requirements of a healing wound, these processes help maintain high levels of anabolic precursors, providing the necessary building blocks for tissue repair. 2~ The effect of small amounts of mechanical stress on the formation and strength of collagen has been described in the orthopedic literature. Healing wounds subjected to continual passive motion manifest increases in wound collagen content, fiber orientation, and wound strength. 21 However, when excessive forces are applied, disruption of both the fragile collagen framework and neovasculature occurs, ultimately delaying the repair process. Electrical gradients have also been measured between the epidermal surface and the underlying connective tissue, but their effects at this time remain undetermined.

Chemotaxis and mitogenesis in wound healing Chemotaxis is the directed migration of cells along a chemical gradient and is a function of specific chemoattractant-cell receptor relationships. 8 This mechanism is responsible for attracting cells necessary for healing to the wound. Leukocytes and mac-

rophages appear shortly after injury, followed by fibroblasts and endothelial cells. Phagocytic leukocytes and macrophages attracted to the wound along chemotactic gradients are responsible for the removal of nonvital debris. Leukocytes precede macrophages by about 24 hours and provide an immediate defense against contamination, s Macrophages are derived from circulating blood monocytes and their delayed presentation represents the time taken to transform themselves into scavengers. Both leucocyte and macrophages share similar receptors. Therefore substances responsible for attracting one cell type may also attract the other, s Several factors present during the initial stages of tissue injury are chemotactic for leukocytes and macrophages. These include factors released during fibrin polymerization (platelet factor 4 and activated Csa, chemical mediators of inflammation (kallikrein) and bacterial phospholipids.9, 22-24 Fibroblasts appear at the wound site between 2 and 4 days after injury. 25 Even though they are pervasive throughout soft tissue, chemoattractants provide a high concentration of these cells where they are required most. TGF-[3 and PDGF, both secreted by macrophages and platelets, have been found to be chemotactic toward fibroblasts in vitro. 26 Other substances that attract fibroblasts in vitro include collagen and its constituents, as well as fibronectin. 27 It must be stressed that not all in vitro effects have been reproduced in vivo, and when a discrepancy occurs, alternative pathways must be responsible. The last Cells to enter a wound are endothelial cells. Recent investigations have shown that a macrophagederived substance, referred to as macrophage-derived angiogeneic activity (MDAA) exerts chemotactic ef-

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Phases of soft tissue wound repair

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fects on endothelial cells (see TNF-a later in review). 9 Wound healing depends on a sufficient supply of cells and cell products. Mitogenesis is the process by

which an adequate cell population is produced to fulfill the requirements of tissue repair. Growth factors are potent mitogens that cause cell division thereby promoting an increase in cell numbers.

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Effects of the environment and gradients on cellular activity The microenvironment within a wound determines and influences constituent cell types and their activities through the effects of different physical and chemical gradients. Chemical gradients affect wound healing by influencing the distribution of cell types, their responsiveness and activities, and by modulating their secretory products. For example, macrophages cultured in high lactate concentrations, approximating the level encountered in a wound, produce the angiogeneic agent bFGF, whereas macrophages incubated in pyruvate or at a low p H do not. 28 Different oxygen tensions between various areas of a wound also promote preferential cell localization. Furthest from the intact vasculature where the PO2 is low (9 to 10 m m Hg), macrophages, developing capillaries, and immature fibroblasts proliferate. In contrast, regions closer to the blood supply with higher oxygen concentrations of 40 to 80 m m Hg are associated with maturing vessels and collagensecreting fibroblasts.17 The consistent pattern of cells distributed within healing wounds prompted Hunt et al. 29 to coin the concept of a "healing module," in recognition of a fundamental unit of tissue repair comprising cells, their products, and the predictable timing of their appearance. The organization of a healing module is summarized in Fig. 3. Although the environment plays an important directional role in determining cell appearance and function, specific biologic mediators expressed by resident ceils guide the proper sequencing of cells as well as their numbers and these will be discussed next.

Growth factors in wound healing Within healing wounds, various proteins have been identified that exhibit special properties important to repair.30, 31 One family of proteins has been called growth factors even though their actions are not restricted to stimulation of growth. In fact, the terminology used to label growth factors is often misleading because the names were derived from tissues in which the factors were initially identified. An often cited example of the anachronistic manner in which growth factors are labeled is PDGF. Platelet-derived growth factor was originally identified in the o~ granules of platelets, but since then has also been found in macrophages, endothelial cells, and smooth muscle cells. 3a, 33 Many of these proteins are capable of both chemotactic and mitogenic functions. They are therefore important in determining both the sequence of cells in the "healing m o d u l e " and their respective functional activities. Several growth factors for epithelial repair are identical t o those responsible for hard tissue regeneration.

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Growth factor receptors Growth factors are believed to exert their specific effects through specific receptors present on the surfaces of target ceils. Receptors may be categorized as direct catalytic receptors and G-protein-coupled receptors. The method of receptor-ligand binding and subsequent cell stimulation is unknown. However, several models describing potential mechanisms have been proposed. 34, 3s It is known that receptors are composed of three parts: a cytoplasmic region, a hydrophobic transmembrane region, and an extracellular ligand-binding domain. One model for the activation of direct catalytic receptors (Fig. 8) suggests that occupation of a receptor site triggers intracellular reactions by the aggregation of separate receptors on the cell surfaceY Because receptors are connected to the intracellular domain through short chains of hydrophobic amino a c i d s Y a pathway is provided through which intracellular enzymes may be activated. Phosphorylation of intracellular serine, threonine, or tyrosine residues by protein kinases (tyrosine kinase) has been has been associated with activation of target cells. These intracellular reactions induce conformational changes in the cell. The process by which extracellular physical activities are convened to intracellular chemical changes is referred to as signal transduction. 36 Activation of G-protein-coupled receptors involves GTP-binding proteins called G proteins. These are present on the inner surfaces of target cells close to receptor sites. G proteins affect a variety of intracellular activities, including regulation of enzymes and ion channels within the cell m e m brane. 25 A third receptor-effector mechanism acts through a set of genes that form the cellular counterparts to viral oncogenes. 37'38 These are known as protooncogenes, and transcription of two genes, c-fos and c-myc, has been reported shortly after exposure of cells to PDGF. 39, 40 The exact function of these genes is unknown, but they are believed to prepare the cell for DNA synthesis and division. 38

Growth factor transmission Growth factors are ubiquitous throughout a wound and can be localized over the three phases of healing. 4~ Because their activity is affected by local concentration levels, the various modes of delivery from source to target become important. There are three ways in which growth factors are transported. Factors secreted into the blood and conveyed to a distant site use an endocrine mode of delivery. Insulin-like factors I and II are examples of this. 41"42 P D G F and TGF-oL and TGF-13 exert their effects through a paracrine mechanism in which the secretory product of one cell acts directly on another. Cell to

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4 CELL ME

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Fig. 8. Growth factors (GF) interact with specific receptors located on external surface of cell membrane causing them to aggregate. Because receptors are connected to intracellular domain by short-chain peptide links, this model proposes means by which growth factors may increase metabolic activities of cell as well as control formation of secretory products. Energy requirements for increased metabolism are provided by GTP. cell distances are therefore important for paracrine factors to act efficiently. Autocrine routes are also known, and these help cells perform self-regulatory functions. TGF-[3 is an example of an autocrine factor whose secretory cell is both the source and target of its activity. 43 Multiple methods of delivery are possible for certain factors with the use of paracrine. endocrine, or autocrine paths to modulate different functions through varying dose-response relationships.

Classification of growth factors according to Lemporal activity Throughout this description of wound healing, a recurrent feature has been the importance of timing for cells and enzymes to exert their influence. Growth factors fit into this scheme by the temporal nature of their activities. Depending on their period of activity, an alternative method of classifying growth factors has emerged. Factors that act on quiescent cells in the Go phase of the cell cycle have been termed competence factors. 44 Examples of these are PDGF and FGF. 45 Other factors are termed progression factors

and include the insulin-like growth factors and a macrophage-derived growth factor. These act later in the cell cycle during the S and G2 phases. This separation of activity creates the impression that factors are limited to fixed stages of cell development whereas in reality, changes in local conditions (e.g,, factor concentration) allow growth factors to act on different periods of cellular growth producing different effects. Timing of activity may have significant pharmacologic relevance as various growth factor therapies are developed to aid wound repair. Studies are currently underway to investigate the benefits of multiple growth factor administration to optimize interactions with cells progressing through different phases of the cell cycle. Other investigations are focusing on repeated dosing of the same growth factor at different periods. Appropriately timed growth factor combinations appear to successfully enhance wound repair. For instance, investigators have shown that combining PDGF and IGF-1 proved more beneficial for wound repair than either factor alone. 46, 47 Purportedly, PDGF promoted cell competence whereas

ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 82, Number 5 IGF-1 stimulated cell progression. This synergistic action helped amplify the respective healing processes.

SELECTED GROWTH FACTORS IN WOUND HEALING Several growth factors have been identified in wounds, and their functional roles have been investigated with both cellular and animal models. W e have chosen to discuss the best understood and most frequently assessed factors involved in the augmentation o f soft tissue repair. Although new activities are constantly being discovered for each o f the factors, some o f the more important features will be discussed. A summary of the pertinent properties o f several growth factors relevant to epithelial repair is presented in Table I and Table II.

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l a n e I. General biologic effects of growth factors Factor

Effects

References

EGF PDGF

General: chemotactic, mitogenic Binds to cell surface receptors: composed of extracellular, transmembrane, and intracellular domains

35 30

IGF FGF TGF-13 TGF-13

Platelet-derived growth factor (PDGF) P D G F is stored in the (x granules of platelets and released after activation of the platelets at sites o f tissue injury, as Macrophages, endothelial cells, vascular smooth muscle cells, and fibroblasts also express P D G F , which is one o f the most potent competence factors present in wounds and which exerts effects 9during the first two phases o f repair. It has been shown to be chemotactic for fibroblasts and monocytes as well as mitogenic for fibroblasts and vascular smooth muscle cells. 39, 48 The production o f P D G F at wound sites is not constant, and increases in concentration have been correlated with augmented connective tissue formation. P D G F acts through paracrine and autocrine mechanisms that enable it to function not only as a stimulator of cellular activity but also in a homeostatic feedback fashion. Stimulation o f the phosphorylative enzyme, tyrosine kinase, and increased transcription o f the proto-oncogenes c-fos and c - m y c have been observed with applications o f PDGF.49, 50

487

PDGF

basic FGF IGF-I EGF

Activationoccurs through tyrosine kinase enzyme system Activationthrough serine-threonine kinases Chemotactic:monocytes, neutrophils. T-cells, fibroblasts, macrophages; Mitogenic: fibroblasts, mesenchymal cells Neovascularization Accelerates collagen deposition Modulates PDGF, FGF, TNK, IL-1, enhances deposition ECM ECM: modulate growth factor activity; ECM: macromoleculesof fibronectin, proteoglycans, and collagens to which cells attach by way of cell surface receptors; integrins Chemotactic;fibroblasts, neutrophils, macrophages Mitogenic; fibroblasts, smooth muscle Activates macrophages Stimulates fibroblasts to secrete types I and III collagens In vitro: stimulates fibronectin, glycosaminoglycans, hyaluronic acid Chemotactic,mitogenic: fibroblasts, endothelial cells Promotesangiogenesis Chemotactic:endothelial ceils Mitogenic: fibroblasts Chemotactic,mitogenic: neuronal cells, epithelial cells neovascularization

61 62 63 55 64 39 63 65 66, 67

68 50 62 39 5, 69, 70 45 35 71 41 31, 35, 72

Transforming growth factor-13 TGF-o~ and TGF-13 were first isolated from tumors and n a m e d for their ability to transform normal cells into malignant phenotypes. 5t-53 TGF-13 has been identified in a wide variety o f cells including platelets, macrophages, bone cells, monocytes, lymphocytes, and platelets. 54'55 Commensurate with its diverse origins, this factor has both mitogenic effects as well as regulatory functions over matrix production. TGF-~3 is chemotactic for fibroblasts and monocytes and is capable o f stimulating or inhibiting fibroblasts. 56 In vivo, applications of TGF-13 promotes cellular influx into the w o u n d and increases synthesis of extracellular matrix proteins. 51-53 In ad-

dition, TGF-13 has been shown to stimulate anglogenesis through the induction o f two monocyterelated polypeptides, interleukin 1 and tumor necrosis factor-(x (llmcrophage-dependent angiogeneic activity). 9 Recently, recombinant D N A techniques have been applied to the study o f TGF-f3. The results indicate that several different proteins comprise the TGF-B superfamily. T w o of these, TGF-13 1 and TGF-[3 2, possess structural and chemical similarities to proteins o f the bone morphogenetic protein group.

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Table II. General clinical effects of growth factors on soft tissue repair Factor EGF

IGF-I

basic FGF

Effects

Reference

Rats: intravenous administration stimulates gastric mucosa Mouse: neuronal regeneration Rabbits: accelerates closure of full-thickness wounds Pigs: accelerates epidermal healing of partial thickness burns Dogs: promotes healing of gastric ulcers Human beings: accelerates donor site healing of partial thickness flaps Stimulates corneal epithelial regeneration Human beings: increases incisional wound tensile strength Rats (hypophysectomized): reverses corticosteroid impaired healing Rats: crush-injured, freeze-injured sciatic nerve; increases neuronal regeneration Pigs: combined with PDGF; increases connective tissue and epithelial differentiation Incisional wounds; accelerates healing Rats: placed into incisional wounds and into wound chambers; increases fibroblasts, macrophagesl angiogenesis diabetic mice: accelerates healing of full thickness cutaneous wounds

73

Source Macrophages

Fibroblasts

Macrophages

74 75 76, 77 78 79 80 79, 81-83 84 85, 86 87 88 89-91

Endothelial cells Fibroblasts

acidic FGF PDGF

TGF-I3

Platelets Endothelial cells Macrophages Monocytes Fibroblasts

Platelets Macrophages Monocytes Lymphocytes

Excisionai wounds: increases granulation tissue, vascularity, decreases collagen, increases epitheliazation Cats: nerve regeneration Diabetic animals: restores normal wound healing Diabetic mice: stimulates wound healing Rats: stimulates granulation tissue Rabbit ear model: accelerated wound closure Pigs: Combined with IGF-I-increased connective tissue and epithelial differentiation Human beings: pressure ulcers-decreased wound volume Human beings: epitheliazation of chronic wounds Rats: placed into incisional wounds and into wound chambers-promotes wound healing

39

92 93 94 1

39 87 95 96, 97 89, 98

Rats: reverses glucocorticoid-induced impaired wound healing

99

Rabbits: increased collagen proliferation in full-thickness skin incisions Guinea pigs: radiation-impaired healing of skin incisions improved Pigs: enhance excisional cutaneous wound repair General: enhances physical properties of wound healing; increases tensile strength and accelerates rate of healing Rats: decreases incisional wound healing strength

100

Fihroblasts

TNF-er

Macrophages Lymphocytes

Epidermal growth factor and TGF-ot These two factors are closely related (35% homology) and possess very similar activities. 57 Both have a molecular weight of 5,700 d and affect mesenchy-

101 102 43, 53, 103 104

mal and epithelial cells: They are derived from transmembrane proteins and act through a paracrine mechanism on the EGF receptor. 57 Wound macrophages contain significant amounts of TGF-oL that add

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to the significance of this cell in the initial tissue response to injury. The main effect of TGF-c~ and EGF appears to be on granulation tissue development with epidermal regrowth and modulation of angiogenesis being unique features of TGF-o~ activity. 58

Basic fibroblast growth factor bFGFs comprise a group of regulatory molecules with a high affinity for heparin. 45 Release of bFGF occurs through the actions of the enzyme heparitinase found in platelets that dissolve the heparin binding. FGFs are found in many different tissues including endothelial cells, macrophages, and fibroblasts and are both chemotactic toward endothelial cells and leukocytes as well as mitogenic for endothelial cells. As expected, bFGF plays a prominent rote in angiogenesis, initiating release of basement membrane-degrading enzymes that liberate endothelial cells before new vessel formation. 45

Tumor necrosis factor-e~ Macrophages can stimulate angiogenesis by expressing TNF-o~, a substance first associated with the necrosis and regression of certain solid tumors. 59 This direct activity, as opposed to the indirect effects of PDGF, was originally assigned to a factor named macrophagederived angiogeneic activity (MDAA). Recent investigations have identified this factor as the same a s TNF-oL. 59

THERAPEUTIC APPLICATIONS OF GROWTH FACTOR TECHNOLOGY IN SOFT TISSUE HEALING The dedicated efforts of both laboratory scientists and clinicians to identify and characterize the different proteins that influence soft tissue healing has been encouraged to a significant degree by the potential for this knowledge to provide the means for augmenting healing in both normal and problematic wounds. However, early hopes for enhanced wound healing through the simple application of topical growth factors have been tempered by inconsistent results and concerns over malignant transformation of treated cells. Several examples of some of the more promising applications of growth factor technology are provided to illustrate both the challenges as well as future directions of exogenous growth factor therapy.

Single factor therapy Some of the earliest attempts at augmented soft tissue healing focused on the effects of topical application o f factors on different types of cutaneous wounds. Experimental partial-thickness burns and skin graft donor sites created in animals epithelialized more rapidly after treatment with EGF. 105 EGF was also capable of increasing the tensile strength of incision wounds in rats 81 and promoting corneal epi-

thelial regeneration in primates. 1~ Successful results have also been reported in human trials. In human beings, split-thickness skin graft donor sites 79 as well as chronic wounds refractory to conventional therapy82 were shown to heal more rapidly after applications of silver sulfadiazine containing EGF (10 lag per ml). Human corneal epithelium was also reported to regenerate faster when treated with EGF.I~ Although accelerated epithetialization after EGF application may be explained by the stimulation of granulation tissue formation, 5s not all growth factors improve healing rates despite their observed in vitro activity. PDGF was described earlier in this article as one of the most potent competence factors present in wounds with significant chemotactic and mitogenic effects on wound healing cells. However, wounds treated with PDGF alone do not demonstrate significant increases in epithelial regeneration. 46 The failure of PDGF to accelerate wound healing has prompted investigations into issues related to local concentrations of growth factors, timing of factor activity, and the interaction of several different factors in a synergistic fashion to benefit healing. Growth factors have also found applications outside of wound healing. Much interest has recently been shown with the anabolic and protein-sparing actions of systemically administered IGF-1 in the treatment of catabolic diseases such as renal failure, burns, malnutrition, or long-term steroid therapy. IGF-1 is also undergoing trials as a substitute for growth hormone to promote linear growth in children with short stature, l~

Combination factor applications A synergistic relationship appears to be necessary between certain growth factors before the expression of a particular function. For instance, a combination of PDGF and IGF- 1 significantly increases the width of epidermis and dermis in regenerating wounds, whereas PDGF alone does not produce appreciable results. 46 Similar benefits are also seen when PDGF is combined with EGF. Short-term applications of gels containing a combination of PDGF and IGF-1 have been found to promote the regeneration of a periodontal attachment in the early phases of healing. 1~ How different proteins interact to improve healing is still unknown, but mechanisms currently under investigation include receptor modulation, upregulation, or downregulation of factor activity, and stimulation of peptide production or release through paracrine or endocrine processes.

Antigrowth factor therapy Characterization of growth factor activity has identified not only those agents that stimulate positive healing effects but also factors associated with wound-

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healing problems. This knowledge has been applied to the development of a class of growth factor antagonists that attempt to diminish the nonbeneficial components of healing by blocking specific factor activity. The principles of this new therapeutic approach are illustrated by examining the role of TGF-[3 and tissue fibrosis. The role of TGF-[3 in the initiation and termination of tissue repair has been well established. However, its sustained activity has also been implicated in the production of pathologic tissue fibrosis. 63 Various categories of TGF-[3 blocking agents including TGF-[3 antiserum isoforms of TGF-[3 that competitively block receptor activity and posttranscriptional gene regulators to decrease TGF-[3 production are currently being tested for their effectiveness at reducing tissue fibrosis. In different experimental models, these "antifibrotic" agents have been effective in reducing the collagen content of dermal wounds, decreasing the amount of scar tissue in brain injury, reducing the amount ofinflammation and synovitis in arthritic joints, and suppressing matrix accumulation that is responsible for vascular intimal hyperplasia. 63 CONCLUSION Patients with problems associated with poor or delayed wound healing pose significant challenges to our understanding of healing mechanisms. Extensive research in this area has already provided benefits through treatments designed to augment deficient processes. Examples of these advances are found in the use of hyperbaric oxygen therapy, nutritional support, and specialized dressing materials that optimize the wound environment. Advances in cell and molecular biology have identified cells and their biologic products that regulate soft tissue repair. Recombinant engineering techniques have made available pharmacologic quantities of wound rePair modulators for clinical assessments. Practitioners may be asked in the future to incorporate the use of these substances in their treatment. Applications of these powerful molecules will have unparalleled effects on the practice of dentistry. By reviewing the mechanisms involved in soft tissue healing, including specific growth factors, potential applications, putative mechanisms, and the cellular interactions that promote soft tissue repair, we have attempted to provide a basis for individuals to appreciate the rationale behind the use of healing supplements and to judge the merits of specific technologies themselves. REFERENCES 1. Steenfos HH. Growth factors and wound healing. Scand J Plast Reconstr Surg Hand Surg 1994;28:95-105.

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