Periodontology 2000, Vol.

22, 2000, 44–50 Printed in Denmark ¡ All rights reserved

Copyright C Munksgaard 2000

ISSN 0906-6713

Biology of wound healing
Periodontal tissues represent a unique system in the human body where epithelial, soft and mineralized connective tissues come together to form a junction. This junction, referred to as the dentogingival junction, is a complex structure, and maintenance of the integrity of this junction is critical for the preservation of underlying bone and periodontal ligament. Unfortunately, with chronic inflammation associated with periodontal diseases, the structure of this junction is lost. Hence, attempts to control the destructive effects of chronic periodontal diseases and to some extent regenerate the lost tissues would require the re-establishment of a dentogingival junction. Conventional periodontal therapy, be it surgical or nonsurgical in nature, usually involves instrumentation in the inflamed dentogingival complex. Such therapies result in wounding of the already inflamed periodontal tissues. Thus, the consequence of such therapeutic procedures depends largely on the cellular and molecular events associated with wound healing. Many of the cellular and molecular events in healing of periodontal wounds are similar to those seen in wounds elsewhere in the body except that, in the periodontal wounds, there is a mineralized tissue interface at the junction of epithelium and connective tissue. Immediately following periodontal surgical procedures, the tissues represent surgically wounded sites and a cascade of cellular and molecular events set in to initiate wound repair. Over the past two decades, numerous advances have been made in understanding the biology of wound healing. At the same time, advances in the biology of the periodontal tissues have led to the development of surgical procedures that facilitate regeneration of the periodontal tissues lost because of chronic inflammation. The newly described principle of guided tissue regeneration (2) for the surgical treatment of periodontal lesions represents a classic example of how basic knowledge of cell biology could be applied to enhance the clinical outcomes of therapy. The purpose of this chapter is to examine some of the current basic concepts of wound healing and is based on two excellent reviews on wound healing that the readers are referred to for more details (10, 21). Since the biological principles of wound repair would almost be the same, no distinction will be made between the healing in different types of periodontal defects. It is hoped that this review will allow the extrapolation of some of the basic principles of wound healing to the various periodontal surgical wounds. As per the classic description of wound healing, initially there is temporary repair characterized by the formation of a clot in the wounded tissues. Inflammatory cells followed by fibroblasts and endothelial cells then invade the clot to form a granulation tissue, while the epithelial cells migrate to cover the denuded surfaces (or form a junction at the tooth interface). Finally, maturation of the healing tissue matrix is seen along with contraction or scarring. It is important to mention that these various phases of wound healing overlap somewhat in time. This oversimplified explanation of wound healing basically summarizes the events at large, but numerous studies in the last two decades have shed light on some of the important cellular and molecular mechanisms. It is hoped that this new knowledge would some day allow therapeutic manipulation to enhance wound healing.

The fibrin clot and inflammatory cells
Injury to the blood vessels during surgery/periodontal therapeutic procedure causes extravasation of blood. A fibrin-rich clot formed by blood coagulation and platelet aggregation plugs the cut blood vessels and also serves to protect the denuded tissues temporarily (10). The clot itself consists of platelets within a network of cross-linked fibrin fibers along with plasma fibronectin, vitronectin and thrombospondin (21). Among the important functions of the clot are its role as a reservoir of growth factors and cytokines that are released by the de-


Biology of wound healing

granulation of activated platelets and serving as a provisional matrix for cell migration. The growth factors and cytokines present in the fibrin clot in fact might be providing the start signals for wound repair. At first, there is recruitment of inflammatory cells to the wound site followed by epithelialization, granulation tissue formation and angiogenesis. Neutrophils and monocytes are usually recruited initially by the signals present in the clot. The neutrophils cleanse the wound of foreign particles and debris and bacteria. It is important to note that neutrophils remove the bacterial debris through the release of enzymes and toxic oxygen products (10). Hence, increasing numbers of contaminating bacteria in the wound increase the potential for neutrophil-mediated tissue destruction. Neutrophils also serve as a source of pro-inflammatory cytokines providing signals that activate adjacent fibroblasts and keratinocytes (17). Neutrophil infiltration ceases after a few days, and they are eventually phagocytosed either by macrophages or fibroblasts (10). Peripheral blood monocytes that continue to be recruited into the wound site become macrophages upon activation. Fibrin along with fibronectin in the clot acts as a provisional matrix for the influx of monocytes and fibroblasts (5). Macrophages continue the task of phagocytosing bacterial, cellular and matrix debris in the wound. Growth factors and cytokines are continually synthesized and secreted into the wound environment by macrophages. Thus, the wound repair signals initiated by degranulating platelets and neutrophils are maintained by macrophages.

Table 1. The integrin family of adhesion receptors
Integrins a, b; a2b1 a3b1 a4b1 a5b1 a6b1; a7b1; a6b4 a8b1 a9b1 avb1; avb5 avb3; a11bb3 Ligands Fibrillar collagen, laminin Fibronectin, entactin, epilgrin, laminin, denatured collagen Fibronectin, VCAM-1 Fibronectin (RGD) Laminin Fibronectin, vitronectin Tenascin Fibronectin, vitronectin Vitronectin (RGD); fibronectin, fibrinogen, von Willenbrand factor, thrombospondin, denatured collagen Fibronectin, tenascin Fibronectin (IIIcs) Vitronectin Factor X, fibrinogen Fibrinogen

avb6 a4b7 avb8 aMb2 aXb2

Re-epithelialization of wounds
In the normal gingival tissues, the basal layer of epithelium is attached to the basal lamina. The keratinocytes use receptors on their surface, known as integrins, to bind to laminin in the basal lamina. Integrins are a family of cell adhesion receptors that mediate cell surface interactions with extracellular matrix and in some cases with other cells (19, 24, 32). There are approximately 20 members of this family of cell surface receptors, and each integrin is a heterodimer made up of one a and one b subunit in a non-covalent complex (33). The current list of the combinations of various integrins and their respective biological ligands is presented in Table 1. Changes in the combinations of the various a and b subunits offer the integrins specificity for the ligand. Since migration of cells is fundamental to wound healing, it is important to

consider changes in the expression of integrins by cells migrating into the wound. In the normal tissues, keratinocytes use the integrins a6b4 to bind to laminin in the basal lamina, and these integrins have intracellular links with the keratin cytoskeletal network (21). In preparation for migration, the keratinocytes at the edge of the surgical wound have to dissolve the hemidesmosome attachment and begin to express other integrins that are more suitable for the wound environment (Fig. 1). The migrating keratinocytes will start expressing the integrins a5b1 and aVb6 (for binding to fibronectin and tenascin respectively), the integrins aVb5 for binding to vitronectin and, finally, reorganize the distribution of the integrins a2b1 (collagen receptor). This mobilization and expression of new integrins facilitates adhesion of keratinocytes to matrix molecules in the provisional matrix as well as the adjacent wound debris. This can explain the lag time between wounding and the initiation of epithelial migration. It is generally believed that the basal cells provide the majority of migrating keratinocytes, although there is some evidence that some of the suprabasal cells may also migrate (13). Once the migration of epithelial cells has begun, cells of the basal layer small distance away from the wound edge undergo proliferation, providing an extra source of basal cells. The apical-basal polarity of the basal cell layer at the wound edge is lost, and instead pseudopodia are seen extending from their free basolateral sides into the wound. Not much is known about the mechanisms that drive the epithelial cell migration, but candidates include chemotactic factors, active



Fig. 1. Diagrammatic illustration of some of the major events during healing of dermal wounds and periodontal surgical wounds. a. The dermal wound. b. A healing periodontal wound with infrabony defects. Zones A represent the borders of the wound where the epithelium (basal layer) will migrate into the wound during epithelialization. At this edge, the cells will have to dissolve the hemidesmosome attachment, downregulate the expression of integrin receptors a6b4 (used to bind to laminin), and upregulate integrin receptors a5b1, aVb6 and aVb5 that are suitable for adhesion to provisional matrix components. Growth factors epidermal growth factor, transforming growth factor-a, heparin-binding epidermal growth factor and keratinocyte growth factor are involved in stimulating the proliferation of the epithelial cells here. Zone B initially represents the fibrin clot consisting of platelets within a network of cross-linked fibrin fibers along with

plasma fibronectin, vitronectin and thrombospondin. This fibrin clot also serves as a reservoir for many of the growth factors listed in Table 2. This is followed by recruitment of inflammatory cells in zone B, resulting in the phagocytosing of debris and bacteria. Again zone B serves as a reservoir of growth factors and cytokines. Zones C represent the borders of the wounds formed by the connective tissue. Here, fibroblasts and endothelial cells show proliferation in response to specific growth signals they receive as wound healing progresses. Matrix degradation is seen in zones B and C in preparation for the migration of these cells into zone B. Finally, granulation tissue forms in zone B followed by contraction of the wound and matrix remodeling. In the case of periodontal wounds, the apical part of zone B may be populated by cells originating from bone and periodontal ligament, while the more coronal part of zone B may get epithelialized.

contact guidance, absence of neighboring cells or a combination of the above (10). Interestingly, epithelial cell migration does not appear to depend on cell proliferation since molecules such as transforming growth factor-b, despite being a potent inhibitor of keratinocyte proliferation, promote the migration of epithelial cells in organ cultures (10). Once re-epithelialization is complete, the components of basal lamina are deposited in a sequential manner starting from the wound margin and the epithelial cells revert to their normal phenotype. Several growth factors seem to be key players in regulating the proliferation of keratinocytes in healing wounds. Among the key growth factors are the

epidermal growth factor, transforming growth factora, heparin-binding epidermal growth factor and keratinocyte growth factor. A list of growth factors active in the healing wounds, their possible sources and biological effects are presented in Table 2.

Matrix degradation and the wound-cleaning process
Migration of epithelial cells through the fibrin clot or along the junction between the clot and underlying connective tissue (mucoperiosteal flap surface in the case of periodontal surgery) requires the creation of


Biology of wound healing

Table 2. Growth factors involved in wound healing
Growth factor Fibroblast growth factors 1, 2 and 4 Transforming growth factor-a Transforming growth factors b1 and b2 Source Macrophages, endothelial cells Macrophages, keratinocytes Platelets, macrophages Effect Fibroblast proliferation and angiogenesis Re-epithelialization Fibroblast and macrophage chemotaxis; extracellular matrix synthesis; secretion of protease inhibitors Re-epithelialization Fibroblast and macrophage chemotaxis, fibroblast proliferation, and matrix synthesis Keratinocyte proliferation Endothelial and fibroblast proliferation Angiogenesis Activate growth factor expression in macrophages, keratinocytes and fibroblasts Activate growth factor expression in macrophages, keratinocytes and fibroblasts

Epidermal growth factor Platelet-derived growth factor (isoforms AA, AB and BB) Keratinocyte growth factor Insulin-like growth factor Vascular endothelial growth factor Interleukin 1a and b Tumor necrosis factor-a

Platelets Platelets, macrophages, keratinocytes Dermal fibroblasts Plasma, platelets Keratinocytes, macrophages Neutrophils Neutrophils

a migrating path. This is achieved by the dissolution of the fibrin barrier by the enzyme plasmin that is derived from the activation of plasminogen in the clot. The two activators, tissue-type plasminogen activator and urokinase-type plasminogen activator along with its receptor, are upregulated in the migrating keratinocytes (21). The importance of plasminogen in wound re-epithelialization is demonstrated by the lack of re-epithelialization of wounds in mice where the plasminogen gene has been knocked out (21). In addition to the fibrinolytic enzyme plasmin that causes lysis of fibrin, several other proteases are also expressed to clear the path for cell migration. Matrix metalloproteinases (MMP) are a family of enzymes with each member of the family specifically cleaving a subset of matrix proteins. MMP-1 (also known as interstitial collagenase) is secreted by those basal cells that have gone past the free edge of the basal lamina. MMP-1 degrades native collagens and aids cell migration by destroying collagens I and III. Similarly, MMP-9 (also known as gelatinase B) can cleave the collagen in basal lamina (type IV) and the collagen that forms the anchoring fibrils (type VII), thereby releasing the basal cells from their adhesion to the basal lamina. MMP10 (also known as stromelysin-2) is also expressed in wounds and is thought to have a wide spectrum of substrate specificity (21).

Granulation tissue and contraction of the wound
Granulation tissue formation usually can begin around day 4 after the wounding and consists

mainly of new capillaries, macrophages, fibroblasts and some loose connective tissue. Needless to say, granulation tissue is a complex reservoir of cytokines possessing chemoattractive, mitogenic and other regulatory activities (21). Growth factors are also present in the granulation tissue and, at this stage of healing, are derived mostly from macrophages. Depending on the stage of the granulation tissue and the predominant cytokines, numerous activities are seen occurring such as cell proliferation, chemotaxis and phenotypic expression of cells (27). It is, however, important to note that the activities of certain cell types and the nature of the extracellular matrix environment influence to a large extent the final phenotype expression by the cells. During the formation of granulation tissue, macrophages, fibroblasts and new blood vessels grow into the wound space in a coordinated manner (Fig. 1). Their interdependence is illustrated by the release of cytokines by macrophages that stimulate fibroblasts to synthesize an extracellular matrix. This extracellular matrix serves to support cell and vascular in-growth carrying nutrients to sustain the cellular functions (21). The term fibroplasia is used to refer to that part of the granulation tissue made up of predominantly fibroblasts and extracellular matrix they make. In addition to macrophages, fibroblasts themselves express many cytokines to which they can also respond in an autocrine manner (27). Cytokines induce proliferation of fibroblasts and regulate the production of extracellular matrix. Similarly, growth factors are also involved in wound fibroplasia. Wounds supplemented with purified growth factors show accelerated granulation tissue formation (20, 28).



The extracellular matrix and fibroblasts function in a reciprocal manner during wound healing. Components of the extracellular matrix such as fibronectin and collagen facilitate the adhesion and migration of fibroblasts in the granulation tissue. Fibroblasts synthesize and decorate the extracellular matrix, and in turn the extracellular matrix regulates gene expression and the general behavior of fibroblasts (10). Fibroblasts adhere to fibronectin, collagen and vitronectin via the integrin receptors listed in Table 1. It is important to note that fibroblasts, like the keratinocytes, have to rearrange their integrin expression profiles in preparation for migration. In the normal tissues, fibroblasts reside in collagenrich matrices. In response to wounding, the fibroblasts in the vicinity of the wound have to downregulate the integrin receptors for collagen and upregulate those needed for adhesion to components of the provisional matrix such as fibrin, fibronectin and vitronectin. The ability of fibroblasts to respond to signals from the extracellular environment is remarkable. For example, when simultaneously challenged by signals from both the provisional matrix (fibrin or fibronectin) as well as growth factors (such as platelet-derived growth factor), fibroblasts respond by upregulating the receptors for provisional matrix components. However, when challenged by the same growth factor (platelet-derived growth factor) in the presence of a collagenous matrix, fibroblasts respond by upregulating the receptors for collagen and not the provisional matrix receptors (31). Not much is known about the effects of extracellular matrix molecules in the wound environment on the regulation of gene expression in fibroblasts. This is complicated by the fact that some of the extracellular matrix molecules occur in different isoforms due to alternative splicing of their transcripts at the pre– messenger RNA level. For example, the well-known molecule fibronectin occurs as different isoforms due to the splicing in or out of the alternatively spliced domains EIIIB and EIIIA in its gene structure. While the fibronectin from plasma does not contain either of the two spliced domains, the fibronectin synthesized by fibroblasts and macrophages in wounds includes the spliced domains EIIIB and EIIIA. Remarkably, the EIIIB and EIIIA inclusive isoform of fibronectin seen in healing wounds is the predominant form of fibronectin in embryonic tissues where cell migration is active (11). The advantages of including the spliced domains EIIIB and EIIIA in fibronectin during embryonic development and wound healing are not clear. Recent studies have suggested that inclusion of these domains in the

fibronectin molecule may be facilitating the migration of cells by modulating the adhesiveness of the substrata (8, 15). Another spliced domain of fibronectin, the CS-III domain, also may be involved in the migration of cells in wounds. Fibroblasts use the integrin receptor a4b1 (Table 1) to bind to the CS-III domain of fibronectin. Interestingly, it has been shown in cell culture studies that the expression of the integrin a4b1 facilitates migration, while the classic fibronectin receptor a5b1 that binds to the RGD sequence can retard movement (7, 14). Fibroblasts in healing wound have been known to over-express the spliced domain CS-III (5, 11). Once cell migration into wounds is completed, fibroblasts show an increased expression of the classic a5b1 integrin (29). Fibroblasts in wounds also use vitronectin and fibrin directly as substrata for adhesion during migration. This is facilitated by the availability of cell membrane receptors for these matrix proteins (Table 1). Finally, the migration of fibroblasts in wounds is also stimulated indirectly by growth factors such as platelet-derived growth factor and transforming growth factor-b by upregulating some of the integrin receptors mentioned above (1, 12). As wound healing progresses, the provisional matrix is replaced by a new, collagen-rich matrix synthesized by fibroblasts migrating into the wound. The synthesis of specific extracellular matrix molecules by fibroblasts in the wound is regulated by transforming growth factor-b1 and some other growth factors listed in Table 2 (10, 21). Cytokines such as interleukin-4 can also induce expression of collagenous matrices in wounds (23). Once the required amount of collagenous matrix is synthesized, the signals that induce down-regulation of collagen synthesis are not clearly known. It is possible that there may exist some kind of regulation by the collagen matrix itself (9). Around 7–10 days after wounding, some of the fibroblasts in the wound transform into myofibroblasts and express a-smooth muscle actin. Such transformation allows these myofibroblasts to generate strong contractile forces that is responsible for wound contraction (21). In the final stages of fibroplasia, the number of fibroblasts and myofibroblasts in the healing wound are decreased by programmed cell death. Compared with wounds in the adult tissues, embryonic wounds heal without much contraction and scarring. In the embryos, there is no conversion of fibroblasts to myofibroblasts and the angiogenic response is also of a lesser magnitude. Compared with wounds in adult tissues, wounds in embryos show low but transient


Biology of wound healing

levels of expression of transforming growth factor-b1 (30) and since transforming growth factor-b1 has been implicated in pathofibrotic conditions, this can explain the tendency for adult wounds to show scarring and contraction. Antibodies that neutralize the effects of transforming growth factor-b1 in healing wounds have been shown to reduce scarring when delivered to wounds (26).

Angiogenesis of wounds
Angiogenesis refers to the formation of new blood vessels and is a crucial event during the healing of wounds. The term granulation tissue in wounds was coined in reference to the red granular appearance of new blood vessels that invade the healing connective tissues. As listed in Table 2, several growth factors are important in the induction of angiogenesis in healing wounds. Fibroblast growth factor-2 is synthesized by macrophages and damaged endothelial cells, while vascular endothelial growth factor is induced in wound-edge keratinocytes and macrophages. Experiments where fibroblast growth factor2 is depleted show blockage of wound angiogenesis (3). As in the case of fibroblasts and keratinocytes, the endothelial cells also have to upregulate specific integrins (aVb3) on their surface in order to respond to angiogenic signals. All the signals that induce proliferation, migration and phenotype expression in endothelial cells have not been completely understood yet. It is clear that angiogenesis is a complex process and relies on the availability of an appropriate matrix in the wound. As endothelial cells migrate into the provisional matrix, they form tubes surrounded by their own provisional matrix initially followed by a true basement membrane. As in the case of fibroblasts, endothelial cells involved with angiogenesis in wounds also undergo programmed cell death during the ultimate maturation of the matrix characterized by regression of capillaries.

Applied clinical aspects and future approaches to enhance wound healing
With the explosion in knowledge of growth factors, cell adhesion molecules and cytokines in the last two decades, understanding of the cellular and molecular biology of wound healing has improved significantly. Some of this new knowledge is already being

applied to manipulate the process of wound healing. For example, application of epidermal growth factor and transforming growth factor-a to burn wounds in animal models has been shown to enhance reepithelialization (4, 25). Similarly, application of keratinocyte growth factor to skin wounds has been shown to have mitogenic effects on the healing epithelium (22). Since cell adhesion and migration are essential to wound healing, attempts have been made in the past to use topical application of fibronectin in periodontal wound healing (18). However, using recombinant molecules as exogenous sources to supplement wound healing has to be approached with caution. For example, in the case of fibronectin, the plasma form of fibronectin (without spliced domains EIIIB and EIIIA) is already available in large quantities in the provisional matrix of a healing wound. Supplementing the same has no biological rationale and enhanced effects should not be expected. The biological functions of the spliced domains EIIIB, EIIIA and the CS-III segments are not known. Should their roles be specific in enhancing cell migration (15), specific isoforms of cell adhesion molecules that allow weak cell adhesion but favor migration of cells would be useful, and studies to this effect are currently in progress. The provisional matrix is rich in growth factors in the normal, healthy subjects. Systemic conditions such as diabetes may be accompanied by reduction in the availability of some of the growth factors; in these cases, supplementing the appropriate growth factor may be beneficial. Since the half-life for some of the growth factors may be short, supplementing wounds with the appropriate growth factors during the later stages of healing via some sort of delayed release mechanism could be useful in the healing of wounds in healthy subjects as well. Preliminary data on the use of recombinant growth factors during periodontal surgery to treat osseous defects shows promising results (16). Future research will have to be directed towards understanding in more detail the molecular mechanisms of differential gene expression in healing wounds. Wound healing is achieved by a series of coordinated efforts by inflammatory cells, keratinocytes, fibroblasts and endothelial cells responding to a complex array of signals. Many of these events have start and stop signals. A thorough understanding of these signals and their consequences (differential gene expression) in responder cells should make possible, hopefully in the near future, therapeutic manipulation of wounds that leads to real regeneration of the damaged tissues.



This work was supported in part by a grant from the National Institutes of Health (DE-07801). I thank Donna Yeager for assistance in the preparation of this manuscript.

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