Periodontology 2000, Vol.

24, 2000, 127152 Printed in Denmark All rights reserved

Copyright C Munksgaard 2000

PERIODONTOLOGY 2000
ISSN 0906-6713

Cell biology of gingival wound healing

L ARI H A KKINEN, V ELI- J UKKA U ITTO & H ANNU L ARJAVA

An overview
Wound healing is a critical process for the organism. The attempt of this chapter is to critically summarize the recent advancements in the wound-healing process. Because of the excessive amount of information (search with the key words wound healing produced 2052 citations within the last 12 months), we will focus on two major events in wound healing, namely re-epithelialization and granulation tissue formation, leaving out many other key events such as clot formation and angiogenesis. At the end, we will also discuss the special features of wound healing in oral cavity. It is commonly stated that oral wounds heal better than other wounds such as dermal wounds. We will explore evidence supporting this assumption. Most of the data presented in this chapter refer to studies with gingival and dermal full-thickness wounds and no special attempts have been made to focus on the healing in tooth-gingiva interphase because only a few studies are available to report special molecular events in humans. In addition, several previous articles have addressed periodontal wound healing and regeneration in great detail (114, 276). It is reasonable to assume, however, that the basic cell biological events of wound healing will follow the same principles at the tooth-gingiva interphase, at least at the supracrestal locations. We will also try to make several back-and-forth citations between in vivo and in vitro studies. This is necessary because functional studies have been difcult to perform with animal models of wound healing in a manner that would allow meaningful conclusions to be drawn. In vitro studies with cultured keratinocytes and broblasts are still the only way to explore functions of various molecules. Recent molecular biology techniques have made it possible to eliminate the expression of molecules of interest and investigate how the function is changed consequently. In vivo studies have dealt with immunolocalization of

extracellular molecules and their expression using in situ hybridization. In addition, transgenic animals provide information how wound healing is altered in the absence or following overexpression of a known molecule (153). Many of these studies have provided new critical information but others have resulted in confusing results, such as showing no alterations even though a molecule has been thought to be critically important for wound healing. It is believed that in these cases there are other related proteins that can provide similar functions and compensate for the absence of the studied molecule. Wound healing has become well protected during evolution because of its critical importance. Numerous molecules appear to overlap each others functions. During wound healing, several molecules that are usually present only during embryonal development are found in the granulation tissue. Epithelial cells start to express extracellular matrix receptors that are not normally present in the resting epithelium. Fibroblastic cells with a special phenotype are found in the healing granulation tissue. Where they come from is still largery unknown but possible sources will be discussed in this chapter. Expression of various proteins by the resident cells is also inuenced by the new environment. For example, in broblasts the expression of hundreds of genes is altered by the exposure to serum alone. Proteolytic enzymes release growth factors from the wounded basement membrane and connective tissue matrix. Matrix degradation produces biologically active peptides from the matrix proteins that could have specic functions in tissue repair. Clarication of the complex interplay between new matrix, growth factors, matrix degradation products and cells during wound healing will be a challenging task. This review focuses on re-epithelialization and granulation tissue formation with the special emphasis on the integrins, since cell adhesion serves as a foundation for cell migration, matrix turnover and differentiation during wound healing.

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Integrins
Integrins play key roles in re-epithelialization and granulation tissue formation during wound healing through their function in cell adhesion and signaling. A brief overview is, therefore, presented describing the structure and function of integrins that we hope will help the reader to better understand how they participate in the regulation of wound healing. Integrins are cell surfaceassociated dimeric glycoproteins that function as cell-to-extracellular matrix adhesion receptors (4, 16, 160). Through binding to the extracellular matrix proteins integrins mediate information transfer from the extracellular matrix to the cell interior leading to alterations in cell functions and ultimately in cell behaviour (100, 160). Integrins are known to play an important role in regulating a wide range of cell functions during growth, development, differentiation, and immune response (4). Integrins are composed of a single a and a single b subunit that are non-covalently linked to each other. At least 17 different a and 8 b subunits are currently known. These subunits can variously combine to form more than 23 different cell surface receptors that have distinct ligand-binding specicities. Both a and b subunits are transmembrane glycoproteins that cooperate in integrin binding to ligands. Ligand binding causes clustering of the receptors, which leads to cytoskeletal organization and signaling (Fig. 1) (160). Integrin-binding sites are targets for therapeutic applications aiming at blocking integrin function. Domain crystal structures are

being used for designing new molecules that have high afnity to the binding site but are irrelevant to the original peptide sequence. Commercial products are already available for cardiovascular applications (146). Several other compounds are in clinical trials and many other similar products will follow and may be used for periodontal applications in the future. The number of proteins known to associate with integrins is rapidly increasing (96). Integrin associated protein, transmembrane-4 superfamily, growth factor receptors and urokinase-type plasminogen activator receptor appear to have a regulatory function on integrins. Growth factor receptors accumulate in the same structures as integrins and regulate integrin functions (Fig. 1) (161). In the context of wound healing, the synergy between integrin and growth factor receptors is probably a key process in the regulation of cell proliferation (100). Integrins avb1 and avb6 can also bind growth factors such as transforming growth factor b1 through its latency-activated peptide that contains an Arg-Gly-Asp (RGD) peptide sequence (166, 167). It has recently been shown that epithelial cells can bind and also activate transforming growth factor b1 through avb6 integrin (Fig. 1); this mechanism may play an important role in the connective tissue bridge formation underneath the epithelium that has covered the wound. It is evident that the epithelium has a much more active role in the regulation of connective tissue formation than previously assumed.

Epithelial wound healing

Re-epithelialization Wound healing is a complex phenomenon that involves series of controlled events including the formation of a provisional extracellular matrix that is mainly composed of brin, bronectin and vitronectin and the migration of epithelial cells from the edges of the wound (33, 108). After the epithelium has been disrupted by tissue injury, re-epithelialization must occur as rapidly as possible in order to re-establish tissue integrity. Keratinocytes start moving into the defect about 24 hours after the injury (277). Epithelial cells from residual epithelial structures dissolve their hemidesmosomal connections and detach from basement membrane and move quickly across the wound defect. Later, as the re-epithelialization proceeds, the proliferation of keratinocytes that feed the advancing epithelial edge becomes more important. It is still somewhat unclear which cells in the epithelium rst move into the wound.

Fig. 1. Integrins and cell signalling. Integrins mediate cell adhesion to extracellular matrix proteins (ECM) leading to organization of cytoskeleton and signaling. Integrins also collaborate with growth factors in regulation of cell growth. Some integrins can also directly activate growth factors such as transforming growth factor b1 (TGFb1). Proteolytic enzymes are also found in cell adhesion sites allowing cells to detach and subsequently migrate.

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There is some evidence that the suprabasal keratinocytes are the rst migratory cells sliding over the basal keratinocytes. Epithelial cell migration on the exposed connective tissue matrix underneath the brin-bronectin clot has been commonly described. In small gingival wounds (Fig. 2), however, the keratinocytes cut their way directly through the clot and may not interact with the connective tissue matrix at all (132). Migrating keratinocytes are highly phagocytic, allowing them to penetrate through tissue debris or the clot (277). Degradation of the brin clot appears to be critical for wound healing, since wounds in animals that lack the plasminogen gene do not re-epithelialize (199). It seems likely that integrins play a role in the brin clot removal. Migrating cells must be able to focalize the proteolysis into the leading edge of epithelium (227). This could be done by activation of proteolytic enzymes at specic sites at the cell membrane. It has been found that urokinase type plasminogen activator receptor is able to associate with integrins (283). This is an example how a cell is able to focalize brinolysis by plasmin and promote subsequent migration by integrins. Modulation of other matrix componets may

also be necessary. Migrating keratinocytes express matrix metalloprotease-9 (type IV collagenase), matrix metalloprotease-1 (interstitial collagenase) and matrix metalloprotease-10 (stromelysin), which all may be required if the cells encounter the exposed matrix (207, 210, 266). Blocking matrix metalloprotease activity prevents keratinocyte migration into the wounds in cell culture (147). This proteolytic modulation of the matrix underneath migrating cells must be well controlled, since overexpression of matrix metalloproteases is a common nding in nonhealing chronic wounds (187). In chronic inammation, matrix metalloproteases such as matrix metalloprotease-9 produced by keratinocytes may bind to the cell surface and into the extracellular matrix resulting in a delayed clearance (147). Thus, regulation of matrix metalloproteases and their inhibitors must be well balanced for normal wound healing (266). During wound healing, keratinocytes function to rapidly cover the exposed connective tissue (236, 277). This process depends upon a variety of interactions between cells and the extracellular matrix. As the basal keratinocytes at the wound margin are exposed to the new provisional matrix, the phenotype

Fig. 2. Stages of healing of full thickness gingival wounds (about 3 mm deep and 2 mm wide). FC: brin clot; GT: granulation tissue.

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of the cells is changed from stationary to migratory (176). In this process, keratinocytes detach themselves from the basement membrane, migrate laterally into the wound bed and nally regenerate the basement membrane (124). In mucosa and in skin, the migration seems to involve similar patterns, although the source of migrating epithelial cells is different. In the mucosa, the basal layers of the wound edge epithelium serves as a major source of the keratinocytes migrating into the wound area. In the skin, however, epithelial cells arise not only from the wound edge but also from hair follicles and sweat glands. During wound healing, most of the components of the basement membrane zone such as type IV and VII collagens, laminin-1 and heparan sulfate proteoglycan are missing underneath migrating keratinocytes (132, 178). Laminin-5, however, appears to be always deposited against the wound bed matrix by keratinocytes during migration (Fig. 3) (132). The role of laminin-5 appears to be critical even for healing of the blister wounds where lamina densa remains intact (110). It is also the rst basement membrane component found against the connective tissue in experimental cyst formation that resembles wound healing (110). Keratinocytes show poor migration in chronic aphthous ulcers and also fail to deposit a proper laminin-5 rich matrix (196).

Laminin-5 can be proteolytically modied (see below) to produce different molecules that function either as a nucleator of the hemidesmosomes (stopping signal) or as a promoter of migration. Laminin5 seems to be the nucleator of a6b4 integrin and therefore the nucleator of basement membrane organization (110). Migrating keratinocytes produce other extracellular matrix molecules that they could use to support their own migration. Fibronectin-EIIIA but not EIIIB (see below) appears to be present underneath the migrating keratinocytes in vivo (Fig. 3) (Hakkinen et al., unpublished). In addition, kera tinocytes in culture deposit bronectin-EIIIA while they migrate on substrates coated with serum bronectin (Koivisto et al., unpublished). TenascinC large and small variants are also found underneath the migrating keratinocytes. As we will discuss elsewhere in this chapter, tenascin-C may function as a modulator of cell adhesion to other matrix components such as bronectin. It appears obvious, therefore, that keratinocytes themselves are capable of making extracellular matrix that they can use to support or modulate their own migration if the provisional matrix is not permissive to migration. One could question whether keratinocytes in healing wounds migrate on the provisional matrix at all. Some of the migration could be intraepithelial, that

Fig. 3. Keratinocyte integrins and extracellular matrix proteins during re-epithelialization. A. Histological representation of a 3-day-old wound. B. Schematic view of the same wound. Keratinocytes express a number of integrin receptors (a2b1 to a6b4) and extracellular matrix proteins

(LM5: laminin-5; TN: tenascin-C; FN ED-A: bronectin isoform EIIIA) while they migrate through wound provisional matrix. FC: brin clot; CT: connective tissue; E: epithelium. refers to the strong immunoreaction at this stage of healing.

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Table 1. Epithelial integrins and their ligands during wound healing

Integrin a2b1 a3b1, a6b4 a5b1, avb1 a9b1 avb5 avb6 Ligand Collagen Tenascin (?) Laminin-5 Serum bronectin Fibronectin EIII/A Tenascin-C Vitronectin Fibronectin EIIIA, bronectin EIIIB Transforming growth factor b1 Ligand source Connective tissue Keratinocytes, granulation tissue Keratinocytes Serum Keratinocytes Keratinocytes, granulation tissue Serum, connective tissue Keratinocytes, granulation tissue Keratinocytes, macrophages, connective tissue cells

is, suprabasal keratinocytes sliding on basal cells at the leading edge. After reaching the provisional matrix, migrating cells could stop moving and become basal stationary keratinocytes. Two models for epithelial sheet migration in wounds have been proposed (236). In the sliding model the cells at the margin are active and pull the cells behind them. In a leap frog model, the migrating cells become nonmotile when they adhere to the provisional wound matrix. Then the cells behind the leading edge crawl over the newly attached keratinocytes. The exact mechanism by which multilayered epithelium migrates remains to be discovered, but it is possible that all three mechanisms described above could function depending on the defect and the state of the healing. Embryonic re-epithelialization appears to occur through a different mechanism. Epidermal cells do not crawl by extending lamellopodia into the wound as they do during, for example, gingival wound healing (132). They rather use the actin lament cables to pull wound edges together (154). In small gingival wounds (23 mm distance between the wound edges), the formation of new basement membrane starts when the migrating epithelial sheets have reached each other and fused (132). The nucleation of the basement membrane appears to occur at multiple sites at the same time. It has been reported that in epidermal wounds the deposition of basement membrane starts from the wound margin in a zipper-like fashion (35). Whether there is a real difference in the sequence of basement membrane organization between gingival and epidermal healing has not been shown. The reorganization of the basement membrane is complete at 4 weeks at which time the localization of all basement membrane components such as type IV and VII collagens, laminin-1 and heparan sulfate proteoglycan appear normal (Hakkinen et al., unpublished). Keratinocytes have been shown to synthesize these main compo-

nents of the basement membrane zone (236). Signicant portion of the basement membrane components are, however, synthesized by the wound broblasts (65). It is obvious that the cross-talk between the wound keratinocytes and broblasts is crucial during the reorganization of the basement membrane zone. Extracellular matrix interactions of keratinocytes during re-epithelialization As the epithelial cells undergo major environmental changes during the wound healing, they have to adjust their cell surface receptors for the new demands. During wound healing keratinocytes have to migrate on the provisional matrix different from their stationary matrix. This requires a change of expression levels and distribution of old integrins and expression of some totally new integrins (Table 1, Fig. 3) (131). As discussed throughout this chapter, bronectin is a critical early component of the clot and the forming granulation tissue. Initially, the blood clot contains plasma bronectin, which is later replaced by cellular bronectin produced by keratinocytes, broblasts and macrophages (33). Fibronectin can vary structurally in a tissue specic manner by alternative splicing of three regions, namely EIIIA, EIIIB and V(IIICS) (284). Expression of the alternatively spliced isoforms of cellular bronectin containing either the EIIIA or EIIIB modules, which are omitted from the plasma bronectin, are upregulated specically during embryonic development and upon wounding (Hakkinen et al., un published). As discussed above, EIIIA bronectin is expressed by migrating keratinocytes in wounds (Hakkinen et al., unpublished). The EIIIB bronectin that is expressed in embryonal tissues but not normally in adult connective tissue is strongly upregulated during granulation tissue formation (Hakkinen

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et al., unpublished, see below). Plasma bronectin and the isoforms containing the EIIIA or EIIIB modules may have different properties in cell adhesion and migration during wound repair. Human wound keratinocytes express two major receptors that are able to bind to bronectins, namely a5b1 and avb6 integrins (Table 1) (91, 132). The classical bronectin binding receptor is a5b1 integrin that binds to the RGD sequence of the bronectin molecule (1, 229). Integrin a5b1 is considered to be a specialized receptor for bronectin and it mediates bronectin-matrix assembly, cell adhesion and migration on bronectin (1). Another bronectin RGD site binding receptor in epithelial cells is the avb6 integrin (17). Integrin avb6 is expressed exclusively in epithelial cells, and it functions as a receptor for the extracellular matrix proteins bronectin (17, 270) and tenascin (191). Neither of the main bronectin receptors, a5b1 or avb6 integrin, appears to be present in keratinocytes residing in the resting epithelium, but the expression of both of these integrins can be induced by wounding or placing epithelial cells in cell culture (Fig. 3). These two bronectin receptors appear in the wound keratinocytes at different times, a5b1 during early migration and avb6 during reorganization of the basement membrane zone suggesting that their function may be different (91, Hakkinen et al., unpublished). As mentioned earlier, epithelial integrin avb6 may play a central role in the reorganization of the basement membrane zone (Fig. 4) as it is expressed by wound keratinocytes after epithelial sheets have joined and because it is able to activate transforming growth factor b (167). In culture, keratinocytes can switch between avb6 and a5b1 integrins in bronectin binding if one receptor is unfunctional (118). Laminin-5 is found in the basement membrane of skin and other epithelial tissues and serves as a component of anchoring laments that span through the basement membrane (19, 203). The extracellular domain of a6b4 integrin interacts with laminin-5 (173, 203). This integrin is known to link the basal keratinocytes to the underlying basement membrane, and this link mainly relies on the interaction with laminin-5 in the anchoring laments. The functional importance of a6b4 integrin is substantiated by experiments in knockout mice that lack either a6 or b4 integrin and cannot form hemidesmosomes, which results in blistering of skin. Laminin-5 is also recognized by a3b1 integrin (19). Transgenic mice lacking a3 integrin also develop localized blistering of the skin (49). In mice and man, a3b1 integrin is occasionally found at the basal surface of

the basal keratinocytes, suggesting that a3b1 integrin collaborates with a6b4 integrin in laminin-5 binding and they may be functionally interchangeable in some circumstances. Integrin a6b4 is present in migratory epithelial cells although hemidesmosomes are absent (132). One possible scenario could be connected to the functions of laminin-5 during wound healing. Intact laminin-5 appears to be a motility factor for keratinocytes, but when proteolytically processed it starts to function as the nucleator of hemidesmosomes and therefore promotes the formation of basement membrane structures (78). This is a good example demonstrating how complex the wound-healing process is, since simple proteolytic cleavage can totally reverse the function of a protein important for the cell migration process. Expression of b1 integrins is strongly upregulated in keratinocytes during wound healing (20, 73, 97, 108, 132). Both a2b1 and a3b1 integrins seem to be equally expressed. Integrin a3b1 is known to be able to bind both bronectin and laminin-5, both of which are present in the provisional matrix (19, 132) although its role as a bronection receptor in keratinocytes is doubtful. Integrin a2b1 is known to be able to bind many types of collagens (Table 1), such as type I, III, V and VI that serve as a substrate for migrating keratinocytes. Direct interactions of keratinocytes with type IV collagen are rare but may occur, for example, during healing of blister wounds, in which case keratinocytes migrate on the components of lamina densa. Keratinocytes are more likely to interact with brillar collagens, such as types I, III and V collagens during healing of full-thickness wounds. In this case, interaction of keratinocytes with collagens is mediated via a2b1 integrin. Integrins are also involved in the regulation of extracellular matrix degradation. Cells in the tissue adapt to their changing environment and sense alterations in the matrix through integrins on the cell surface. Recognition of an altered matrix by integrins lead changes in gene expression. For example, epithelial and connective tissue cells attach to collagen by a2b1 integrin which process induces the expression of collagenase (matrix metalloproteinase-1) that can cleave the exposed collagen matrix and facilitate the migration of wound keratinocytes (186, 187, 278). Vitronectin is an abundant protein present in blood plasma, extracellular matrices and brin clots. Vitronectin is known to be an active cell adhesion mediator as well as playing a role in cell migration and invasion (190). The avb1, avb3, avIIb3 and avb5 integrins are all vitronectin receptors binding to the RGD cell attachment sequence of the protein (24).

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Fig. 4. Hypothetical role of keratinocyte avb6 integrin in the regulation of granulation tissue formation. A. Fibrin staining of a 7-day-old gingival wound. Arrowheads mark the wound area. B. Immunolocalization of avb6 integrin in a 7-day-old wound. Basal keratinocytes of fused epi-

thelium that cover the granulation tissue (GT) express avb6 integrin. C. Schematic representation of the putative role of avb6 integrin in wound healing. TGFb1: transforming growth factor b1.

Migrating keratinocytes in porcine epidermis have been reported to express the avb5 integrin (73). The avb5 integrin is present in cultured keratinocytes and has been shown to mediate keratinocyte migration on vitronectin (116). Vitronectin is present in the provisional wound bed matrix and it is, therefore, reasonable to assume that an avb5-vitronectin interaction can exist during wound healing (Table 1) (36). Tenascins are a family of large extracellular matrix proteins whose expression is tightly regulated, making this protein particularly interesting. The expression of tenascin is closely associated with morphogenetic events, including embryonic induction and migration, wound healing and tumorigenesis (28, 122, 275). Tenascin-C knockout mice develop normally (208), suggesting that there are other mol-

ecules that can replace its function and compensate for its absence. Tenascin-C is found in developing brain and in mesenchyme associated with epithelialmesenchymal interactions (28, 247). In adult tissues tenascin-C is found in the connective tissue underneath epithelium (138), but its expression is strongly upregulated during wound healing (31, 144, 275). As mentioned above, the large and small tenascin-C variants are found under migrating keratinocytes, suggesting their active role in re-epithelialization. Whether tenascins modulate keratinocyte adhesion to other matrix components or support directly keratinocyte adhesion remains to be shown. Several keratinocyte integrins have been found to bind tenascin-C, namely a2b1, avb6 and a9b1 integrins (14, 191, 290, 291). Integrin a9b1 in actively migrating,

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and avb6 integrin in nonmigrating wound keratinocytes, colocalize in the same area as tenascins (Table 1) (Hakkinen et al., unpublished). These two epi thelial integrins may, therefore, have a different role in cell signaling caused by tenascin-C variants. Upregulation of a9b1 and tenascin-C is also observed during corneal wound healing (239). Transforming growth factor b and re-epithelialization In wounds, specic regulatory signals are required for normal repair. Currently it is believed that transforming growth factors-b have a central role in the wound healing process. Transforming growth factors b are a family of polypeptides that have multiple regulatory actions in cell growth, differentiation, and developmental processes (155, 234, 244). Three highly homologous transforming growth factor-b genes have been identied in mammals, representing transforming growth factor b1, transforming growth factor b2 and transforming growth factor b3 polypeptides. Human keratinocytes of intact skin express transforming growth factor b3 (216). This growth factor seems to play an important role in epidermal maintenance. In animal studies, only small amounts of transforming growth factor b2 and transforming growth factor b3 messenger RNA have been found in keratinocytes of intact dermis (215). All transforming growth factor b isoforms are found in healing wounds of animals (111, 137). However, Schmid et al. (216) did not nd any detectable levels of transforming growth factor b2 messenger RNA in human wound keratinocytes. Wound broblasts and macrophages are known to express both transforming growth factor b1 and transforming growth factor b2 in cells adjacent to the wound (175, 267). Transforming growth factor b1 is the major isoform in wound keratinocytes, and induction of transforming growth factor b1 in migrating keratinocytes is crucial for the successful re-epithelialization of skin wounds. In vitro studies have shown that transforming growth factor b3 inhibits the growth of primary human keratinocytes, while transforming growth factor b1 seems to stimulate keratinocyte motility by switching the cells from the differentiating to regenerative phenotype (152) and by inducing their production of bronectin (172) and laminin-5 (110). In cultured human keratinocytes, transforming growth factor b1 has also been shown to increase the levels of messenger RNA for some integrins, such as a5, av and b5, that may facilitate migration of wound keratinocytes (73, 292). In cultured

HaCaT keratinocytes, transforming growth factor b1 and 2 specically stimulate the expression of avb6 integrin, and promote both haptotactic and epithelial sheet migration (118). As we have discussed earlier, the most important role for avb6 integrin may be the activation of transforming growth factor b1 that then promotes the formation of the connective tissue bridge formation under the joined wound epithelium. The rst collagen brils are, indeed, laid down under the epithelium in areas where the expression of avb6 is strong and transforming growth factor b1 is present in active form. Transforming growth factor b1 may also stimulate the proliferation of wound keratinocytes at the wound margins indirectly by inducing the expression of other polypeptide growth factors, such as platelet-derived growth factor (3, 22). In addition to transforming growth factors b, many other growth factors regulate wound healing. This review will not try to list all possible factors involved in re-epithelialization and the readers are advised to review other sources for the roles of plateletderived growth factor, epidermal growth factor, keratinocyte growth factor, hepatocyte growth factor and others in wound healing.

Connective tissue repair

Activation of broblasts The resolution of a tissue defect after wounding requires not only re-epithelialization but also proliferation and migration of broblasts into the wound bed where they participate in the formation of granulation tissue and synthesize, deposit and reorganize various extracellular matrix molecules. Wound repair involves phenotypic change of broblasts from quiescent to proliferating cells, and subsequently to migratory, and then to stationary matrix producing and contractile cells. In normal connective tissue, broblasts reside in a quiescent state and have a slow proliferation rate and metabolic activity. Upon wounding, broblasts become activated and change their gene expression. This is supported by a recent nding with quiescent broblasts that show a rapid change in expression of hundreds of genes when stimulated by serum. Interestingly, many of the serum-activated genes are known to be involved in the physiology of wound repair, including control of cell cycle and proliferation, coagulation and hemostasis, inammation, angiogenesis, tissue remodeling, cytoskeletal reorganization and re-epithelialization (107). This indicates that exposure of

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broblasts to serum that is present in the blood clot in the wound initiates not only a rapid general stimulus for cell proliferation but also a more specic gene expression that controls function of other cells involved in inammation, angiogenesis and reepithelialization. Therefore, it is likely that broblasts play a more important role in the physiology of wound repair than has been previously realized (107). In addition to serum stimulation, cell adhesion to different matrix molecules can also alter gene expression and protein synthesis (4, 120, 121, 143). Cell adhesion also modulates signaling initiated by growth factors (109, 163). During tissue repair, broblasts are exposed to a variety of extracellular matrix molecules and soluble growth factors that can exert multiple signals at the same time. Additionally, cells experience tensional forces from the extracellular matrix during cell migration and reorganization of the wound matrix, which can modulate signaling inside the cell (27, 205, 228). The function of extracellular matrix proteins may be further modulated by proteolytic modication (13, 143, 227), and by conformational changes induced by tensional forces (295). Therefore, the regulation of broblast gene expression upon wounding is likely to be very complex and dependent on the balance between different activated signaling pathways (41). Origin of wound broblasts The multiple functions of wound broblasts raise the question of whether all of the tasks are performed by a single cell type or multiple different phenotypes are involved (Table 2). If the latter case is correct, does the heterogeneity result from migration of phenotypically different cells into wound or does it result from differentiation of the broblasts that have populated the wound? These questions are still largely unanswered. There is evidence, however, that connective tissue broblasts are heterogeneous in several properties, including, responsiveness to growth factors and in the ability to produce specic extracellular matrix proteins (93, 94,

185, 188, 219, 225) suggesting that signals initiated by wounding may stimulate certain subpopulations to enter the wound space. On the other hand, there is also evidence that some of the broblasts that have migrated into wound actually change phenotype and differentiate into myobroblasts (see below). Progenitor broblast populations have been identied in wounds for decades using autoradiographic labeling techniques. Based on these studies, the main source of wound broblasts seems to be the surrounding connective tissue. Typically, 2 or 3 days after wounding, the label-retaining cells are located at the subepithelial connective tissue underlying the wound edge and at the perivascular location. It seems, however, that only a subpopulation of the local broblasts proliferate in response to tissue injury (225), suggesting that certain broblast subsets are more responsive to growth stimulation than others. Local broblasts are not the only source of wound broblasts. After wounding, pericytes that are residents of the surroundings of the vascular endothelium of capillaries and venules are also induced to proliferate and migrate into the wound (79, 106, 158). Although broblasts have multiple functions in wound repair, their hallmark characteristic is the property to produce collagen and other structural extracellular matrix proteins that replace the blood clot. This property separates wound broblasts from inammatory and vascular endothelial cells that are also involved in the formation of granulation tissue. Accordingly, upon wounding, the pattern of gene expression in pericytes is reprogrammed from a contractile to a migratory collagen-producing cell, suggesting that these cells are able to function as matrix-producing wound broblasts. Could some of the wound broblasts also arise from bone marrow stem cells? Interestingly, early work by Cohnheim in 1867 suggested that some of the wound broblasts may originate from bone marrow. This was further supported by recent ndings showing that genetically marked bone marrow stromal cells serve as a source of progenitor cells for various mesenchymal tissues. Most importantly,

Table 2. Origin of wound broblasts

Possible sources of wound broblasts Surrounding connective tissue Pericytes Bone marrow Steps involved Migration, differentiation Proliferation, migration Systemic control, homing, differentiation

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these cells apparently translocate to the target tissue where they acquire the phenotype of the resident cells (183, 193). These peripheral blood brocytes accumulate in the wound already in the early acute phase but are also present in the scar tissue (15). Fibrocytes function as antigen presenting cells and can prime naive T cells (25) and secrete a number of cytokines involved in immune response, hematopoesis and extracellular matrix synthesis (25, 26), suggesting that brocytes may play a role in the regulation of the acute inammatory reaction as well as in the matrix deposition in wounds. Taken together, the matrix-producing cells that populate wound granulation tissue originate from diverse tissue sources and, depending on their origin, they may serve different functions. Integrin expression and function in broblasts during wound repair In the connective tissue, broblasts are surrounded by a matrix that contains collagen and cellular bronectin as the major components. Consequently, quiescent broblasts express collagen receptors a1b1 and a2b1 and the major bronectin receptor a5b1 integrin which they use for adhesion to the matrix (74, 271, 281). Fibroblasts express a3b1 (281) and integrin heterodimers of the av subfamily (Hakkinen et al., unpublished) that can also be used to bind bronectin (4). Although cultured broblasts are able to express avb1, avb3 and avb5 integrins (70, 73, 92), it is not completely clear with which b subunits av actually combines to form integrin heterodimers in quiescent broblasts in vivo. In cell culture, broblasts also express a4b1 integrin that binds to bronectin (72), but it is unclear whether this integrin is actually expressed by broblasts in vivo. The b1 integrins in quiescent broblasts are partially in an inactive state and become activated after wounding in broblasts that are located near the defect (Hakkinen et al., unpublished). Several lines of evidence show that integrin activation and integrinmediated cell adhesion to extracellular matrix is important for induction of DNA synthesis (179). Specic integrin-ligand binding is required to commence DNA synthesis of cells. Cell adhesion to extracellular matrix via integrins strongly inuences the ability of normal cells to respond to soluble mitogens (43, 76, 109, 221). Activated integrins can physically complex with growth factor receptors (see above) which provides a mechanism of how different signaling molecules may be targeted to cell-matrix interaction sites inside the cell (161, 218). It is, therefore, prob-

able that the decision of the broblast to enter the cell cycle depends on the repertoire of integrins that actively interact with specic extracellular matrix proteins and on the combination of growth factors that the cells are exposed to at the particular phase of wound repair. Cell migration is needed for broblasts to enter the wound provisional matrix. Fibroblast migration into the blood clot occurs only after a lag period of 3 to 5 days, the time required for cells to proliferate (32, 157). The lag phase may also be needed for the cells to be activated from quiescent state to be fully active migratory cells and to be recruited from their original location in the tissue or bone marrow to the wound site. It is believed that, in order to detach from a collagen-rich matrix, broblasts have to downregulate their expression of collagen receptors and upregulate integrins that bind bronectin, brin, brinogen and vitronectin in the provisional wound matrix. Accordingly, about 3 to 5 days after injury, broblasts that migrate into the blood clot express the primary bronectin receptor a5b1 integrin and a3b1 integrin while they show downregulation of collagen-binding a1 and a2 integrin expression at the wound margin (74, 281). Platelet-derived growth factor, which is abundantly expressed during early wound repair, stimulates cell migration towards bronectin by increasing the synthesis of a5 and a3 integrins (74, 117). On the other hand, platelet-derived growth factor downregulates a1 (74) while it stimulates a2 integrin expression by broblasts in culture. Therefore, the regulation of a2 integrin expression in vivo and in vitro is different, possibly because of the multiple signals in addition to plateletderived growth factor that modulate integrin expression in the tissue. One of the factors that modulates integrin expression during wound repair is the composition of the extracellular matrix. If broblasts are cultured in brin and bronectin-rich matrices that resemble the early wound matrix, they show selective upregulation of a3 and a5 integrin mRNA as opposed to cells that are in collagen-rich matrix, which show upregulation of a2 integrin (281). This suggests that signals initiated by growth factors and extracellular matrix molecules collaborate to induce appropriate integrin expression to support cell migration on brin-bronectin matrix in the early wound repair and to allow cell adhesion on collagen later during scar formation. In the early wound, broblast migration seems to be primarily mediated by bronectin because broblast migration from a three-dimensional collagen matrix into a brin clot depends on the presence

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of bronectin in the matrix and can be blocked with antibodies against a5b1 and avb3 integrins in vitro (84). Fibroblasts can also use avb3 and avb5 integrins for cell adhesion on vitronectin present in the clot (70) and avb3 for cell adhesion and migration on brin and brinogen, respectively (71, 84). Cell migration is regulated by the structural organization of the bronectin matrix. In the normal connective tissue, cells deposit and arrange bronectin into a brillar network between the cells and other matrix molecules using primarily a5b1 and possibly avb3 integrins (279, 280, 287). In contrast, in the blood clot, plasma bronectin is cross-linked with brin to form a three-dimensional scaffold (224). The variation in the architecture of the bronectin network can regulate multiple cell functions, suggesting that bronectin in the connective tissue and in the blood clot may elicit different signals (224, 233). During wound repair, cells are induced to deposit alternatively spliced isoforms of cellular bronectin containing either the EIIIA or EIIIB modules (see above). The EIIIA or EIIIB modules have different properties to support cell adhesion, migration, and proliferation (95, 150, 151), suggesting that they may have different functions in wound repair. Wounding induces deposition of extracellular matrix proteins that modulate cell adhesion. Among these molecules, tenascin-C, thrombospondin and secreted protein, rich in cysteine/osteonectin (SPARC) are multifunctional molecules that can serve as ligands for multiple cell surface receptors (83). Typically, the expression of tenascin-C and thrombospondin is potently stimulated early during granulation tissue formation, while SPARC is expressed from middle to late stages of repair. Expression of tenascin-C and SPARC remains elevated during tissue reorganization while the expression of thrombospondin is only transient (50, 144). Interestingly, wounding specically induces the expression of the large tenascin-C splice isoform (144, Hakkinen et al., unpublished) that is normally expressed predominantly during embryonic development (156, 192). Tenascin-C contains both adhesive and counteradhesive domains, although mostly it is believed to reduce the adhesion of cells to bronectin and therefore enhance cell migration (253). The effect of tenascin-C on cell adhesion to bronectin can also further modulate gene expression by the cells (253). Interestingly, the large tenascin-C splice isoform functions differently in regulation of cell adhesion and in binding bronectin as compared with the more ubiquitously expressed small isoform (29, 63, 64, 113, 169, 184). Tenascin-C can serve as ligand

for several cell surface receptors, including avb3 integrin in broblasts that binds to the RGD sequence found in the third bronectin type repeat of the tenascin molecule. Ligand binding by integrins to this repeat is modulated by other counteradhesive domains in the molecule (40). Thrombospondin can also, like tenascin-C, modulate cell adhesion, migration and proliferation (83). It contains an RGD sequence that is recognized by avb3 integrin expressed by broblasts (240, 255), but it also contains, like tenascin-C, additional binding sites for other cell surface receptors (240). SPARC is also able to interfere with integrin-mediated cell adhesion on extracellular matrix, and this is believed to occur via interaction with other cell surface molecules that are able to bind SPARC and not with steric disruption of integrinextracellular matrix interaction (168). Because of their different temporal expression pattern during wound repair, it is possible that tenascin-C, thrompospondin and SPARC regulate different cellular functions. Indeed, gene knockout animal studies have indicated that thrombospondin plays a role in regulation of the activity of transforming growth factor-b, collagen ber organization and vascularization in the connective tissue (39, 126), which are all important processes in the physiology of wound repair. Wound contraction One of the tasks of broblasts in granulation tissue is to bring the wound margins closer together to allow rapid wound closure. It is not completely clear how wound contraction actually happens in vivo. The most widely held concepts suggest that wound closure follows when central granulation tissue is contracted either by myobroblasts pulling the new collagen matrix (69) and/or by broblasts changing the organization of the wound matrix by migrating through it (54, 67). On the other hand, experimental removal of central granulation tissue does not seem to prevent wound contraction, suggesting an alternative mechanism in which polarized coordinated migration of a rim of densely packed broblasts pull forward the wound edges (89). Myobroblasts are likely to play a role in wound contraction and matrix deposition. This is supported by ndings that myobroblasts are typically present in tissues that are under mechanical stress but they are only occasionally found in normal tissue (46). Myobroblasts become especially prominent in pathological conditions including wound repair, tissue brosis and tumor stroma (42, 46, 201). These

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cells are characterized by presence of abundant cytoplasmic microlament bundles and a-smooth muscle actin. They differ from normal broblasts morphologically, in growth potential, expression of proteoglycans and collagen, responsiveness to transforming growth factor b, organization of focal adhesions, and in their ability to organize cellular bronectin in culture (51, 93, 94, 212). Fibroblasts cultured from wound granulation tissue are rich in myobroblasts and express a similar repertoire of integrins as normal connective tissue broblasts. They are, however, phenotypically unique in that they show reduced cell spreading on bronectin as a result of altered interaction of a5b1 integrin and bronectin (92). The differentiation of myobroblasts occurs between 6 and 15 days after wounding (42, 217). After 15 days, about 70% of broblasts in granulation tissue express a-smooth muscle actin. Differentiation of myobroblasts coincides with wound contraction, which has led to the idea that these cells are actually involved in this process. The differentiation of myobroblasts is induced by transforming growth factor b in vivo and in cell culture but not with other probrotic growth factors and cytokines (47, 204). Importantly, myobroblasts differentiation is controlled by integrin mediated mechanisms and depends on the composition of the extracellular matrix and the tensional forces mediated from the matrix. Recent studies have shown that the induction of myobroblast differentiation by transforming growth factor-b requires deposition of cellular EIIIA bronectin in the pericellular matrix (226). In tissue, differentiation of wound broblasts into myobroblasts follows induction of EIIIA bronectin expression (226). The induction of the expression of asmooth muscle actin in broblasts by transforming growth factor b is also regulated by the changes in the collagen matrix around the cells and on the development of intracellular tension (5, 171). This is in turn regulated by interaction between a2b1 integrins and collagen (171). The induction of a-smooth muscle actin expression during wound repair is thought to provide a mechanism to stop broblast migration when cells reach their destination. Termination of migration and formation of prominent focal adhesions may be needed to promote stable (200) cell adhesion to matrix, which is required for myobroblasts to acquire a matrix-synthesizing and contractile phenotype. In ideal wound repair, scar tissue is remodeled and reorganized to form structurally and functionally normal connective tissue. When contraction stops, the

myobroblasts disappear because of apoptosis and the scar becomes less cellular and new broblasts with properties typical to normal connective tissue broblasts emerge (42, 48, 217). Apoptosis of myobroblasts begins at day 12, peaks at day 20 and resolves by day 60 after wounding (48). Factors that inhibit a-smooth muscle actin expression and differentiation of myobroblasts are not well characterized. It has been noted that interferon-g has an antibrotic effect, which probably results from its ability to inhibit broblast differentiation into myobroblasts (217). Additionally, myobroblasts are more sensitive to basic broblast growth factorinduced apoptosis than normal connective tissue broblasts (68), which may provide a mechanism to reduce the amount of myobroblasts in the tissue when they are no longer needed. Interestingly, myobroblasts persist in certain pathological conditions, including hyperthrophic and brotic lesions of many organs, suggesting that they are involved in the accumulation of extracellular matrix that is characteristic for these lesions (45, 217). Therefore, for normal wound repair it is important that the myobroblasts undergo apoptosis and are replaced by normal broblasts when wound contraction is completed. An interesting question is where the broblasts that replace myobroblasts and that eventually maintain the connective tissue structure during tissue homeostasis originate from. Because these cells have phenotypic properties characteristic to normal connective tissue broblasts, it is possible that they are derived from the connective tissue next to the wound or, alternatively, they differentiate from more primitive broblasts that initially populate the wound. Regardless of their origin, it seems that replacement of the wound broblasts with cells of normal connective tissue broblast phenotype is necessary for the complete regeneration of the tissue. Mechanical tension The interactions between cells and extracellular matrix during granulation tissue formation can be studied in cell culture using cell-populated three-dimensional brin or collagen gels (86, 256). In principle, the brin gels correspond to the situation in early wound repair when broblasts have migrated into the brin containing provisional matrix while the collagen gel model resembles later phase of wound repair when granulation tissue broblasts reside in a newly deposited collagen-rich matrix that undergoes remodeling. In these models, broblasts attach to the proximal brin or collagen bers and

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in an attempt to migrate pull the bers together to form a dense tissue-like structure. When broblasts are allowed to grow in brin gels they eventually replace the brin matrix with newly deposited collagen (256). If the gel is not relaxed, broblasts trying to pull the bers experience high tension from the matrix that resembles wound granulation tissue. Contraction of three-dimensional collagen gel is mediated by integrins and can be stimulated by serum and growth factors present in wound, including lysophosphatidic acid, transforming growth factor b and platelet-derived growth factor (34, 87, 90, 164, 197, 250). Currently, there is some controversy over which specic integrin-ligand interactions actually mediate collagen contraction. Binding between broblasts and collagen brils is evidently the rst and most important step in the collagen gel contraction. Most data show that in human connective tissue broblasts, collagen gel contraction is mediated by the collagen receptor a2b1 integrin (214, 238). The rate of contraction depends on the amount of a2b1 integrins expressed by the cells (197). This is in accordance with simultaneous induction of a2 integrin expression by wound broblasts with formation of collagenous scar and beginning of wound contraction in vivo (282). Role of integrins in regulation of cell proliferation and survival in granulation tissue During the formation of granulation tissue, broblasts that emerge into the provisional wound matrix stop migrating and undergo cell proliferation. Accordingly, about 7 to 10 days after wounding, the granulation tissue becomes hypercellular containing abundant broblasts and endothelial cells (157, 271). After that, during tissue maturation, the cell number in the granulation tissue gradually decreases (48, 157, 271). It appears that the organization of collagen and mechanical tension mediated from the extracellular matrix are important factors that modulate DNA synthesis and cell survival. This is evidenced by the nding that broblasts cultured under mechanical tension proliferate actively, but after stress relaxation the DNA synthesis is rapidly downregulated and cells start to undergo apoptosis (66, 87, 139, 162). Stress relaxation also downregulates autophosphorylation of growth factor receptors, which contributes to the growth reduction (139, 159, 241). In a mechanically stressed collagen gel, cells develop an organized cell surface bronectin network (162, 251) that disappears after stress relaxation. Since a5b1 integrin can probably serve as a mechanoreceptor

and mediate signals that promote cell growth (269, 294), differences in bronectin receptor engagement may be a mechanism of how cell proliferation is regulated by mechanical signals. Therefore, reduction of mechanical strain during tissue maturation may provide a mechanism to downregulate cell growth and induce apoptosis to normalize the cellularity in the tissue. Additionally, in the later phases of wound repair the activity of growth factors is gradually downregulated (231, 288), which will further reduce the number of growth stimulatory signals for broblasts. In the regulation of cell growth and apoptosis in the granulation tissue, integrins probably play an important role because they mediate the signals initiated by mechanical tension and collaborate with growth factor signaling and mediate signals that regulate cell survival (109, 205, 206, 228). Interestingly, broblasts undergo apoptosis in contractile collagen gels but not in brin gels (66), suggesting that specic types of integrinextracellular matrix interactions are needed for initiation of apoptosis. This may provide a mechanism to prevent broblasts from undergoing apoptosis in the early brin-rich provisional wound matrix and to allow their proliferation, which is needed to populate the granulation tissue with broblasts. An additional mechanism that is likely to regulate cell growth in the granulation tissue is the structural organization of the extracellular matrix. During granulation tissue formation, broblasts rst synthesize bronectin and then type I collagen that is gradually organized from monomeric molecules to form a brillar collagen matrix (125). Monomeric collagen through a2b1 integrin promotes cell proliferation while brillar collagen downregulates it (123). Additionally, studies using bronectin decient cells have shown that assembly of bronectin matrix by the cells is required for bronectin to promote cell growth on different extracellular matrix molecules (233). Therefore, it is probable that the deposition and organization of extracellular matrix by broblasts provides a feedback mechanism to regulate cell growth during wound repair. Regulation of protein synthesis and matrix degradation in the granulation tissue Remodeling of tissue during wound repair requires controlled synthesis and degradation of extracellular matrix. The most important factors that regulate synthesis and secretion of these molecules include growth factors and signals from the extracellular matrix. There is evidence that integrin binding to its

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ligand can directly induce gene expression (102, 254, 272). Most importantly, there seems to be a specic integrin-ligand interaction that is needed for stimulation of specic type of protein synthesis (254, 272). In this process, the signals mediated by integrins are further modulated by growth factors, the structural architecture of the extracellular matrix as well as by tensional forces (109, 202, 228). During granulation tissue formation, broblasts are stimulated by transforming growth factor-b to deposit new extracellular matrix proteins. Interestingly, early matrix deposition seems to occur rst at about day 7 after injury in the granulation tissue immediately under the newly formed epithelium (178). This coincides with the peak in activation of latent transforming growth factor-b from storages in the blood clot provisonal matrix and in the matrix of the granulation tissue (81, 288) and with induction of epithelial avb6 integrin which may regulate the activation of latent transforming growth factor-b (see above, 91, Hakkinen et al., unpublished). Once acti vated, transforming growth factor-b can induce its own production by broblasts (139), which may be a mechanism to maintain high activity of transforming growth factor-b in the granulation tissue. As discussed earlier, a potent modulator of protein synthesis is mechanical tension. Generally, when cells are grown in collagen gels under tension they show a relatively high protein synthesis rate, while after stress relaxation the protein synthesis is downregulated (162). The signals initiated by tension in a three-dimensional collagen matrix require specic integrin-collagen interactions and may lead to specic gene activation. Additionally, platelet-derived growth factorinduced upregulation of a2 integrin expression is stimulated if broblasts are cultured in collagen gel while expression of a5 and a3 integrins is attenuated (281). Transforming growth factor-b can also potently stimulate expression of a2 integrin in broblasts that interact with collagen, and this depends on the tensional forces that the cells experience (5). It seems that tension mediated from the matrix can effectively regulate the balance between matrix production and degradation. For example, upon stress relaxation the expression of type I collagen is downregulated while that of matrix metalloproteinase-1 and -13 is upregulated (30, 195, 197, 252). The regulation of collagen and matrix metalloproteinase genes by collagen matrix is partially regulated by different collagen receptors. The downregulation of collagen synthesis after stress relaxation is mediated by a1b1 integrin and the upregulation of matrix metalloproteinase-1 and matrix metallopro-

teinase-13 expression occurs by a2b1 and by both a1b1 and a2b1 integrins, respectively (195, 197). Integrin a1b1 seems to be able to function as a feedback regulator of collagen synthesis in broblasts (75). Gingival wound repair: similarities to scarless fetal wound repair There has been a general observation that wounds in the oral mucosa heal faster and with less scarring than extraoral wounds (Table 3). Scar tissue is characterized by excessive accumulation of disorderly arranged collagen, mostly type I and III (219), proteoglycans (274, 289) and persistent myobroblasts (42, 46, 201), which leads to aberrant function of the tissue. Surprisingly, there are only a few reports that have quantitatively tested this hypothesis. Nevertheless, based mostly on animal studies it seems that wound healing in oral mucosa, indeed, is faster and results in less scarring than in skin (223, 286). It is probable that oral wound healing is enhanced partly because of factors present in the saliva and by the specic microora of the oral cavity (see below). Additionally, the properties of the cells involved in tissue regeneration in oral mucosa are unique and share properties of fetal cells (219). Scar formation is developmentally regulated because, in early gestation, fetal wound repair occurs without scar formation and the transition to healing with scar formation occurs late in the gestation (56, 104). Interestingly, the initiation of scar formation parallels the induction of myobroblast differentiation during wound repair at different stages of embryonal development (56), suggesting that phenotypic modulation of wound broblasts into myobroblasts may be involved in scar formation. As compared with adult wounds, fetal wounds are characterized by the absence of clot formation and inammatory reaction. They also show unique spatial and temporal organization of extracellular matrix and reduced expression of transforming growth factor-b (274). Several investigations have compared the properties of adult skin and gingival broblasts with fetal broblasts. The ndings indicate that, unlike adult skin broblasts, adult gingival broblasts located in the papillary connective tissue share many properties with fetal broblasts, including their growth and migration properties, cell morphology, production of migration stimulating factor, and production and response to cytokines (219). Additionally, similar to fetal broblasts, oral mucosal or gingival broblasts are able to contract three-dimen-

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Table 3. Special features of oral wound healing

Factor Saliva Mechanism Moisture, ionic strength Growth factors (EGF, TGFb, FGF, IGF etc.) Unknown factors Stimulation of macrophage inux Direct stimulative action on keratinocytes and broblasts Fetal-like broblasts with unique response Specialized epithelium and connective tissue

Bacteria Phenotype of cells

sional collagen matrix faster as dermal broblasts (105, 237). They also populate experimental wounds faster than their dermal counterparts in culture (2). Gingival broblasts also differ from dermal broblasts in their ability to secrete proteolytic enzymes. Matrix metalloproteinase-13, a potent collagenase (273), is expressed in the granulation tissue during acute wound repair at 7- and 14-day-old human gingival wounds but not in dermal wounds (296). Additionally, when gingival broblasts are inoculated into three-dimensional brin matrix they are able to reorganize and degrade the matrix rapidly. This is because of high expression of tissue plasminogen activator. Matrix reorganization and brinolysis is less advanced in dermal broblast-inoculated matrices (141, 142). The regulation of brinolysis by gingival broblasts depends on the tensional forces that the cells experience from the matrix (140). The various ndings described above suggest that several cell functions important in tissue repair are shared by fetal and gingival broblasts and differ from dermal broblasts. It is possible that gingival broblasts are phenotypically unique cells in adult tissue that may contribute to the rapid healing of oral wounds with minimal scarring in the gingiva.

Role of saliva and gingival crevicular uid in oral wound healing

While it is clear that the excellent healing potential of oral mucosa results to a large extent from the intrinsic tissue factors such as the presence of structural cells with potential for tissue regeneration, dense vasculature and high turnover rate of connective tissue and epithelium, it is also apparent that saliva provides a unique environment in the mouth conducive for rapid tissue repair (Table 3). This becomes obvious from the studies showing delayed

healing of oral wounds in people with xerostomia or sialadenectomized animals (12, 55, 103). Animals instinctly lick their wounds, which appears to result in faster healing (11). There are several physicochemical factors in saliva favoring gingival healing. These include an appropriate pH, ionic strength, and presence of ions such as calcium and magnesium required for healing (53). Saliva also has an efcient capacity to reduce redox activity caused by transitional metal ions and inhibit the production of free radicals that may be benecial for the healing process (170). Lubrication of oral mucosa provided by saliva is benecial for wound healing. The advantageous effects of maintaining a moist wound environment include prevention of tissue dehydration and cell death, accelerated angiogenesis and increased breakdown of brin and tissue debris. Hence, the use of hydrocolloid occlusive dressing is a useful adjunct in facilitating cutaneous wound healing (61). The therapeutic effect of saliva on healing of skin wounds has been demonstrated experimentally in calves. Compared to saline-treated wounds, saliva-treated wounds had shorter inammatory reaction and faster epithelial coverage and connective tissue regeneration (268). It appears that moisture and ionic strength are not the primary factors in saliva that promote tissue repair. This potential is probably due to the presence of several factors in saliva including various growth factors and bacteria (293). Growth factors found in saliva are synthesized by salivary glands or derived from plasma through gingival crevice. Epithelial cells and connective tissue cells also produce their own growth factors that act either in a paracrine or autocrine manner. Because many of the growth factors are transported to saliva along with gingival crevicular uid, it is conceivable that their concentrations in gingival tissue are higher than elsewhere in the oral cavity. Therefore the periodontium is in a favorable position with respect to tissue healing.

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As discussed above, wound healing is a complex phenomenon involving increased proliferation, adhesion and migration of cells of connective tissue and epithelium, inammatory reactions and remodeling of extracellular matrix. All these phenomena are directed by growth factors (9, 10, 82). Different growth factors have specic functions and target cells in wound healing, and their delicate balance is required for optimal tissue repair. The actual roles of each growth factor in saliva are not known. The best studied salivary growth factor, and possibly the most important one, is epidermal growth factor. In his pioneer studies, Cohen found that a protein component of mouse submandibular gland induced precocious eyelid opening and incisor tooth eruption. Later it was discovered that the factor termed epidermal growth factor has a multitude of effects on cell proliferation, cell migration and extracellular matrix metabolism (21, 37, 220). It has now become obvious that epidermal growth factor is needed for the normal maintenance and repair of oral mucosa (174). Interestingly, salivary epidermal growth factor also plays a role in the maintenance of gastric and ileal mucosal integrity (194, 213). In humans and many other animals, salivary glands are the major epidermal growth factorproducing organs. In humans epidermal growth factor is synthesized by both parotid and submandibular glands. During oral wound healing, the concentration of epidermal growth factor is increased in saliva (99, 180, 181). Another growth factor found in saliva is the vascular endothelial growth factor (243). This protein is important in many aspects of angiogenesis and inammation such as endothelial growth, permeability and leukocyte adherence (52). A number of other growth factors are also present in saliva. These include nerve growth factor and members of transforming growth factor b, broblast growth factor and insulin-like growth factor families (230). As these substances have specic regulatory roles in cell growth and extracellular matrix formation, they are important in maintaining health of the oral cavity and in healing of oral mucosal tissues (293). The presence of growth factors in crevicular uid has received limited attention. The major focus of the studies has been pro-inammatory cytokines, such as interleukin-1 and tumor necrosis factor a (177). It is important to recognize that while one action of the crevicular uid cytokines is promotion of inammation, another function is to facilitate tissue repair. Maintenance of the normal tissue turnover results from a delicate balance of the cytokines. An alteration in this balance results either in persistence

of inammation and tissue destruction or increased cell growth and tissue repair. Integrin ligands such as collagen, vitronectin, bronectins and laminins are involved in directing the adhesion and migration of cells (261). It is therefore possible that these molecules, when present in gingival crevicular uid or saliva, may modulate wound healing of gingiva. Indeed, bronectin has been found both in crevicular uid and saliva (112, 129, 257). Even though not yet reported, it is reasonable to assume that other adhesion molecules may be present in saliva. Extracellular matrix remodeling is an essential part of wound repair. In this process matrix metalloproteinases and other neutral proteinases have an important function. During periodontal diseases gingival crevicular uid and saliva are rich in collagenases, gelatinases and elastases (133136, 149, 232, 263265). Most of these enzymes appear to derive from neutrophils migrating from inamed periodontium. However, salivary gland cells and activated epithelial cells and broblasts of periodontium produce their own matrix metalloproteases including collagenase-1 (matrix metalloprotease-1), collagenase-3 (matrix metalloprotease-13) and gelatinase A (matrix metalloprotease-2) (209, 211, 258). In concert the matrix metalloproteases and elastases are capable of cleaving all extracellular matrix proteins and proteoglycans. As discussed earlier, proteolytic enzymes are necessary for proper wound healing. These matrix metalloproteases could also release cryptic bioactive domains from matrix molecules that may regulate cell proliferation and migration (285). For example, bronectin and collagen fragments have been demonstrated in gingival crevicular uid (245) and saliva (77). Practically no information presently exists on the possible role of these extracellular matrix fragments in promoting healing of oral wounds. Role of bacteria in oral wound healing The oral cavity harbors considerable amounts of bacteria. More than 500 bacterial species have been so far identied in the oral cavity (165). Recognizing the limitation of the present detection methods, in reality several times more species may colonize in the mouth. It is clear that bacteria affect wound healing in the oral cavity, and it is well established that wounds colonized by pathogenic bacteria have delayed healing (198, 249). Clinicians are aware of the painful complications in extraction wound repair that result from bacterial infection (38, 57). Much

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less attention has been given to the fact that small concentrations of bacteria may increase the rate of wound healing. In 1921, Carrel reported that wounds of dogs treated with certain concentrations of Staphylococcus aureus healed faster than untreated wounds. Since then several studies have conrmed that nding, also using other bacterial species. Many factors may contribute to this effect. Inammatory reaction that is a prerequisite for tissue repair is accentuated by bacterial contamination. Bacteria present in a wound will attract macrophages into the area and induce their cytokine secretion. As a consequence, blood supply and granulation tissue formation are accentuated in the wound. Proliferation of mesenchymal cells is increased and synthesis rate of connective tissue components is stimulated, leading to greater tensile strength of the contaminated wounds in the course of healing (115, 127, 128, 136). Bacteria contain a variety of substances, some stimulating host cell proliferation and others exerting toxic effects. In addition, the same substance can be either stimulatory or inhibitory, depending on its concentration in tissue. Bacteria may act directly on epithelial cells and connective tissue cells in wounds and, depending on the type and concentration, either accelerate or delay wound regeneration. We found that proliferation of gingival broblats in culture was increased by Prevotella intermedius but decreased by the same concentrations of Porphyromonas gingivalis (133). Interestingly, there was a great variation in this effect between broblast populations obtained from different patients. These ndings imply that the potential for periodontal repair depends both on the bacterial ora and the individual cell populations of the periodontal wounds. Some bacterial factors have direct broblast and epithelial cell stimulating properties. Lipopolysaccharide of both Actinobacillus actinomycetemcomitans and P gingivalis slightly increases . cell growth in vitro (98, 189). At higher concentrations lipopolysaccharide from different bacterial species inhibits cell proliferation (8, 134). Lipopolysaccharide as well as bacterial plaque extracts increase hyaluronan production in cultured gingival broblasts (7, 130). Hyaluronan is a high-molecularweight polyanionic glycosaminoglycan that plays an important role in wound healing through specic interaction with other matrix molecules and cells (23). S. aureus lipoteichoic acid and protein A induce broblast production of hepatocyte growth factor, which stimulates epithelial proliferation (6). There are numerous other factors present in bacteria that

could modulate oral wound healing. For example, A. actinomycetemcomitans GroEL-like heat-shock protein and phospholipase C are able to modulate epithelial cell growth and cell migration (62, 80, 294). Specic mechanisms of these effects remain to be explored.

Conclusion
Wound healing in the oral cavity is a very complex process. We are just starting to uncover the complex interplay between various cell types, growth factors and salivary components. The focus of this chapter was to summarize the two major events of gingival wound healing, namely re-epithelialization and the formation of granulation tissue. Because of the unique environment of oral cavity, we also reviewed the special features of oral healing. The interplay between oral cells and their extracellular matrix, bacteria, saliva and gingival crevicular uid involves myriad factors dictating the nature of the tissue repair process. It would be an enormous if not impossible task to sort out all these factors. Some of the factors are already relatively well understood and could be used for practical applications. Active studies on certain growth factors are underway in order to provide new tools for periodontal therapy (101). Similar studies utilizing other factors benecial for wound healing can be expected when more basic research has been done elucidating their specic functions. Only then we can design precision tools to speed-up (or slow-down) re-epithelialization, granulation tissue formation and scarless wound healing. Modulation of celltoextracellular matrix adhesion will be the key target for computer designed drugs engineered to guide optimal tissue repair.

Acknowledgments
We thank Colin Wiebe for critical reading of the manuscript. Wound-healing studies performed in the laboratories of the authors are supported by the Medical Research Council of Canada.

References
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