Molecular factors in periodontal wound healing

The periodontium is composed of both soft as well as mineralized tissues and form a unique junction of epithelial and soft connective tissues against mineralized tissue. Chronic periodontal diseases are characterized by destruction of both the soft as well as the mineralized tissue components of the periodontium. While some of these components such as the junctional epithelium and lamina propria of gingival have a good potential for self renewal and repair, others such as cementum and to some extent alveolar bone have limited potentials. Complicating the healing/repair process in the periodontium is the ubiquitous presence of microorganisms. Conventional periodontal therapy focuses, in a step-wise manner, first on eliminating the etiologic factors for chronic periodontal disease (initial therapy phase) followed by the definitive/reconstructive phase and the eventual maintenance phase. It is important to recognize that healing of the periodontal tissues, albeit in increments, occurs at each one of these therapeutic phases. A good understanding of the healing events in the periodontium following periodontal therapy requires that molecular factors involved in healing be examined at the initial and reconstructive phases of periodontal therapy. Maintenance phase of periodontal therapy relies more on the control of etiologic factors and hence will not be discussed here.

Initial phase of periodontal therapy

The most often used therapeutic procedures during initial phase of periodontal therapy are scaling and root planing. While the objective of these procedures is the removal of calculus and the contaminated root surface so as to make the root surface biologically compatible for periodontal tissue healing, in principle the site represents a surgical wound. In biologic terms, surgical wounds are created by therapeutic procedures and are characterized by the removal of diseased tissues, loss of continuity/tissue integrity in tissues and the formation of a blood clot. A closer look at the periodontium following scaling and root planing reveals that there is removal of diseased tissue (pocket epithelium, granulation tissues, contaminated root surface layer, etc.), loss of continuity/tissue integrity (no junctional epithelium immediately after scaling/root planing) and a blood clot.

Definitive/reconstructive phase of periodontal therapy

Once the etiologic factors have been controlled and healing allowed to occur following initial therapy, the reconstructive phase involves surgical procedures aimed at either further eliminating etiologic factors and/or restoring tissues lost due to disease or surgical resection. The surgical procedures in this phase usually have well defined goals and involve facilitating the biologic restoration of tissue architecture. To achieve this, either the biologic factors alone or in combination with natural or engineered substitutes are utilized. For example, in the treatment of infrabony defects, bone grafts (autogenous/allogenic/synthetic substitutes) are used to enhance the extent of bone regeneration. Regardless of the use of tissue substitutes, the final outcome of the surgical procedure depends mainly on the biologic factors operating in the given surgical wound.

Whatever the objective of periodontal therapy is, the initial and reconstructive phases of periodontal therapy culminate in a surgical wound, the outcome of which depends on a variety of molecular factors. Based on their biologic activities, these molecular factors can be broadly classified into growth factors, molecules involved in cell adhesion and migration, proteolytic enzymes and angiogenic factors. Within the growth and cell adhesion categories, there are subdivisions such as factors that elicit cellular response, cellular receptors, signal transducers and transcription factors. Before analyzing these factors and their roles in detail, it is important to briefly review the different aspects of wound healing following periodontal therapy. Immediately following periodontal treatment, the periodontal wound is characterized by the presence of a blood clot, the size of which depends on the extent of tissue destruction caused by periodontal disease and the adaptation of flaps (^{Aukhil, 2000}). This blood clot serves as a transitional matrix and a reservoir of molecular factors (^{Clark, 1996}). As healing progresses, cells from the adjacent areas, epithelial as well as mesenchymal in origin, migrate into the wound in paths created by matrix-degrading enzymes. Once cell migration occurs, there is further modification in the composition of molecular factors in the healing wound, leading to angiogenesis, matrix synthesis and matrix maturation. In its final stages, there may be mineralization if new bone is being formed. It is important to note that during the various stages of periodontal wound healing, several molecules acting either singly or in conjunction with other molecules, are serving important functions. The healing periodontal wound can be best described as a dynamic matrix wherein there is ordered succession of molecular factors that is most suited to restore tissue architecture. This ordered succession of the expression of molecular factors varies in different areas of the wound with time and with the different cell types. The molecular factors in periodontal wound healing will be discussed in the context of their roles played.

Growth factors

Following periodontal therapeutic procedures, there is a need for replacement of epithelial and connective tissues and this occurs by the proliferation and migration of cells from the adjacent tissues. Growth factors are important since they induce proliferation of cells in the adjacent compartments and cell proliferation is usually a pre-requisite to cell migration from the borders of the wounds (^{Ahlen and Rubin, 1994} ; ^{Broadley et al., 1989} ; ^{Brown et al., 1986} ; ^{Gailit et al., 1996} ; ^{Pierce et al., 1994} ; ^{Schultz et al., 1987}, ^{Sporn and Roberts, 1986} ; ^{Sporn et al., 1983}). A partial list of the growth factors involved in periodontal wound healing is shown in ^{table 1}.

Platelet-derived growth factor (PDGF) is synthesized by a number of cell types and also acts on many different cell types (^{Heldin and Westmark, 1996}). PDGF stimulates the growth and chemotaxis of cells. PDGF is present at the sites of wounds and contributes to healing by its effects on growth, chemotaxis and matrix synthesis. PDGF is a family of isoforms made up of disulfide - bonded homo- or heterodimers of products of two genes - the PDGF A-chain gene and PDGF B-chain gene (^{Clark, 1996}). PDGF-AB is the most common isoform while homodimers (AA or BB) can also exist. Since the different isoforms interact with different receptors, they can serve different functions although there can be some overlap. PDGF has been known to stimulate the healing of soft tissue wounds (^{Heldin and Westmark, 1996} ; ^{Lynch et al., 1989}). It is important to note that healing wounds have different growth factors that influence the different phases in wound healing in different ways.

Transforming growth factor-ß (TGF-ß) is probably the cytokine with the broadest range of activities in repair of wounded tissues (^{Clark, 1996}). TGF-ß is produced by a variety of cell types and has a broad spectrum of cellular responses in a variety of cell types (^{Roberts and Sporn, 1996}). It affects nearly every aspect of wound healing and acts initially by inducing chemotaxis followed by the stimulation of the formation of granulation tissue. TGF-ß is a very potent stimulator of chemotaxis for monocytes, lymphocytes, neutrophils and fibroblasts. A variety of cell types (such as keratinocytes, osteoblasts, endothelial cells, fibroblasts and smooth muscle cells) are also stimulated by TGF-ß to produce extracellular matrix. Application of neutralizing antibody to TGF-ß1 and -2 isoforms in healing rat incisional skin wounds reduces cutaneous scarring (^{Shah et al., 1992}) suggesting that TGF-ß overexpression may be contributing to scarring. This possibility is also suggested by the low levels of TGF-ß1 in fetal wounds which are characterized by absence of scarring (^{Longaker et al., 1994}).

Fibroblast growth factors (FGFs) are a family of about 9 polypeptides that are expressed in a variety of cells. FGFs are capable of stimulating the proliferation and migration of fibroblasts, endothelial cells and keratinocytes (^{Abraham and Klagsbrun, 1996}). Exogenous application of FGFs in models of impaired healing has shown increased granulation tissue formation, cellularity and angiogenesis (^{Abraham and Klagsbrun, 1996}).

Molecular factors regulating cell adhesion and migration

Cell adhesion is a critical biologic event during wound healing. Epithelial and mesenchymal cells that need to migrate into the healing wound must adhere to the substrata they would be migrating on. A large number of extracellular matrix molecules mediate cell adhesion and many of these are adhesive glycoproteins. These adhesive glycoproteins have certain features in common. All adhesive glycoproteins are multidomain and multifunctional proteins, each bearing various binding regions for cells and other matrix components. These glycoprotein subunits have discrete structural domains having specific functional characteristics. Many of them contain the versatile cell-binding site, RGD (Arg-Gly-Asp) in their amino acid sequence (^{Yamada, 1991}). A third important feature of these adhesive glycoproteins is that their interactions with cells is mediated by a superfamily of transmembrane receptors, the integrins (^{Yamada et al., 1996}). A partial list of adhesive glycoproteins and the integrin receptors are shown in ^{tables 2} and ³.

Among the various cell adhesion molelcules, fibronectin is the most extensively studied (^{Hynes, 1990}). Hepatocytes secrete into human plasma the soluble form of fibronectin. In addition, fibroblasts and many other cell types synthesize the cellular fibronectin. Alternative splicing of the RNA transcript at three regions leads to several different splicing variants and the role of these splicing variants is not clear yet. The spliced domains EIIIB and EIIIA are usually present during embryonic development and are absent in adult plasma fibronectin. It is interesting to note that in adult wound healing, the splicing variant to include the spliced domains EIIIB and EIIIA reappear at the base of the wound, suggesting that some of the molecular events during wound healing may be mimicking those seen during embryonic development (^{French-Constant et al., 1989} ; ^{Brown et al., 1993}). Tenascin is a large extracellular matrix glycoprotein showing transient expression in some tissues (embryonic tissues and tumors) and a permanent expression in other tissues. While tenascin allows the adhesion of some cell types, the spreading of cells is limited. Tenascin also has antiadhesive effects in the region of the EGF-like repeats. In adults, tenascin appears at the edges of healing wounds beneath migrating, proliferating epithelial cells, suggesting that it may be involved in the migration of cells (^{Clark, 1996}). Growth factors such as TGF-ß, FGF and TNF-ß induce the expression of tenascin implying that the presence of these growth factors in wounds may in part be responsible for the upregulation of tenascin expression in wounds. Vitronectin is a multifunctional glycoprotein found in plasma and fibrin clots. Vitronectin mediates cell adhesion and migration. Thrombospondin is also a large multifunctional glycoporotein that is released upon the activation of platelets. Thrombospondin interacts with cells and binds to several extracellular matrix molecules. In addition to mediating the adhesion of cells, thrombospondin also regulates the growth of certain cells. Laminin is a large adhesive glycoprotein forming the major glycoprotein of the basement membrane and modulates diverse biologic functions. Like other adhesive glycoproteins, laminin is a multifunctional molecule and promotes adhesion, polarization, proliferation, differentiation and locomotion of many cell types.

Most of the adhesive glycoproteins described above and in ^{table 2} bind to specific transmembrane receptors from a superfamily of cell surface proteins called the integrins. Integrins are heterodimeric polypeptides made up of an a and a b subunit (^{Yamada et al., 1996}). Numerous types of α and ß transmembrane glycoprotein chains exist and ligand specificity is conferred by the mixing of particular a and b subunits. ^{Table 3} lists many of the integrin subunits and the ligands for the various heterodimeric glycoproteins.

Cell migration in wound healing is accompanied by changes in the expression profiles of integrins (^{Clark, 1996}). When cells are resting in their native matrices, they use certain integrins but in response to chemotactic signals, they prepare for migration. For example, keratinocytes use the integrin α6-ß4 to bind to laminin in the basal lamina and develop intracellular links with the cytoskeletal network. In response to wounding and in preparation for migration, the keratinocytes at the edge of the wound will have to dissolve the hemidesmosome attachment and begin the expression of other integrins (α5-ß1, αv-ß6 and αv-ß5) to bind to fibronectin, tenascin and vitronectin. As wound healing progresses, the fibroblasts, like the keratinocytes, also have to reorganize the integrin receptors in preparation for cell migration in to the wound. In the resting stages, fibroblasts are expressing integrins to bind to collagen but in preparation for cell migration, they will have to down-regulate the collagen integrins and up-regulate the integrins for fibronectin, vitronectin and tenascin. As one can see, the migration of cells is complicated and involves the preparation of cells by reducing the resting receptors (integrins) and reorganizing/increasing the receptors for molecules in the provisional matrix (clot) such as fibronectin, tenascin, vitronectin. Any measures to enhance cell migration should keep these issues in mind.

Matrix-degrading enzymes

Migration of cells through the fibrin clot or between the junction of clot and underlying connective tissue requires that clear migrating paths be created and this is achieved by the dissolution of the fibrin barrier by the enzyme plasmin. Plasmin is derived by the activation of plasminogen in clots by two activators-the tissue-type plasminogen activator and urokinase-type plasminogen activator (^{Martin, 1997}). Absence of plasminogen significantly delays wound healing. In addition to the fibrinolytic enzyme plasmin that causes lysis of plasmin, there are other proteases that degrade the matrix and clear pathways for cell migration. These include the matrix metalloproteinases (MMPs). Examples of these are MMP-1 (interstitial collagenase) that degrades native collagens at the borders of wounds, MMP-9 (gelatinase B) that cleaves collagens in basal lamina and MMP-10 (stromelysin) that has a wide spectrum of substrate specificity.

Angiogenesis of wounds

Formation of new blood vessels (angiogenesis) is crucial for successful wound healing. Several growth factors listed in ^{table 1} are important in the induction of angiogenesis in healing wounds. Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) are two important molecules involved in angiogenesis. FGF-2 is synthesized by macrophages and damaged endothelial cells while VEGF is synthesized by macrophages and wound-edge keratinocytes. The endothelial cells also have to change the expression profiles of their integrins in preparation for cell migration, just as the epithelial and fibroblast cells do. Angiogenesis is a very complicated process depending heavily on signals for endothelial cell proliferation, migration and formation of blood vessels. These angiogenic signals have not been completely understood yet.

Conclusion

In summary, wound healing is a very complicated molecular and cellular event where a very intricate series of overlapping events regulate how certain molecules direct the activities of certain cells and how certain cells by expressing some molecules, change the composition of this dynamic wound matrix. The end result of this coordinated series of events is a healed tissue architecture which may partly resemble the original tissue. Given the limited potential of periodontal tissues to regenerate, complete restoration of tissue architecture is, if not impossible, difficult at present. Recent advances have led to the identification of the structure and functions of these important molecules. Future studies should focus on how this new knowledge on molecular factors can be used to enhance wound healing and tissue regeneration in the periodontium.

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