Macromolecules in periodontal wound healing

Introduction

The healthy periodontium provides the necessary support to maintain teeth in adequate function and is comprised of four principal components namely the gingivae, periodontal ligament, alveolar bone and cementum (^{Bartold and Narayanan, 1998}). Each of these periodontal components is distinct in its location, biochemical composition and tissue architecture, yet together they function as a single unit. Recent research has revealed that various extracellular matrix components of one periodontal compartment can influence the cellular activities of adjacent structures. Thus, pathological changes occurring in one periodontal component may have significant ramifications for the maintenance, repair and regeneration of other components of the periodontium.

The connective tissues of the periodontium consist of both fibrous and nonfibrous elements including collagens, elastin, fibronectin, laminin, osteopontin, bone sialoprotein, proteoglycans and other noncollagenous proteins, a variety of growth factors, lipids, minerals and water. In this review, advances in our knowledge of the distribution of these components in normal and diseased periodontal tissues will be discussed and the principles that underlie the likely biochemical mechanisms necessary for the regeneration of the connective tissues destroyed by inflammatory diseases will be summarized.

Connective tissues in normal periodontium

Gingiva

Within the gingival connective tissues, type I and type III collagens are the main collagen species present (^{Narayanan et al., 1980}). These collagens are arranged as fibers in two patterns of organization. Large, dense bundles of thick fibers, consist predominantly of type I collagen while the loose, short and thin fibers mixed with a fine reticular network are composed mainly of type III collagen (^{Chavrier et al., 1984} ; ^{Narayanan et al., 1985}). Other collagens present in gingival connective tissues include types IV, V, VI. Type V collagen appears to coat dense fibers composed of type I and III collagens (^{Narayanan et al., 1985} ; ^{Romanos et al., 1991}). Type VI collagen is found near the basement membrane and is distributed in a diffuse microfibrillar pattern (^{Romanos et al., 1991}). Type IV collagen is found within the basement membranes located at the junctions of connective tissue with epithelium and cementum, in rete pegs and around blood vessels and nerves (^{Chavrier et al., 1984} ; ^{Narayanan et al., 1985} ; ^{Romanos et al., 1991}). The presence of type IV collagen appears to be with both the external and internal basal lamina of basement membranes (^{Sawada et al., 1990}).

Proteoglycans, which are also ubiquitous constituents of the periodontal tissues, are very heterogeneous in structure, function and location. This family of matrix macromolecules is classified into three groups depending upon their location and include :

- matrix organizers and tissue space fillers ;

- cell surface components ;

- intracellular proteoglycans of the hemopoietic cells.

All proteoglycans are composed of at least one glycosaminoglycan chain covalently bound to a core protein. Both the glycosaminoglycan chains and the core proteins confer various biologic and structural features on the various proteoglycans.

The glycosaminoglycans found in the gingival connective tissue include hyaluronan, heparan sulfate, dermatan sulfate and chondroitin sulfate of which dermatan sulfate and chondroitin sulfate are the predominant species (^{Bartold, 1987}). Gingival proteoglycans have been identified as decorin, biglycan, versican and several syndecans (^{Bartold et al., 2000}). Decorin is present within the gingival tissues closely associated with collagen fibers, especially in the subepithelial region. Biglycan is a relatively minor constituent of gingiva, but it appears to be localized in the matrix near the oral epithelium. Syndecan and CD-44 have been localized mainly to epithelial cells. In the gingival, the glycosaminoglycans are largely made by fibroblasts, which may synthesize up to six different proteoglycans including decorin, biglycan, versican and syndecan.

The gingival connective tissues also contain fibronectin (^{Pitaru et al., 1987}), osteonectin (^{Salonen et al., 1990}), tenascin (^{Steffensen et al., 1992}) and elastin (^{Bourke et al., 2000}). Tenascin is present diffusely in the gingival connective tissue, and prominently near the subepithelial basement membrane in the upper connective tissue and capillary blood vessels. Although elastin is a minor constituent of gingival connective tissue, it is more prominent in the submucosal tissues of the more movable and flexible alveolar mucosa.

Cells within the gingival connective tissues interact with their surrounding matrix through specific cell surface-associated molecules termed « integrins ». These are heterodimeric molecules composed of α and β-subunits and are classified on the basis of their β-subunit composition. Integrins, which are integral cell membrane components, are implicated in cell migration, cell-cell interactions, and clot formation. Accordingly, integrins are expressed in high proportions during wound healing (^{Ruoslahti, 1991}). Fibroblasts express specific integrins (express α1, α2, α5, αv, αv-β3) which can bind to matrix proteins such as fibronectin, vitronectin, collagen, laminin and fibrinogen (^{Ruoslahti, 1991}).

Periodontal ligament

In mature periodontal ligament, collagen fibers are classified as « principal fibers » and « secondary fibers ». The principal fibers traverse the periodontal space obliquely and insert into cementum and alveolar bone as Sharpey's fibers, while the secondary fibers are randomly oriented and located between the principal fibers investing nerves and blood vessels. The principal collagen species in Sharpey's (and other collagen) fibers of the periodontal ligament is type I and this collagen also constitutes the fibrous component of endosteal spaces (^{Rao et al., 1979}). Type III collagen coats the Sharpey's fibers. These two collagen types are codistributed with types V, XII (^{Karimbux and Nishimura, 1995}), and type VI collagen which is present as microfibrils (^{Romanos et al., 1991}). The type XII collagen has been implicated in the three-dimensional organization of the ligament extracellular matrix and expression of this protein appears to be restricted to terminally differentiated stages and associated with its regeneration. The ligament also contains undulin (type XIV collagen) tightly associated with the fibrils (^{Zhang et al., 1993}). Tenascin is also found in the periodontal ligament, where it is present in attachment zones along cementum and bone (^{Lukinmaa et al., 1991}). Fibronectin and vitronectin are found on collagen fibrils and the fibronectin is localized between cross-striated collagen fibrils and surrounding individual fibrils. Although small amounts of elastin and associated glycoproteins are present in the periodontal ligament connective tissue (^{Fullmer et al., 1974}), this component has been poorly studied.

The glycosaminoglycans present in the periodontal ligament are similar to those found in gingiva and include hyaluronan, heparan sulfate, dermatan sulfate and chondroitin sulfate, of which dermatan sulfate is the principal species (^{Gibson and Pearson, 1982}). Several proteoglycans have been identified in the periodontal ligament connective tissue including versican, decorin and biglycan (^{Häkkinen et al., 1993}). Immunohistochemical localization studies have shown these proteoglycans to be intimately associated with the collagen fibers of the periodontal ligament. Another proteoglycan, CD-44, has been found to localize to the surface of the resident fibroblasts (^{Hakkinen et al., 1993}). Syndecan 1 and 2 have been located in both the developing periodontal ligament as well as within the ligament of adults (^{Worapamorn et al., 2001}). Although yet to be identified, it would also be expected that other proteoglycans such as perlecan (in vessel basement membranes) would also be present in periodontal ligament.

Cementum

Cementum, one of the five calcified tissues found in mammals, has been a difficult tissue to characterize due to its limited quantity and distribution. Histologically cementum is similar to bone and dentin containing an organic matrix composed primarily of collagens and glycoproteins embedded within a granular mineralized matrix (^{Selvig, 1965}). Light microscopic studies have shown that fine, randomly oriented collagen fibers are embedded within the granular matrix of primary cementum, while in secondary cementum coarse collagen fibrils are oriented at right angles to Sharpey's fibers and parallel to the root surface. At least four different regions and types of cementum have been described on the basis of its cellularity and fiber content (^{Jones, 1981} ; ^{Schroeder, 1992}). At the dentoenamel junction « acellular afibrillar cementum » lacks both collagen fibers and cells, while in the cervical to mid root region, « acellular extrinsic fiber cementum » does not contain cells but does have a fibrous component (Sharpey's fibers). At the apical and interradicular root surfaces « cellular cementum » contains both extrinsic (Sharpey's) and intrinsic collagen fibers. « Reparative cementum » has only the intrinsic fiber system. Chemical and biochemical analyses of cementum have shown that approximately 50 % of the inorganic matrix is hydroxyapatite, whereas up to 90 % of the organic matrix is composed of types I and III collagens (^{Birkedal-Hansen et al., 1977}). Although the inserting Sharpey's fibers are made up from collagen types I, III and XIV (^{Zhang et al., 1993}), cementum also contains type V and VI collagens at pericellular locations (^{Romanos et al., 1991}). In addition, nonfibrous proteins such as bone sialoprotein (BSP), osteopontin, tenascin, fibronectin, osteonectin and several proteoglycans have also been identified in the organic matrix of cementum (^{Somerman et al., 1991}). Biochemical and immunohistochemical studies have identified a number of proteoglycans in the matrix of cementum (^{Bartold et al., 1990}). In particular, chondroitin sulfate and keratan sulfate are the predominant glycosaminoglycan components of cementum proteoglycans, although moderate levels of dermatan sulfate and hyaluronan have also been identified (^{Ababneh et al., 1998}). These glycosaminoglycan and proteoglycan components are closely associated with cementoblasts and are lightly distributed throughout the matrix (^{Bartold et al., 1988} ; ^{Ababneh et al., 1998}). More recently, syndecan-2 but not syndecan-1 has been localized in acellular cementum (^{Worapamorn et al., 2001}). A number of newly identified biologically active polypeptides which are relevant to connective tissue formation and tissue regeneration are also present in cementum as minor biochemical components (^{Somerman et al., 1989} ; ^{Narayanan et al., 1995}).

Alveolar bone

Although one would not expect the alveolar bone of the periodontium to differ significantly from similar bone elsewhere in the body it is remarkable that this tissue has not been studied in more detail. As for all of the other tissues of the periodontium, the collagens comprise the bulk of the organic constituents of bone with types I and III being the principal collagens identified (^{Rao et al., 1979} ; ^{Wang et al., 1980}). The collagen fibers of the periodontal ligament insert into alveolar bone through Sharpey's fibers. In addition to the collagens, biochemical analyses of alveolar bone extracts have revealed the presence of biologically active polypeptides including bone sialoprotein and osteopontin (^{Chen et al., 1993}). The distribution of integrins in alveolar bone has been studied with osteoclasts being found to express the integrin subunits αV integrin strongly and α5 to a lesser extent, while osteoblasts express the α5 and α2 subunits (^{Steffensen et al., 1992}). Although chondroitin sulfate is the major glycosaminoglycan in alveolar bone, heparan sulfate, dermatan sulfate and hyaluronan have also been identified in extracts of alveolar bone biochemically and in histological sections by immunohistochemistry (^{Bartold, 1990}). The sulfated glycosaminoglycans are located on osteocytes in their lacunae as well as in the surrounding mineralized matrix. The major proteoglycans identified in alveolar bone are chondroitin sulfate rich (^{Bartold, 1990} ; ^{Waddington and Embery, 1991}), and are most likely a mixture of decorin and biglycan.

The constituent collagen and proteoglycan types and their locations in various periodontal components of the periodontium are summarized in ^{table 1} .

Alterations to the periodontal connective tissues with the development of periodontal inflammation

Amongst the most prominent features of the inflammatory periodontal diseases are the qualitative and quantitative changes in the molecular composition of the periodontal connective tissues, especially in the gingiva. Following the initial accumulation of dental plaque adjacent to the gingival margin there is a very rapid establishment of an inflammatory infiltrate within the subjacent connective tissue. Within three to four days following this initial inflammatory response, connective tissue destruction occurs (^{Payne et al., 1975}). Within this time approximately 70 % of collagen within the foci of inflammation is lost. With further development of the inflammatory response, the destruction expands deeper towards the periodontal ligament and alveolar bone. Simultaneously with destruction, a form of frustrated repair is initiated with fibrosis and scarring coexisting at foci of inflammation (^{Bartold and Narayanan, 1998}).

As the lesion progresses from a contained gingivitis into a more progressive periodontitis lesion, biochemical analyses have demonstrated that numerous quantitative and qualitative changes occur to the gingival collagens (^{Narayanan et al., 1983}). Not only do the gingival collagens become more soluble, but the ratios of collagen types are altered. For example, the amount of type V collagen increases and may exceed type III, and a new collagen, type I trimer, may appear (^{Narayanan et al., 1985}).

Quantitative and qualitative changes also occur to the gingival proteoglycans, but these are not as marked as those noted for the collagens. There is a reduction in the amount of dermatan sulfate with a concomitant increase in the content of chondroitin sulfate. Furthermore, there is evidence of degradation of both proteoglycan core proteins and hyaluronic acid during the development of periodontitis (^{Bartold and Page, 1986}). Although there is no overall quantitative depletion of total proteoglycans within inflamed periodontal tissues it is very likely that there is a significant change in the types of proteoglycans present. For example in inflamed human gingiva changes in the distributions of decorin and syndecans have been reported (^{Oksala et al., 1997} ; ^{Manakil et al., 2001}).

Within other compartments of the periodontium (ligament, bone and cementum) the changes associated with the development of inflammatory periodontal disease have been less well characterized. The periodontal ligament manifests subtle topographical changes in the distribution of various glycosaminoglycan with the major change noted being an increase in the presence of chondroitin sulfate (^{Kirkham et al., 1992}). Changes to the hard tissue matrices of bone and cementum differ significantly due to their different anatomical location. Cementum may become altered due to its exposure to the oral or pocket environment in which there is a loss of collagenous attachment and changes in both the organic and inorganic content (^{Stepnick et al., 1975}).

As the lesion of periodontitis develops, the junctional epithelium migrates apically and is responsible for the formation of the pocket epithelium. This process requires not only cell proliferation but also migration of the cells over the connective tissue substratum which has been modified by the inflammatory process. Recent studies have identified variable expression of integrins and other adhesion molecules at the epithelial/connective tissue interface during the inflammatory process and subsequent migration of the junctional epithelium (^{Haapasalami et al., 1995}). In particular changes were noted in the distribution of type VII collagen, laminin-5, fibronectin, tenascin and β1 integrins in inflamed periodontal tissues when compared to healthy tissues.

Degradation and remodeling of connective tissue matrix

Matrix components also undergo degradation and remodeling during development, inflammation and wound repair, and during resorption in bone. Degradation occurs via the activity of matrix metalloproteinases (MMP) and reactive oxygen species or through phagocytosis of matrix components. Of particular interest to the molecular pathways of tissue remodelling, repair and regeneration are the processes associated with MMP activity and cellular phagocytosis of matrix components.

The MMP are a family of 20 or more metal-dependent enzymes which have a highly conserved and related gene structure and broad specificity (^{Nagase and Woessner, 1999}). MMP relevant to periodontal connective tissue remodeling and repair have been reviewed elsewhere (^{Birkedal-Hansen, 1993} ; ^{Reynolds, 1996}). Although the MMP have broad specificities, only the interstitial collagenases (MMP-1 and -8) cleave native collagen molecules. The various collagen types also differ in the rate of their susceptibility to different MMPs, and this may be one reason why differences occur in collagen type ratios in the diseased gingiva. For example, greater susceptibility of type III and resistance of type V and type I trimer to proteinases may be one reason why the amount of these collagens are less and more, respectively, in inflamed tissues.

MMP activity is controlled in vivo in three ways. The enzymes are synthesized and secreted as inactive precursors and conversion to the active form requires activation by plasmin, trypsin or other proteinases (^{Nagase et al., 1990}). Second, production of MMP is regulated by several growth factors and cytokines. Interleukin-1 (IL-1) and TGF-β are key regulators of MMP production in inflamed tissues whereby IL-1 increases, and TGF-β decreases MMP synthesis (^{Overall et al., 1991}). Finally, activity of MMP is neutralized by serum and tissue inhibitors. The major serum inhibitor is a α2-macroglobulin, which covalently crosslinks with and inactivates target MMPs. The α2-macroglobulin is a potent inhibitor to MMP-1 as it binds to the enzyme with even greater avidity than its substrate, collagen. Tissues contain another group of protein inhibitors to MMP known as tissue inhibitors of metalloproteinases (TIMP). At least four (TIMP-1, TIMP-2, TIMP-3, TIMP-4) have been characterized (^{Birkedal-Hansen et al., 1993}). These inhibitors inactivate MMPs by preventing the conversion of precursor forms of MMP to their active forms.

In the periodontium fibroblasts, epithelial cells, endothelial cells as well as inflammatory cells express a battery of MMP (^{Sodek and Overall, 1992}). However, differences exist among these cells in the type of MMP produced. For example, PMN-type collagenase (MMP-8) and 92 kDa gelatinase (MMP-9) are produced by the PMN. In contrast, fibroblasts express several MMPs including collagenase (MMP-1), but they do not produce MMP-8 or MMP-9 (^{Birkedal-Hansen, 1993}). The major MMP types present in the GCF are MMP-8 and MMP-9, but not 72 kDa gelatinase (MMP-2) or SL-1 (MMP-3) (^{Golub et al., 1976} ; ^{Gangbar et al., 1990} ; ^{Villela et al., 1987} ; ^{Sorsa et al., 1989} ; ^{Uitto et al., 1990} ; ^{Ingman et al., 1993}). Enzymes from fibroblasts and epithelial cells are believed to be involved in tissue remodeling of the periodontium (^{Meikle et al., 1994}).

Phagocytosis is also a significant pathway of collagen degradation in the physiological turnover and remodelling of periodontal connective tissues (^{Ten Cate and Deporter, 1974}). Cells which are defective in phagocytosis may contribute to gingival over-growth and fibrosis as well as compromise normal wound repair and tissue regeneration (^{McCulloch and Knowles, 1993}).

Effects of soluble mediators on connective tissue cells

In soft connective tissues, fibroblasts are largely responsible for producing and maintaining the matrix constituents. Under healthy conditions, the fibroblasts are embedded in a matrix composed of collagen and noncollagenous components and are sparsely distributed. The cells assume a flattened morphology indicating low metabolic turnover. However, unlike in most other tissues, the matrix of the gingiva and periodontal ligament has a high turnover rate presumably in response to tooth movement and inflammatory challenge (^{Sodek, 1976}). Following injury and inflammation, the fibroblasts are activated by factors present in the local environment. These factors are very potent and have a wide ranging effects on the cells which can affect migration, adhesion, proliferation and matrix synthesis (^{Clark and Henson, 1988}).

Amongst the myriad cytokines and growth factors known to affect fibroblast function, PDGF, IGF, TGF-β, IL-1, PGE2, IFN-γ and TNF-α are amongst the most potent.

Cytokines and growth factors influence cellular activities in several ways. These substances first bind to specific cell surface receptors ; the binding activates a variety of signaling events that include Ca ⁺⁺ mobilization, receptor phosphorylation, inositol phosphate hydrolysis, activation of protein kinase C, and tyrosine phosphorylation of focal adhesion kinase. These reactions result in activating cell migration, attachment, DNA synthesis and other cell functions. Frequently the expression of integrins and other cell surface receptors is affected, resulting in modification of cell-matrix and the cell surface interactions. The target genes could also be genes for other cytokines or growth factors, which in turn influence and regulate the activities of cells and cell to cell interactions (^{Page et al., 1997}). The various substances that affect matrix synthesis and degradation have been recently reviewed elsewhere (^{Slack et al., 1993}) and are listed in ^{table 2} .

Various cell types respond to specific cytokines differently. For example, IL-1 enhances type VII collagen synthesis significantly, while type I is not affected (^{Mauviel et al., 1994}). This difference may also be manifested by different subpopulations of the same cell type because not all the resident cells in a tissue react in the same manner to the inflammatory mediators. Evidence indicates that fibroblast cultures obtained from the same tissue explants consist of subtypes which differ in functional properties such as growth rate and collagen synthesis, and they respond differently to TGF-β, IFN-γ, PGE2 and other substances (^{McCulloch and Bordin, 1991}). Selective interactions between fibroblast subpopulations and inflammatory mediators have been shown to give rise to selection and enrichment of fibroblast subtypes and presence of such subtypes is believed to be one factor contributing to the selection of certain phenotypes in inflammation and repair (^{McCulloch and Bordin, 1991}).

Repair and regeneration of periodontal connective tissues

Tissue damage caused by gingivitis is reversible provided the causative agent(s) are removed. Indeed the gingival tissues have a remarkable capacity to regenerate to their original form and function following inflammatory or traumatic injury (^{Bartold and Narayanan, 1998}). However, with periodontitis, once the destructive phase reaches the deeper periodontal structures, regeneration does not usually happen on a clinically predictable basis. One of the major challenges in contemporary periodontal therapy is to reestablish soft tissue attachment to the root surface and to restore lost bone ; this requires regeneration of gingival connective tissues destroyed by inflammation, formation of new cementum and restoration of bone loss, as well as attachment of connective tissue fibers to previously diseased root surfaces (^{Schroeder, 1992}). The molecular and cellular events associated with periodontal regeneration are extraordinarily complex and require the participation of all components of the periodontium. These include an initial inflammatory component, recruitment of cell populations, their proliferation, differentiation and synthesis of matrix constituents. The periodontal system is unique because in order for new connective tissue fibers to insert into in cementum and bone, the healing components of both the soft tissues and the hard tissues of the periodontium need to be coordinated and integrated. Periodontal regeneration presumably involves several cell types, fibroblasts for soft connective tissues, cemento-blasts for cementogenesis, osteoblasts for bone and endothelial cells for angiogenesis (^{Pitaru et al., 1994}). During the regenerative process these cells must interact with a variety of soluble mediators such that the course of regeneration is dictated by cell-factor, cell-matrix and cell-cell interactions. Very little is known about the signals which regulate these interactions. In the adult patient, periodontal connective tissues contain heterogeneous populations of cells with diverse properties and functions (^{McCulloch and Bordin, 1991}). The fibroblasts originate from gingival and periodontal ligament connective tissues, while cementoblasts are believed to originate from ancestral cells in the ligament and bone (^{Pitaru et al., 1994}). Cementoblasts may be derived from precursor cells located paravascularly in periodontal ligament and in endosteal spaces of alveolar bone (^{McCulloch, 1985}). In humans such cells may be present as subpopulations of cells in all periodontal tissues (^{Pitaru et al., 1994}). Whether these represent populations of stem cells within the adult periodontium remains to be established. Irrespective of their genesis, for regeneration to occur, the cells responsible for each process must be able to participate at the right location and temporal sequence. More importantly, for successful regeneration certain cells, especially the epithelial cells, should be excluded from the healing site. This selection process is presumably dictated by signaling systems in which soluble agonists, the matrix and cell surface receptors participate.

For many years attempts have been made to exploit emerging principles of wound repair for periodontal therapy (^{table 3}). In one approach, conditions were created to allow selective repopulation on root surfaces by cells responsible for regeneration. For this reason root surfaces were « conditioned » in early attempts either by demineralization of root surfaces, or by coating root surfaces with chemical agents such as fibronectin, or both. The demineralization procedure was believed to reverse periodontitis-induced hypermineralization and to expose old collagen fibers with which newly formed fibers could interdigitate. Exposed collagen fibers were also expected to discourage the attachment of unwanted epithelial cells. However, this procedure did not yield predictable regeneration, and often caused ankylosis and root resorption as side effects instead. The advantage of using fibronectin root surface coating was also unclear because serum contains high fibronectin levels and providing additional protein is unlikely to have any beneficial effect.

More recently, a novel procedure has been utilized in which a physical barrier was introduced by surgically placing a membrane between connective tissue of periodontal flap and curetted root-surface. The membrane was expected to prevent apical migration of gingival (epithelial) cells onto the root surface, to exclude unwanted gingival connective tissue and facilitate the repopulation of the wound site with periodontal ligament cells (^{Nyman et al., 1982} ; ^{Gottlow et al., 1986} ; ^{Karring et al., 1993}). This « guided tissue regeneration » procedure demonstrated, for the first time, that regeneration of root cementum, alveolar bone and periodontal ligament, and new attachment formation are possible. Although the clinical results of this procedure are variable, it has gained wide acceptance (^{Minabe, 1991} ; Wikesjo et al., 1992 ; ^{Karring et al., 1993}), and currently several resorbable membranes are being evaluated as physical barriers. Several studies have reported that this technique results in tissues forming underneath barrier membranes containing cells and matrix macromolecules found in bone and cementum (Amar et al., 1995 ; ^{Amar et al., 1997} ; ^{Ivanovski et al., 2000} ; Kawaguchi et al., 2001). Clonal analysis of cells populating these tissues have shown the presence of several subsets of cells which have a regenerative phenotype with capabilities to respond to growth factors and produce mineralized tissue macromolecules such as bone sialoprotein, bone morphogenetic proteins, osteopontin and osteocalcin (^{Ivanovski et al., 2001}).

In another approach to induce periodontal regeneration, polypeptide growth factors have been locally applied to the root surface in order to facilitate the cascade of wound healing events that lead to new cementum and connective tissue formation. Among the myriad growth factors currently characterized and available, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulinlike growth factor (IGF), PDGF and TGF-β have been proposed to be of potential use in relation to their regulatory effects on immune function, cells of epithelium, bone and soft connective tissues. Two of these growth factors, PDGF and IGF-1, enhance regeneration in beagle dogs and monkeys with periodontal disease (^{Lynch et al., 1991} ; ^{Rutherford et al., 1993}). Another promising group of polypeptide growth factors is the bone morphogenetic proteins (BMP) which offer good potential for stimulating bone and cementum regeneration (^{Ripamonti and Reddi,1994}).

Enamel matrix proteins, produced by Hertwig's epithelial sheath, are known to play an important role in cementogenesis, as well as in the development of the periodontal attachment apparatus (^{Ten Cate, 1996} ; ^{Hammarström, 1997}). There is some evidence that these proteins also play a role in regeneration of periodontal tissues after periodontal therapy (^{Hammarström et al., 1997} ; ^{Wilson 1999}). In vitro studies have demonstrated that EMD addition to cultures of periodontal fibroblasts results in enhanced proliferation, protein and collagen production as well as promotion of mineralization (^{Gestrelius et al., 1997} ; ^{van der Pauw et al., 2000}). As no specific growth factors have been identified in EMD preparations (^{Gestrelius et al., 1997}), it is postulated that EMD acts as a matrix enhancement factor, creating a positive environment for cell proliferation, differentiation and matrix synthesis (^{Haase and Barthold, 2001}).

The importance of cementum in the formation and regeneration of periodontal tissues cannot be underestimated. New attachment requires new cementum formation to replace diseased root contaminated with bacterial endotoxins and which is removed during therapy. Although some information is available on the biology of cementogenesis during development (^{Saygin et al., 2000}), how cementum formation is regulated in adult humans is not clear. The connective tissue matrix of cementum sequesters FGFs and a battery of other growth factors, including osteopontin, BSP and other polypeptides which mediate cell adhesion and spreading (Bosshardt et al., 1998 ; ^{Saygin et al., 2000}). These molecules affect the migration, attachment and proliferation of periodontal cells and their matrix synthesis and, more importantly, they manifest cell specificity, and tissue specificity among the same cell type (^{Somerman et al., 1989} ; ^{Narayanan et al., 1995}). The extracellular matrix of cementum has the potential to regulate the differentiation of precursor cells into cementoblasts and the subsequent formation of cementum matrix and fiber insertion (^{Diekwisch, 2001}). Thus, cementum components are capable of providing informational signals for the recruitment, proliferation and differentiation of periodontal cells, and regulate the regeneration of cementum as well as adjacent periodontal components.

Notwithstanding these elegant developments over the past 40 years, there is no doubt that the desired clinical endpoint is predictable regeneration of the periodontal tissues damaged by inflammation to their original form and function. To date, this has been elusive. In order for regeneration to occur there will need to be the coordinated deposition of specific matrix molecules consistent with both soft and hard connective tissue formation and this will be largely driven by soluble cytokines and growth factors. A summary of the molecular requirements for periodontal regeneration is shown in ^{table 4} .

New perspectives

As the etiologic and pathologic process of the inflammatory periodontal diseases are better understood new vistas in management of these problems will evolve. Most likely these will develop along three fronts and address issues relating to the etiology of the disease, the host response to the disease and the anatomic changes occurring as a result of the disease. With respect to the issues discussed in this review, future developments in connective tissue biochemistry will have a significant impact on dealing with the host response to disease as well as managing the anatomic changes due to the disease process.

With the emerging principles of inflammation indicating the important relationship between polypeptide signalling mechanisms and tissue destruction, the control of tissue destruction via control of these processes will become an important therapeutic goal in periodontics. Recently advances have been made in the development of agents which block enzymatic activity, cytokine activity and other inflammatory agents such as prostaglandins and free radicals and indicate this to be a fruitful avenue for future investigations. Nonetheless, one should not forget that the potential shortfall of such approaches is that they deal with the « effect » of the disease, rather than the « cause » of the disease. Thus, any therapy which aims to modulate host responses should be used in conjunction with other modes directed to modifying the causative agent(s) of the disease.

To understand the rational basis of regenerative procedures, more information is needed on the variety of molecular and cellular processes associated with the formation of each periodontal component. In particular, very little is known about cementogenesis and the mechanisms necessary for reattachment. While the use of growth factors shows some promise in this area, these suffer from being very broad in their range of activity and thus lack a degree of tissue specificity. Therefore, it seems reasonable to continue to probe local factors which may be specific for the development (and therefore regeneration) of the periodontal tissues. To this end efforts to characterize cementum components have provided new perspectives and possibilities in the role that local factors may play in periodontal regeneration.

Recent developments in the field of tissue engineering, which is the science of developing techniques for the fabrication of new tissues to replace damaged tissues, provide new horizons in the field of periodontal regeneration (^{Bartold et al., 2000}). The concept of tissue engineering takes into account the need for regenerative treatment of periodontal defects with an agent or procedure requires that each functional stage of reconstruction be grounded in a biologically directed process. This biological technology, together with the emerging field of nanotechnology (the science of bioengineering at the molecular level to produce materials of hitherto unknown, and unthought of properties) will pave the future of periodontal regeneration. To this end the rational use of biodegradable scaffolds, informed use of instructive molecular messengers and the selection of specific cell phenotypes or even stem cells will form the basis of periodontal regeneration in the new millennium.

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