j.1600-0757.1997.tb00094.x

Periodontology 2000. Vol. 13, 1997, 20-40 Printed in Denmark. All rights reserved

Coavricrht 0 Munkscraard 1997

PER 10 Do NTO LO GY 2 0 0 0 ISSN 0906-6713

The periodontal ligament: a unique, multifunctional connective tissue WOUTER BEERTSEN, CHRISTOPHER A. G. MCCULLOCH & JAROSLAV SODEK

The periodontal ligament is the soft connective tissue interposed between the roots of our teeth and the inner wall of the alveolar socket. Its fibers form a meshwork that stretches out between the cementum and the bone and is firmly anchored by Sharpeys fibers (Fig. 1). The periodontal ligament links the teeth to the alveolar bone proper, providing support, protection and provision of sensory input to the masticatory system. The fibroblasts of the ligament originate in part from the ectomesenchyme of the investing layer of the dental papilla (181, 1831, and this developmental origin may give these cells specialized properties. In a number of respects they are different from cells in other connective tissues. Their properties are addressed in this chapter, both from a morphological and a functional point of view. Our objective is to illustrate how the unique properties of the periodontal ligament endow this tissue with functional attributes that are not replicated by other tissues. Where possible, reference will be made to studies on the periodontal ligament of human teeth. The reader should always keep in mind, however, that very little information about the periodontal ligament has come from direct observations in humans.

How unique is the periodontal ligament?

Experiments in dogs (92), monkeys (4, 127) and rodents (201) have shown that when periodontal ligament cells are removed from the cementum by mechanical means or displaced or altered under the influence of certain compounds, such as the bisphosphonate 1 -hydroxyethylidene- 1,l-bisphosphonate, ankylosis may occur. Bone tissue invades the peri-

odontal ligament space and establishes a direct con- nection between the tooth and the wall of the alveolar socket. Although such ankylosis can exist for some time, this nonresilient type of tooth support usually leads to loss of function and to resorption of the tooth root. Thus, migration and eruption of teeth can no longer occur, and the adaptability of the periodontium is greatly impaired.

Sometimes localized ankylotic areas can be removed and the integrity of the ligament restored when periodontal ligament fibroblasts or their progenitors are allowed to gain access to the root and repopulate the area. These cells may come from the adjacent periodontal ligament and perhaps also from contiguous endosteal spaces in the alveolar process but probably cannot differentiate from gingival cell populations. Invasion of periodontal ligament fibroblasts in an ankylosed site must be pre- ceded by cells that have the capacity to resorb bone and/or cementum (such as osteoclasts). A new periodontal ligament space may be created at the cost of the cementum (and sometimes part of the dentin) and also of the alveolar process. Shortly thereafter, this space is colonized by periodontal ligament cells that form new periodontal ligament fibers, new cementum and a new alveolar wall in which the fibers insert (201). Neither the nature of the signals that initiate this repair process nor the molecular mechanism associated with cellular interactions is clearly understood. However, it is known that under certain conditions bone can be replaced by a functionally oriented periodontal ligament of normal structure and architecture, provided that the progenitors for periodontal ligament fibroblasts can repopulate the site. Moreover, masticatory function can accelerate the resolution of ankylotic areas and restoration of normal periodontal ligament width (3, 56, 199, 201).

The notion that periodontal regeneration specifi-

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The periodontal ligament: a unique, multifunctional connective tissue

Fig. 1. Light micrograph of human periodontal ligament showing the collagenous fiber meshwork interposed between the root cementum (C) and the socket wall (B).

Note the presence of Sharpeys fibers in the bone (arrows). Azan stain, x250.

cally requires periodontal ligament cells is under- lined by an experiment of Boyko et al. (401, who seeded fibroblasts from either periodontal ligament or gingiva onto the surface of tooth roots, which

were subsequently inserted in artificial sockets pre- pared in edentulous areas of dog mandibles. Apart from ankylotic areas, they observed after some time areas with newly formed periodontal ligament fibers

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Fig. 2. Electron micrograph of periodontal ligament of rodent molar showing fibroblasts oriented parallel to the collagen fibers. Cementum (C); socket wall (B); nucleus (N), x2000.

Fig. 3. High-power electron micrograph of collagen fibrils in human periodontal ligament inserting into acellular extrinsic fiber cementum [ x 25,000).

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Fig. 4. Light micrograph of distal aspect of human premolar. Note the presence of many cement lines (arrowheads) in the socket wall (B) indicating sequential periods

of bone formation. Cementum (C); periodontal ligament (P); Sharpeys fibers (arrows). Hematoxylin and eosin stain, x 130.

inserted in newly formed cementum. This outcome was observed only when periodontal ligament fibroblasts were used as seeding cells: gingival cells could not induce regeneration of the periodontal ligament (98).

So far, no one has succeeded in establishing a new and normally functioning periodontal ligament without periodontal ligament cells, indicating that these cells have specialized properties. Below we de- scribe some of these properties in terms of structure, architecture, functioning and regenerative potential, with special emphasis on cellular and molecular regulation. Given the scope of this chapter, we do not cover every aspect of the biology and physiology of the periodontal ligament. More complete reviews of the periodontal ligament are available elsewhere (31-33, 157).

cementoblasts. The sensory system and the vasculature of the periodontal ligament have been reviewed (97, 101, 167). The predominant cell type is the fibroblast (Fig. 21, which (in rodent molars) occupies about 35% of the volume of the periodontal ligament space (blood vessels excluded) (14) and in sheep incisors about 20% (33). For adult human premolar teeth a comparable cell density (ca 25%) has been found (W. Beertsen, unpublished observations).

Matrix

The major fibers of the ligament are mainly collagenous in nature and consist of bundles of cross- banded fibrils. In the adult human the individual fibrils are slightly thicker than those in several other mammalian species, such as rodents (169), and measure about 54-59 nm in diameter (107). They are firmly anchored in the cementum and the bone by Sharpeys fibers (Fig. 3). Along appositional surfaces of the socket wall (tension side), Sharpeys fibers can often be followed over great distances, far exceeding the width of the ligament. As they traverse the bone they cross many cement lines, representing sequential periods of active bone formation (Fig. 4). The demarcating lines are relatively rich in the noncolla- genous proteins osteopontin and bone sialoprotein

Compositional and structural aspects

The healthy periodontal ligament contains several cell populations comprising fibroblasts, endothelial cells, epithelial cell rests of Malassez, cells associated with the sensory system, bone-associated cells and

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Fig. 5. High power electron micrographs of intercellular junctions between periodontal ligament fibroblasts. a. Gap junction. b. Adherence type junction. Collagen fibrils (c); oxytalan fiber (ox). a: ~100,000; b: ~60,000.

(118, 119) which are thought to play a role in the local regulation of bone metabolism. The great length of the Sharpeys fibers on the appositional side of the socket wall suggests interstitial fiber growth where the fiber bundles are incorporated into the bone. Evidence for this arises from labeling studies in mouse molars showing uptake of considerable amounts of the collagen precursor H-proline (72).

The collagen fiber bundles in the ligament and the fibers of Sharpey are mainly composed of interstitial collagens I and I11 (43, 84, 196), which form banded fibrils. Collagen V (one of the minor collagen molecules) is associated with these fibrils (1031, and is either buried within the core of the fibrils (35) or is found in the spaces between the fibril bundles (12). All of these collagens are essential for the normal architecture of the ligament, and none is restricted to particular regions of the periodontal ligament (12, 48, 146). In addition to collagen V, the fibrous meshwork of the mature periodontal ligament has been reported to contain several other minor collagens: collagens VI and XI1 (12, 41, 53, 90, 170). Whereas

collagen VI is a microfibrillar component, not directly associated with the major banded collagen fibrils (but with the oxytalan fiber system (64)), collagen XI1 belongs to the fibril-associated collagens with interrupted triple helices that contribute to the construction of the three-dimensional fibril arrange- ment (130). Temporal and spatial expressions of collagens I and XI1 in the remodeling periodontal ligament during experimental tooth movement suggest that collagen XI1 is closely associated with regeneration of periodontal ligament function (89). Besides collagens, several other proteins occur in the extracellular matrix of the ligament that may have a relationship with the macromolecular organization. These include several proteoglycans (57, 81) and glycoproteins (such as undulin and fibronectin (207)).

Fibroblasts

The fibroblasts of the periodontal ligament are interconnected by numerous junctions which can be cat- egorized as gap- and adherence-type junctions (Fig. 5) (18, 161). From observations on the rodent molar ligament, it was estimated that each fibroblast is in direct contact and communication with neighboring cells by about 20 of such intercellular junctions (18). Although communication between cells and mutual interaction are facilitated via junctional complexes and electrical coupling, chemical signals (paracrine factors) are also believed to be important. The fibroblasts in the ligament are oriented more or less parallel to the collagen fibers (Fig. 21, whereas in cross-sections they may exhibit a stellate appearance, with cytoplasmic processes segregating individual bundles of collagen fibers (Fig. 6). The cells may attach to the collagen via a fibronexus-type of attachment plaque (74, 85, 168) and are likely to have the capacity to orient the extracellular matrix (82). Together with their interaction with the collagenous framework, periodontal ligament fibroblasts may also interact with an entirely distinct system, the oxytalan fibers (21) which are predominantly oriented in the apicocoronal direction, seemingly unrelated to the collagen fiber system (Fig. 7). These fibers were described initially by Fullmer (68) and resemble pre-elastic fibers both histochemically (69, 145) and ultrastructurally. They contain a glyco- protein with a molecular weight of 140 kDa (162) and are associated with type VI collagen (64), one of the minor collagens that can mediate cell attachment (10). Although their resemblance to elastin suggests that the oxytalan fibers may add to the elastic properties of the periodontal ligament, their function and

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extracellular matrix, properties that depend on the level of expression of a-smooth muscle actin (6, 7). Migration and contraction are in fact basic properties of many connective tissue cells and are of great importance during developmental processes and wound healing. For these activities the cytoskeletal apparatus is a prerequisite. Drugs that dis- rupt the cytoskeleton also interfere with periodontal ligament cell migration and eruption of rodent incisors (27, 30, 45). Such drugs prevent the contraction of collagen gels in which periodontal ligament fibroblasts are seeded and the movement of pieces of dentin attached to it (6,27). Further, the migration of periodontal ligament fibroblasts into wounded rat molar ligament is impaired when the alkaloid colchicine is locally administered to the rat lower jaw via

Fig. 6. Light micrograph of periodontal ligament of human premolar. Note that collagen fiber bundles (blue) are cut transversely and segregated by cytoplasmic processes of connective tissue cells (red). Azan stain, x 500.

cellular origin are unknown. Several functions have been tentatively ascribed to this system, such as regulation of vascular flow (165, 166) and facilitation of fibroblast attachment and migration (21).

Fibroblast function

Although the migratory and contractile properties of periodontal ligament fibroblasts have been documented in several animal and in uitro studies, little if any is known about the migration and contraction of cells in the healthy and functioning periodontal ligament in humans. Migration of fibroblasts has been shown in the periodontal ligament of continuously erupting teeth (13, 14, 22, 206) and in teeth of limited eruption during normal function (116, 136) and during wound healing (76). There is also evidence that cells in the vicinity of the alveolar wall and the tooth surface may arise from precursor cells that have divided in other locations (116). Peri- odontal ligament fibroblasts, as most connective tissue cells, are quite rich in cytoplasmic microfilament systems which are indispensable for contrac- Fig. 7. Oxytalan fibers (stained dark purple) running in

and movement (211 25J 134). et (259 the anico-occlusal direction. Note that their course does 26, 28) have shown that Periodontal ligament fibre- blasts in vitro strongly contract and can orient their

not ckespond with that of the collagen fibers in the periodontal ligament. Cementum (C), x 500.

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a mini-pump (W. Beertsen L? T. Van den Bos, unpublished). How the cells direct their migratory and contractile activities and what signals are important are largely unknown. Perhaps chemoattractants produced locally or by the hard tissue cells bordering the ligament may have a role in these processes (129). Directed migration of cells (and perhaps also other functional activities) is associated with polarity of organelles: the nucleus is usually in the trailing portion of the cell, and the Golgi apparatus and cen- triolar region are just in front of the nucleus towards the leading edge of the cell, as is seen for instance in migratory neutrophils (110). In this context, it is intriguing that fibroblasts in rodent molar periodontal ligament are polarized (19, 71). Although the biological rationale of this phenomenon is still not understood, it could have a bearing on directed activities of the cells, such as directed migration or contraction or directed synthesis and breakdown of matrix components (19, 71, 73). Structural polarity is perhaps also fundamental to fibril orientation in the ligament, a phenomenon that to date has received very little attention in periodontal literature. In fact, the mechanisms by which fibrils and fibers in the ligament (both collagen and oxytalan) are oriented are unclear, except that fibroblasts are likely to be important in this process.

Periodontal ligament fibroblasts have several other characteristics which may help in distinguishing them from other connective tissue cells in the body. For instance, they appear to be rich in alkaline phosphatase activity (23,77,79). This enzyme is notable in that it plays a key role in phosphate metabolism, probably in mineralization processes (24, 184, 191) and perhaps also in acellular (afibrillar) cementum formation (78, 79). In this respect it is of interest that alkaline phosphatase activity in rat molar periodontal ligament shows a high correlation with acellular cementum thickness (79) and that in patients suffering from hypophosphatasia - a recessive hereditary disease in humans characterized by very low concentrations of alkaline phosphatase in the blood and in calcifying tissues - cementum formation is greatly impaired (202). The enzyme is not restricted to cementoblasts and osteoblasts but is found in all periodontal ligament fibroblasts, especially along their outer plasma membrane face. Some alkaline phosphatase (about 10%) is strongly associated with the extracellular collagenous fiber framework (80). What is not clear from these data is why the entire periodontal ligament does not mineralize in normal function in view of the relatively high constitutive expression of alkaline phosphatase activity.

Other cells

Apart from fibroblasts, the periodontal ligament contains the epithelial rests of Malassez (Fig. 8) which are derived from Hertwigs epithelial root sheath. The root sheath breaks up after initial root formation and persists in the ligament as a wide- meshed network (basket) of cells close to the tooth surface. The cells have characteristics typical of epithelial cells in that they are interconnected by de- smosomes, contain tonofilaments and are sur- rounded by a basal lamina. Although there has been considerable speculation about their physiological role, no evidence has emerged to support a specific function (177, 200). In humans, rests of Malassez are more frequent on the mesial side of molars than on the distal side (188). In the mouse, rests of Malassez are 3-4 times more frequent in the periodontal ligament of the mesial root of the first molar as compared with all other sites (200). In humans and other mammals, their number tends to decrease with age along all aspects of the root (147, 164, 188,200). Dur- ing repair of molar periodontium in the mouse after prolonged administration of the bisphosphonate 1 - hydroxyethylidene- 1,l -bisphosphonate (which induces localized ankylosis and narrowing of the ligament space), re-formation of the periodontal ligament does occur without the re-appearance of rests of Malassez (200), suggesting that these cells do not play a critical role in maintaining the normal width of the ligament and in cementum formation and do not prevent ankylosis and root resorption, as has been suggested by several groups of workers (102, 105).

The cementoblast is of central importance in periodontal ligament physiology and function. However, since cementum is extensively dealt with elsewhere in this volume (391, we will be very brief on this. For a comprehensive review, see Schroeder (157) and Freeman (67).

Although it is widely held that cementoblasts are responsible for the formation of all types of cementum, there is only convincing evidence for their involvement in the formation of cellular cementum, which is usually found in the apical two thirds of the root (37, 98, 157). The cementoblast forms a ce- mentoid layer, which soon after its deposition un- dergoes mineralization. Some of these cells are buried deeply in their own matrix and remain behind as cementocytes, not unlike osteocytes in bone. It seems doubtful whether specialized cementoblasts occur adjacent to the acellular cementum at all developmental stages in all mammalian species. At


Fig. 8. Rest of Malassez in rat molar periodontal ligament. Basal lamina (arrowheads); nucleus (N); X5000.

least in adult rodents (except for the growing ends of the continuously erupting incisors) and in humans no connective tissue cells, distinct from periodontal ligament fibroblasts, can be seen in the vicinity of acellular cementum layers. Only during the initial stages of acellular cementum formation in rat molars is there demonstrable evidence for a specialized cell type (46). Cho & Garant (46) believe that cementoblasts, derived from the ectomesenchy- mal cells of the dental follicle, detach from the cementum surface and contribute to early periodontal ligament fibroblast populations.

Matrix remodeling and adaptability Numerous studies have indicated that matrix proteins of the periodontal ligament have an extremely high turnover and remodeling rate, much higher than in gingiva, skin and bone (171). Although these studies have been performed in animal species other than humans, there is evidence, albeit indirect, that human periodontal ligament also has a high turnover rate and can easily adapt to changing local conditions. This section discusses some of the cellular

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Fig. 9. Diagrammatic representation of collagen phagocytosis by fibroblasts. 1. Cytoplasmic protrusions of fibroblast (Fi) surrounding a collagen fibril (co). 2. Lyso- some (Ly) fusing with collagen-containing phagosome and releasing its proteolytic content. 3. Phagolysosome (PL). Reproduced with permission from Davidovitch Z, ed. Biological mechanisms of tooth eruption and root resorption. Birmingham AL: EBSCO Media, 1988.

characteristics that are fundamental to the adaptability of the periodontal ligament.

Both turnover and remodeling are characterized by the coordinated breakdown and synthesis of extracellular matrix components. In contrast to remodeling, turnover describes a process in which the structural organization of the tissue remains un- changed. During remodeling the three-dimensional organization of the fiber meshwork is adapted to ac- commodate for positional changes of the tooth in its socket or changes in functional state (such as hypofunction (15)). Both processes can occur simul- taneously and may therefore be indistinguishable. It has been suggested that the rapid remodeling is a unique characteristic of the periodontal ligament that relates to the adaptability of the periodontal tissues (171).

Turnover and remodeling in the periodontal ligament involve rapid synthesis and breakdown of matrix components, most notably the collagenous meshwork that stretches out between cementum and bone. As mentioned above, the fibroblasts of the periodontal ligament form a highly structured and interconnected network, which strongly suggests the coordinated action of cells. The collagen fibers of the

ligament form a meshwork of smaller fibers, each of which is composed of unbranched collagen fibrils, which may run from one fiber strand into another. Although Sicher (163) suggested that remodeling in the ligament is particularly confined to an intermediate plexus in the mid-region of the periodontal ligament, where fibers from the bone and fibers from the tooth intermingle, turnover and remodeling in teeth of limited eruption, like the molars of rodents, appear to occur throughout the periodontal ligament from cementum to bone (16, 17, 150). Even Sharpey's fibers during their incorporation into alveolar bone and cementum seem to be exposed to considerable remodeling activity, as indicated by 3H- proline incorporation (72).

In order to adapt to positional changes of teeth, the fiber systems in the periodontal ligament must be broken down. Because the collagen in the periodontal ligament appears to form a complex meshwork, not unlike a stretched fishing net, breakdown processes can occur at different sites without com- promising tissue integrity. Thus, there is flexibility in the system to permit adaptive changes by breaking down short stretches of collagen fiber bundles or single fibrils while leaving others intact. Although it is still widely thought that degradation of collagen requires the activity of collagenase in the extracellular space, biochemical studies have shed some doubt on the role of this enzyme under steady state conditions (60). First, despite the extremely short half life of the collagens in the periodontal ligament (1721, which has been estimated to be on the order of several days in rodents (1711, collagenase has not been detected in periodontal ligament tissues. Sec- ond, substantial evidence indicates that collagen degradation in the ligament occurs intracellularly following a process of phagocytosis by periodontal ligament fibroblasts (Fig. 9, 10) (21, 59-64, 66, 70, 104, 160, 180). Third, in vitro studies have shown that collagen fibrils taken up by fibroblasts can be broken down in lysosomal structures by the activity of cysteine proteinases (61, 194) without the involvement of collagenase (63). Lysosomes isolated from pig periodontal ligament fibroblasts degrade collagen and contain cathepsins B, D, H and N (195). In- deed there is substantial evidence to indicate that the intracellular and extracellular routes of collagen breakdown constitute separate pathways (62, 192, 193). Studies by Svoboda et al. (179) have shown a relationship between turnover rate of collagen in the various tissues constituting the periodontium and the amount of collagen ingested by fibroblasts, the highest amount being found in the tissues with the

The Deriodontul ligament: a unique, multifunctional connective tissue

Fig. 10. Collagen fibrils internalized by human periodontal ligament fibroblasts (arrows). A. electron trans- lucent vacuoles representing an early stage of collagen di-

gestion. B. The same, in cross section. C. Collagen-containing vacuole fused with lysosome representing a later stage in the digestive process. A, B, x 15,000; C, X60,OOO.

highest turnover, most notably the periodontal ligament.

What advantage could there be in degrading collagen intracellularly instead of extracellularly? Phago- cytosis allows a more precise and selective control for the collagen fibers to be degraded, whereas the release of extracellular collagenolytic enzymes af- fords a more rapid, extensive degradation around the cells, as observed during inflammation (173). Al- though the work of Everts et al. (63) indicates that collagenase is not important in collagen phagocytosis, the involvement of other members of the

class of metalloproteinases (presumably the gela- tinases) cannot be ruled out (62).

Although little is known about collagen degradation in the healthy human periodontium, electron microscopic studies (71, 203) have shown that collagen phagocytosis by fibroblasts is a normal feature (Fig. 10). Thus, we adhere to the view that, in the human periodontal ligament, collagen remodeling and collagen breakdown do not differ essentially from what is known about these processes in other animal species.

Taken together, all these studies indicate that the

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periodontal ligament is characterized by a rapid turnover and a high remodeling capacity. Under steady-state conditions, collagen degradation in this tissue is carried out via a process of collagen phagocytosis without the involvement of collagenase.

Force distribution and dynamic role in bone remodeling

Periodontal ligament and alveolar bone cells are exposed to physical forces in uivo in response to masti- cation, speech, and orthodontic tooth movement. Physiological loading of teeth or orthodontically induced tooth movements involve remodeling of the periodontal and gingival connective tissue matrices (54, 148, 149, 182). As long as ankylosis does not occur, the general trend after application of physical forces to teeth is preservation of the width of the periodontal ligament, a remarkable process involv- ing precisely controlled osteogenic resorption and deposition at specific sites in the tissues. Although the histological and some of the biochemical effects of orthodontic force application have been described (50, 148, 204), the mechanisms by which applied forces produce reactive changes in periodontal ligament and bone cells are poorly understood. Thus, while it is known that applied mechanical force leads to more rapid bone remodeling in vivo (151), knowledge of exactly how force distribution from the periodontal ligament to the alveolar bone regulates bone remodeling is limited. In spite of these caveats, morphological observations of bone and periodontal ligament after application of applied forces to mammalian teeth have lead to the following general conclusions:

The periodontal ligament distributes applied forces to the contiguous alveolar bone. The direction, frequency, duration and size of the forces determines in part the extent and rapidity of bone remodeling. When forces are applied to teeth devoid of a periodontal ligament, the rate and extent of bone remodeling is very limited.

These conclusions suggest that the periodontal ligament may be both the medium of force transfer and the means by which alveolar bone - at least the wall of the socket - remodels in response to applied forces. Further, they strongly suggest that the periodontal ligament is an irreplaceable tissue in the context of force distribution and bone remodeling.

Indeed, osseointegrated implants, with no interven- ing periodontal ligament, are used as immobile an- chors because applied orthodontic forces induce only very limited bone remodeling (83). However, the periodontal ligament is a complex tissue containing diverse mixtures of matrix proteins, cells and vascular elements that are enclosed in very narrow, yet precisely regulated dimensions. Thus, investigat- ing directly and modeling the biophysical attributes of the tissue are extremely difficult. In spite of these limitations, progress over the last 5 years on force transduction in biological systems has now been applied to bone remodeling and to the role of the periodontal ligament. For example, Andersen et al. (2) examined stress and strain levels and their distribution within the periodontium in a model system based on human autopsy material. This model system permitted an estimate of the stress levels that may be distributed across the periodontal ligament under applied loads. Below we discuss data relating to basic mechanisms of force transduction and how the periodontal ligament may be involved in bone remodeling. Notably, much of these data are from in vitro studies or from in vivo rodent models, and the direct application of these findings to the human periodontal ligament is uncertain.

A wide range of approaches to model external force application in vitro have been taken, including subjecting cells to shear stress, hydrostatic pressure, and strain of deformable substrates (126, 189). Cur- rent evidence suggests that cells have a mechanism to respond directly to mechanical forces by activation of mechanosensory signaling systems, including adenylate cyclase and stretch-activated ion channels, and by changes in cytoskeletal organization. These alterations result in the generation of intracellular second messengers such as intracellular calcium ion concentration ([Ca2+Ii), inositol phosphate (IP3) and CAMP For example, [CaZ+li oscillations are seen in periodontal cells responding to substrate tension (6), and increased IP3 has been observed after physical stretching of osteoblasts (44, 88). Intermediate-term responses to applied force include generation of arachidonic acid metabolites. For example, in uitro work shows that there is increased prostaglandin release (176, 205), and in vivo experiments of applied force to cat teeth also demonstrated that there was increased immunoreactivity for prostaglandin E2 in expected tension areas of the periodontal ligament (154). Interleukin- 1 p may also be involved in periodontal ligament regulation of bone remodeling in that cyclic-tension force causes increased interleukin- 1 p production by human peri-

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odontal ligament cells (159), and a higher interleukin- lj3 level was observed at tension sites of cat periodontium in vivo (154). Longer-term responses to mechanical loading in vitro include stimulation of cell division (42, 491, altered collagen synthesis (88, 99), promotion of collagenase activity (96) and stimulated release of transforming growth factor- p (94). Intermittent pressure application to periodontal ligament cells in vitro increases bone resorption, which may involve the previously described increased synthesis of prostaglandin Ez (155). Collectively, these data indicate that there are many potential routes by which applied loads to periodontal ligament may lead either directly or in- directly to alveolar bone remodeling. Current research on mechanotransduction has focused on signaling mechanisms and the identification of mech- anosensors in periodontal ligament and bone cells.

Signaling mechanisms

The most rapid responses documented in periodontal fibroblasts or osteoblasts subject to mechanical strain in vitro involve an elevation in [Ca2+Ii (6,88), and changes in actin filament polymerization (134), which implies a fundamental role for their modulation of subsequent intracellular events. An increase in calcium-channel or nonspecific cation- channel conductance would permit such a rapid elevation in [Ca2+Ii due to influx down a strong electrochemical gradient. Although earlier studies into the mechanisms of physical force transduction in periodontal tissues concentrated on the role of piezoelectric charges, the vasculature, cytokines and inflammatory mediators in regulating the response of bone cells and fibroblasts to mechanical forces, more recent studies have investigated the ability of these cell types to respond directly to membrane perturbation. Watson (197) has proposed that a transducer of mechanical forces into intracellular events would likely possess both multiple membrane-spanning domains stabilized by electrostatic attractions, and a functional association with the cytoskeleton (such as actin microfilaments) to focus the physical forces onto the mechanosensor. Other authors suggest that the membrane itself has a low bending modulus; physical forces such as shear stress and direct stimulation may therefore cause a conformational change of membrane-associated protein complexes, leading to protein activation or altered substrate-enzyme interactions (34). Stretch of the cell membrane, which may be induced by cell

volume increase, can activate specific stretch activated ion channels, leading to an influx of calcium ions down the electrochemical gradient (47, 5 1, 93). Concurrent alterations in cell membrane structure may expose and precipitate membrane phospho- lipids (1 1, 197). The effect of physical forces on cyclic AMP levels has also been well documented in orthodontic tooth movement in vivo (44, 205) and following cell stretching in vitro (124). However, the mechanism of the mechanosensitive transduction is still speculative.

In addition to their structural role in cell shape determination, microfilaments may transduce applied forces into intracellular signals (87), which is perhaps mediated by local alterations in pressure or volume. This, in turn, could alter molecular polymerization and subsequently modify cellular function (86, 87). Most models of cytoskeletal force transduction focus on the effects of cytoskeletal interactions with kinases, actin- and guanosine triphosphate-binding proteins, and other regulatory enzymes. With specific regard to intracellular calcium metabolism, it has been proposed that microfilaments regulate stretch-activated ion channels (153), functionally affect membrane enzymes and substrates (such as phospholipase C and phosphoti- dylinositol (52, 7511, and influence internal Ca2+ release by IPS (190). Microfilaments in turn are highly regulated by [Ca2+Ii.

The ability of actin filaments to reorganize rapidly in response to diverse external signals has been demonstrated in cultured stromal cells in vitro. In relation to physical stimuli, mechanical strain of attached periodontal cells via a flexible substrate re- duces F-actin content within 10 seconds, which is followed by rapid polymerization (134). The polymerization state of the sub-membrane cortical actin meshwork may affect the stretch-activated cation channel current (153).

Collectively, these data indicate a dynamic, re- ciprocal relationship between the activation of membrane-localized cation-permeable channels and the structure of the cytoskeleton and also that stromal cells (and, in particular, the fibroblasts and osteoblasts that populate the periodontal ligament) have the necessary signaling and effector mechanisms to both sense applied physical force and to mount a remodeling response. In the instance of the periodontal ligament and the alveolar bone, these cellular characteristics have an important conse- quence: the periodontal ligament is an absolute re- quirement for rapid remodeling of alveolar bone when physical forces are applied to teeth.

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Regenerative potential

One of the major objectives of periodontal therapy is to restore the lost fibrous attachment and bone that occurs as a result of periodontitis and other periodontal diseases. This objective requires the existence of a coherent biological program to restore the destroyed connective tissue, form new cementum and bone and induce attachment of new connective tissue fibers (9, 55, 143). This program must also integrate these processes, and the periodontal ligament likely plays a central, integrative role in achieving periodontal regeneration. Con- versely, it is difficult to conceive of how periodontal ligament can be restored without the concomitant production of new cementum and new alveolar bone. Many reports indicate that restoration of destroyed periodontal ligament is at least possible, although their effectiveness is unclear and success is unpredictable (108, 127, 128, 141, 142). Notably, repair but not complete restoration of the lost periodontal ligament is a frequent result.

Challenges of periodontal ligament regeneration

Peculiar to the healing of periodontitis lesions is the fact that the various types of cementum are not re- newed during normal function (158). Cementum and the root surfaces of teeth that are exposed to periodontal pathogens and their metabolites under- go significant pathological alterations (1). Even after debridement, the roots appear to favor the attachment of epithelial cells compared with connective tissue fibroblasts (186, 187). Although it may be ad- vantageous to retain a long junctional epithelium for protection against root resorption, it is not con- ducive to connective tissue regeneration and cementum formation (185). As regeneration of periodontal ligament necessitates direct contact between denuded root surfaces and regeneration- competent connective tissue cells, it is of considerable importance that cells of the periodontal ligament can interact with a biologically acceptable root surface and not be blocked by epithelium. Indeed, recent work (141) suggests the use of combinations of extracellular matrix proteins to prevent specifi- cally the migration of epithelium during periodontal wound healing.

Cells contributing to regeneration

The periodontal ligament has long been suggested to be of central importance in determining the

outcome of periodontal regenerative procedures (120). In periodontal wound healing, the periodontal ligament provides the following important functions: 1) generates new fibers and continuity of fibers from root to bone; 2) prevents apical migration of epithelium; 3) contributes cells to restore lost bone and cementum; 4) acts as a biological sensor and activator to regulate its own width. The importance of the periodontal ligament is perhaps best demonstrated by the considerable interest that has been generated following the sug- gestion of guided tissue regeneration (109). Briefly, wound-healing experiments using occlusive mern- branes to separate gingival from periodontal ligament and bone compartments have indicated that preferential colonization of periodontal wounds by cells derived from the periodontal ligament may result in enhanced healing (109). Although no definitive studies on the origin of cells repopulating wounded periodontal ligament have been under- taken in humans, separate investigations using cell kinetic (117) and cell culture methods (121) indicate that periodontal ligament fibroblast populations in adult rodents are derived from precursor cells of both the periodontal ligament and endosteal spaces. Although it is not possible to ex- trapolate directly from the mouse or rat to the human, these data do suggest that there is considerable mixing of cell populations in periodontal tissues. Thus, the basis for periodontal regeneration by selective cell repopulation 191) remains theoretical: it is not known whether anatomical boundaries constrain cell populations with different origins and phenotypes or whether separation of cells by physical means will actually result in the repopulation of a wound by a predetermined phenotype. This point leads to an important ques- tion: what cellular phenotypes are required for regeneration? For example, several features of periodontal ligament cells indicate that they may be- long to a separate population distinct from connective tissue cells in the gingival domain. In addition to the features mentioned already, there is type XI1 collagen expression (901, initiation of mineralized nodule formation in vitro (5) and vari- ations of proliferative responses and matrix synthesis profiles (111, 131, 174). None of these markers alone is absolutely specific for periodontal ligament cells, but collectively they indicate some important functional attributes of these cells that are important for regeneration. For example, the expression of alkaline phosphatase by periodontal ligament cells may be required for cementogenesis

32


(78, 79). As the identity of the various periodontal ligament cell populations is not certain, and as it is recognized that several types of cell populations inhabit this tissue, we will restrict our discussion to the fibroblastic series. In the context of periodontal ligament regeneration, as the formation of functionally oriented fiber systems is a widely cited indicator of periodontal regeneration (8, 55, 143), it may be appropriate to examine this attribute as a marker for regeneration-competent fibroblasts.

Fiber formation

In regenerating periodontal ligament, the fibroblast population must first produce oriented collagen bundles and then maintain the orientation of these fibers during the development of normal function. In uitro studies indicate that periodontal ligament fibroblasts have the capacity to form fibrillar arrays that are morphologically similar to their in uivo counterparts (139, 140). Indeed periodontal fibroblasts have a constitutive capacity to orient them- selves between two attachment points (29). Directed cell migration is likely to play an important role in this process (139) which, in turn, is affected by the biochemical nature of the substrate at the attachment points (106, 138). During migration, fibroblasts are able to either deposit oriented extracellular matrix or to orient existent extracellular matrix (25, 178). Directed cell migration and matrix secretion (73) and the existence of tension (137) have been demonstrated in periodontal tissues during development and homeostasis, thus providing support for in vitro models. However, despite the interesting information from these in uitro models, it is not understood how these data apply to the regenerating periodontal ligament, how fibrillar arrays around teeth are maintained at such large distances from teeth or how physical forces acting on teeth in viuo regulate the formation of the arrays.

Regulation of periodontal ligament cell differentiation

The hierarchy of periodontal ligament cell populations is not well understood. Cell kinetic experiments in mice and rats (114, 116) have shown that periodontal ligament fibroblast populations are a renewal cell system in steady state: the number of new cells generated by mitosis is equal to the number of cells lost through apoptosis and migration (115, 156). Consequently, periodontal ligament fibroblast populations are renewal cell systems. In other renewal

systems, the most primitive cell is classified as a stem cell, characterized by extensive self-renewal, re- sponsiveness to regulatory factors, generation of multiple types of different specialized cells (58) and restriction to a specific niche within the tissue (144). By analogy, progenitor cell populations of mouse molar periodontal ligament are located adjacent to blood vessels and exhibit some of these same features of stem cells (76, 113). Although it is not known whether the daughter cells of these paravascular progenitors actually migrate and contribute to periodontal ligament cell populations in steady state (1 161, they do repopulate wounded periodontal ligament (76). The periodontal ligament may also be the source of other cells, including tooth-related sites for cementoblasts (1 16) and bone-related portions for the osteoblasts.

The precise relationship between putative stem cells in regenerating periodontal ligament and normally functioning (steady-state) tissues is not clear (144). Studies in normal and wounded mouse periodontal ligament (761, normal rat gingiva (135) and inflamed monkey gingiva (123) have identified a common paravascular location for fibroblast progenitors. The differentiation control systems of periodontal ligament cells are poorly understood but are likely to be affected by the proximity of the tooth root (100). Several different factors may coor- dinate the control of periodontal ligament fibroblastic cell differentiation. Physical forces (1341, lectins (132), cell shape and extracellular matrix (198) and a variety of fibrogenic cytokines (95) modulate the form and function of fibroblasts. As there are no unambiguous differentiation markers for fibroblast populations, there is little definitive evidence on which populations of fibroblasts are regulated by specific factors, particularly in the periodontal ligament. Similarly, the locations of the control points in the differentiation pathway are unknown. However, in spite of these obstacles, there has been considerable interest in assessing the regulation of periodontal ligament function by cytokines that are known to influence fibroblast metabolism and wound healing (36, 112, 125, 152). Collectively, these reports show that wound healing cytokines, such as platelet-derived growth factor, are mitogenic and that there is considerable interaction between cytokines when applied simul- taneously (152, 192). There are no convincing data yet that a single cytokine or multiple cytokines will induce selective growth and differentiation of periodontal ligament fibroblasts and not other stromal cells in the wounded periodontium.

33

Beertsen et al.

Towards a rational approach for the regeneration of periodontal ligament in uiuo

Short-term application of platelet-derived and insulin-like growth factors can enhance new attachment procedures in dogs (108, 133). A possible short- coming in the application of cytokines to improve periodontal wound healing is the nonspecific activities of some cytokines on different cell lineages in time and space (65) and the rapid loss of topically applied factors over time (108). Currently, we do not have a detailed knowledge of the temporal distribution of these substances, their concentrations or how they interact with each other. In contrast, recent experimental data on cementum-bound mitogenic, migration and attachment factors (8, 122, 175) indicate that informational molecules produced during cementum formation may be stored in the ce- mental matrix and that their regulation of periodontal ligament regeneration is interfered with by endotoxin and other molecules present on diseased roots. Conceivably, the appropriately timed release of cementum-bound proliferation and differentiation factors may promote periodontal ligament re- formation, although they certainly are not a prerequisite under every condition, since regenerative cementum formation can occur directly over exposed dentin surfaces in both humans and other mammals (38).

Conclusion

Over the past decade, insight into the physiology of the periodontal ligament and the properties of its cells has increased. This insight has come from the synthesis of research results from sources as varied as in uiuo rodent models, in uitro studies of cells and tissues and protein biochemistry. Based on these studies, we conclude that the periodontal ligament is a unique connective tissue: it cannot readily be replaced by cell populations other than those that have their origin in the ligament itself.

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