PERIODONTIUM.pdf

CHAPTER OUTLINE Cementum, 193 Biochemical Composition, 193 Initiation of Cementum Formation, 194 Origin of Periodontal Cells and Differentiation of Cementoblasts, 194 Molecular Factors Regulating Cementogenesis, 196 B...

CHAPTER OUTLINE Cementum, 193 Biochemical Composition, 193 Initiation of Cementum Formation, 194 Origin of Periodontal Cells and Differentiation of Cementoblasts, 194 Molecular Factors Regulating Cementogenesis, 196 Bone Morphogenetic Proteins, 196 Epithelial Factors, 197 Major Matrix Proteins With Cell Adhesion Motifs, 197 Gla Proteins, 198 Transcription Factors, 198 Wnt Signaling, 199 Other Factors, 199 Cementum Varieties, 199 Acellular Extrinsic Fiber Cementum (Primary Cementum), 199 Cellular Intrinsic Fiber Cementum (Secondary Cementum), 202  Acellular Afibrillar Cementum, 203 Distribution of Cementum Varieties Along the Root, 204 Cementoenamel Junction, 204 Attachment of Cementum Onto Dentin, 204 Alveolar Process, 205 Periodontal Ligament, 206 Fibroblasts, 208 Epithelial Cells, 210 Undifferentiated Mesenchymal Cells, 210 Stem Cells, 210 Bone and Cementum Cells, 210 Fibers, 210 Elastic Fibers, 212 Ground Substance, 213 Blood Supply, 213 Nerve Supply, 213 Adaptation to Functional Demand, 215 The periodontium is defined as those tissues supporting and investing the tooth and consists of cementum, periodontal ligament (PDL), bone lining the alveolus (socket), and that part of the gingiva facing the tooth. Proper functioning of the periodontium is achieved only through structural integrity and interaction between these various tissues. Together, these tissues form a specialized fibrous joint, a gomphosis, the components of which are of ectomesenchymal origin (except the gingiva). The widespread occurrence of periodontal diseases and the realization that periodontal tissues lost to disease can be repaired has resulted in considerable effort to understand the factors and cells regulating the formation, maintenance, and regeneration of the periodontium. This chapter describes the histologic events leading to the formation of supporting tissues (Figure 9-1), except for the dentogingival junction, which is covered under oral mucosa (see Chapter 12).  FIGURE 9-1 Summary of (1) the differentiation of odontoblasts from ectomesenchymal cells in the radicular pulp, (2) the fragmentation of Hertwig's epithelial root sheath with residual portions forming the epithelial rests of Malassez, and (3) the ensuing differentiation of cementoblasts from Hertwig's epithelial root sheath cells or follicle cells, and the follicle contribution to the formation of  the fiber bundles of the periodontal ligament (PDL) and, possibly, osteoblasts. Cementum Cementum is a hard, avascular connective tissue that covers the roots of teeth (Figure 9-2). Cementum is classified according to the presence or absence of cells within its matrix and the origin of the collagen fibers of the matrix. The development of cementum has been subdivided into a prefunctional stage, which occurs throughout root formation, and a functional stage, which starts when the tooth is in occlusion and continues throughout life. Several varieties of cementum exist. The beginning student, however, needs only to think of the two main forms of cementum that have different structural and functional characteristics: acellular cementum, which provides attachment for the tooth, and cellular cementum, which has an adaptive role in response to tooth wear and movement and is associated with repair of periodontal tissues.  FIGURE 9-2 Ground section of a premolar showing the distribution of cementum around the root. Increasing amounts of cementum occur around the apex. Biochemical Composition Four mineralized tissues are found in the oral cavity, and three of these—enamel, dentin, and cementum—are components of the tooth. Their characteristics and biochemical composition are summarized in Table 1-1. The composition of cementum is similar to that of bone. Cementum is approximately 45% to 50% hydroxyapatite by weight, and the remaining portion is collagen and noncollagenous matrix proteins. Type I collagen is the predominant collagen of cementum; in cellular intrinsic fiber cementum, it constitutes up to 90% of the organic components and, just as in bone, it accommodates a substantial part of mineral deposition. Type I collagen is also the major collagen within the PDL region, and its main function is to structure the fiber bundles that anchor the tooth to the bone and distribute masticatory forces. Other collagens associated with cementum include type III, a less crosslinked collagen found in high concentrations during development and during repair and regeneration of mineralized tissues but is reduced with maturation of this tissue, and type XII collagen, a fibril-associated collagen with interrupted triple helices that bind to type I collagen and to noncollagenous proteins. Type XII collagen is found in high concentrations in ligamentous tissues, including the PDL, with lower levels noted in cementum. This nonfibrillar collagen interacts with type I collagen and may assist in maintaining a functional and mature PDL that can withstand the forces of occlusion. Trace amounts of other collagens, including types V, VI, and XIV, also are found in extracts of mature cementum; however, these may be contaminants from the PDL region, produced by PDL fibroblasts associated with collagen fibers inserted into cementum. Noncollagenous proteins identified in cementum also are associated with bone and include the following: alkaline phosphatase, bone sialoprotein, dentin matrix protein 1, dentin sialoprotein, fibronectin, osteocalcin, osteonectin, osteopontin, proteoglycans, proteolipids, tenascin, and several growth factors. Two apparently unique cementum molecules, an adhesion molecule (cementum attachment protein) and an insulin-like growth factor, have been identified, but further studies are warranted to confirm their existence and function. In addition to producing the listed common matrix proteins, the expression of the osteoblast-specific membrane protein Bril, a member of the interferon-inducible transmembrane protein family, adds additional support for similarity between these two cell types. Initiation of Cementum Formation Although cementum formation takes place along the entire root, its initiation is limited to the advancing root edge (Figure 9-3). At this site, Hertwig's epithelial root sheath (HERS), which derives from the coronoapical extension of the inner and outer enamel epithelium (see Chapter 5), is believed to send an inductive message, possibly by secreting some enamel proteins or other epithelial product, to the facing ectomesenchymal pulp cells. These cells differentiate into odontoblasts and produce a layer of predentin (Figures 9-3 and 9-4). The next series of events results in formation of cementum on the root surface; however, the specific cells and trigger factors responsible for promoting its formation still are unresolved. Current theories include the following: (1) Soon after deposition of dentin, HERS becomes interrupted, and ectomesenchymal cells from the inner portion of the dental follicle then can come in contact with the predentin; (2) infiltrating dental follicle cells receive a reciprocal inductive signal from the dentin and/or the surrounding HERS cells and differentiate into cementoblasts; or (3) HERS cells transform into cementoblasts (a process discussed subsequently). During these processes, some cells from the fragmented root sheath form discrete masses surrounded by a basal lamina, known as epithelial cell rests of Malassez (ERMs), which persist in the mature PDL (Figures 9-5 and Figure 9-6). Evidence is increasing that these rests are not simply residual cells but instead may participate in maintenance and regeneration of periodontal tissues. If some HERS cells remain attached to the forming root surface, they can produce focal deposits of enamel-like material called enamel pearls (Figure 9-7), most commonly found in the area of furcation of roots.  FIGURE 9-3 Histologic sections of the advancing root edge in (A) a rat during acellular extrinsic fiber cementum (AEFC) formation and (B) a human during cellular intrinsic fiber cementum (CIFC) formation. In the rat, Hertwig's epithelial root sheet (HERS) is still present when radicular dentin (D) calcifies, and in fact, deposition of acellular cementum starts on mineralized dentin, often in the presence of cells with epithelial characteristics (arrows). In human teeth, acellular and cellular cementum are deposited before the surface layer of dentin mineralizes. Cb, Cementoblast; Od, odontoblasts; PC, precementum; PD, predentin.  FIGURE 9-4 Electron micrograph of early root dentinogenesis. The large collagen fibril bundles are first deposited parallel and at a distance from the basal lamina (BL) that supports Hertwig's epithelial root sheath (HERS). N, Nucleus.  FIGURE 9-5 Initial cementum formation. The first increment of cementum forms against the root dentin surface. Epithelial cell rests of Malassez (remnants of the root sheath) can be seen within the follicular tissue.  FIGURE 9-6 Light micrographs taken along the forming root in (A) a human tooth and (B) a porcine one. Epithelial rests of Malassez (ERM) are seen close to the tooth surface. These can appear as long strands or more discrete elongated or spherical groups of cells. The size of the cells and their staining density may vary. C, Electron micrograph of an epithelial rest. The scarcity of cytoplasmic organelles and basal lamina (BL) surrounding it are notable. AEFC, Acellular extrinsic fiber cementum; Cb, cementoblast; Cc, cementocyte; CIFC, cellular intrinsic fiber cementum; Coll, collagen fibrils; PDL, periodontal ligament.  FIGURE 9-7 Enamel pearls appear as spherical masses and develop ectopically in the area of root furcations. Origin of Periodontal Cells and Differentiation of Cementoblasts Several factors need to be fully determined to better understand the periodontium, including the following: 1. What are the precursors of cementoblasts and PDL fibroblasts? 2. Do cementoblasts express unique genes products, or are they simply positional osteoblasts? 3. Are acellular and cellular cementum phenotypically distinct tissues? 4. What factors promote cementoblast differentiation? 5. What regulates formation of the PDL versus cementogenesis, thus providing a balance between cementum, PDL, and alveolar bone? Answers to these questions are important not only to understand normal formation processes but also to envisage novel, targeted therapeutic approaches for periodontal diseases. The long-standing view is that precursor cells for cementoblasts and PDL fibroblasts reside in the dental follicle and that factors within the local environment regulate their ability to function as cementoblasts that form root cementum or as fibroblasts of the PDL. Cells involved in regenerating periodontal tissues include stem cells migrating from the vascular region, as well as local progenitor cells. The precise location of the progenitor cells and whether there exists a common progenitor or distinct progenitors for each cell type remain to be defined. In addition, there is now increasing evidence that epithelial cells from HERS may undergo epithelial-mesenchymal transformation into cementoblasts during development. Such transformation is a fundamental process in developmental biology; as seen in Chapter 2, it takes place during neural crest cell migration, and in Chapter 3 during medial edge fusion of the palatal shelves. Structural and immunocytochemical data support the possibility that at least in part cementoblasts are transformed from epithelial cells of HERS. In rodents, initial formation of acellular cementum takes place in the presence of epithelial cells, and some studies have shown that enamel organ–derived cells are capable of producing mesenchymal products, such as type I collagen, bone sialoprotein, and osteopontin. Uncertainty still exists as to whether acellular (primary) and cellular (secondary) cementum are produced by distinct populations of cells expressing spatiotemporal behaviors that result in the characteristic histologic differences between these tissues. This potential cellular and formative distinctiveness is highlighted in mice null for the tissue- nonspecific alkaline phosphatase gene or rats treated with bisphosphonates. In these animals, acellular cementum formation is affected significantly, whereas cellular cementum appears to develop normally. In hypophosphatemic mice, formation of acellular cementum can be rescued by enzyme replacement therapy for tissue- nonspecific alkaline phosphatase. This suggests differences in cell types or factors controlling development of these two varieties of cementum. In the human counterpart, hypophosphatasia, characterized by low levels of alkaline phosphatase, cementum formation appears to be limited or nonexistent, not exclusive to acellular versus cellular. In contrast, in mice with mutations in genes that maintain extracellular pyrophosphate levels, such as ank and PC-1, resulting in limited levels of pyrophosphate, formation of cellular cementum occurs even at early stages of root development. These findings suggest an important role for phosphate in controlling the rate of cementum formation. Molecular Factors Regulating Cementogenesis To understand the specific role of phosphate and other molecules, additional studies that focus on defining the cells and factors controlling development, maintenance, and regeneration of periodontal tissues are required. Some of the factors known to be involved in controlling these events are discussed next and are summarized in Table 9-1. TABLE 9-1 Some Key Molecules in the Periodontium Suggested Function Related to Cementogenesis Growth Factors Transforming Reported to promote cell differentiation and growth factor β subsequently cementogenesis during development superfamily and regeneration. (including bone morphogenetic proteins) Platelet-derived Existing data suggest that platelet-derived growth factor and growth factor alone or in combination with insulin-like growth insulin-like growth factor promotes cementum factor formation by altering cell cycle activities. Fibroblast growth Suggested roles for these factors are promoting factors cell proliferation and migration and also vasculogenesis—all key events for formation and regeneration of periodontal tissues. Adhesion Molecules These molecules may promote adhesion of Bone sialoprotein selected cells to the newly forming root. Bone sialoprotein may be involved in promoting Osteopontin mineralization, whereas osteopontin may regulate the extent of crystal growth. Epithelial/Enamel Proteins Epithelial-mesenchymal interactions may be involved in promoting follicle cells along a cementoblast pathway. Some epithelial molecules may promote periodontal repair directly or indirectly. Collagens Collagens, especially types I and III, play key roles in regulating periodontal tissues during development and regeneration. In addition, type XII may assist in maintaining the periodontal ligament space versus continuous formation of cementum. Gla Proteins Matrix Gla These proteins contain γ-carboxyglutamic acid, protein/bone Gla hence the name Gla proteins. Osteocalcin is a protein marker for cells associated with mineralization— (osteocalcin) that is, osteoblasts, cementoblasts, and odontoblasts—and is considered to be a regulator of crystal growth. It has also been proposed to act as a hormone regulating energy metabolism through several synergistic functions favoring pancreatic β-cell proliferation, increasing insulin secretion (in the pancreas) and sensitivity in peripheral tissues, and promoting energy expenditure (in brown adipose tissue) and testosterone production by Leydig cells in testes. Matrix Gla protein appears to play a significant role in preventing abnormal ectopic calcification. Transcription Factors As for osteoblasts, these may be involved in Runt-related cementoblast differentiation. transcription factor 2 (Runx-2) Osterix Signaling Molecules Osteoprotegerin These molecules mediate bone and root Receptor- resorption by osteoclasts. activated nuclear factor κB ligand Receptor- activated nuclear factor κB Sclerostin Antagonist of Wnt and promotes cementum formation Wingless-related Regulates stem cell populations and integration site differentiation of cementoblasts (Wnt) Cementum-Specific Proteins Cementum May play a role as a local regulator of cell protein 1 differentiation and extracellular matrix (cementum- mineralization. derived protein 23) Bone Morphogenetic Proteins Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β superfamily that act through transmembrane serine and threonine protein kinase receptors. These signaling molecules have a variety of functions during morphogenesis and cell differentiation, and in teeth they are considered to be part of the network of epithelial-mesenchymal signaling molecules regulating initiation of crown formation. The roles for BMPs in root development, including whether they are implicated in epithelial- mesenchymal signaling, and the signal pathways and transcription factors involved in modulating their behavior remain to be defined. However, several of the BMPs, including BMP-2, BMP-4, and BMP- 7, are known to promote differentiation of preosteoblasts and putative cementoblast precursor cells. In addition, BMPs have been used successfully to induce periodontal regeneration in a number of experimental models and in certain clinical situations. Epithelial Factors Epithelial-mesenchymal interactions are required for formation of the tooth crown, and epithelial factors are implicated. The same two populations of cells involved in crown morphogenesis—that is, epithelial and ectomesenchymal cells—also take part in root formation. The possibility that such interactions also are required for development of periodontal tissues and that some of the same signaling molecules are involved is thus a logical assumption. Prospective candidates include enamel proteins, parathyroid hormone–related protein, and basal lamina constituents. In the case of enamel proteins, the debate centers around the fact that enamel proteins have not been detected consistently along forming roots. However, this does not rule out a transient expression at early stages of root formation where they could influence odontoblast and/or cementoblast differentiation. Along this line, an enamel matrix derivative, consisting predominantly of amelogenin molecules, is used clinically to stimulate repair and regeneration, and although much has been discovered over the past 20 years, its mechanism of action remains to be determined (see Chapter 15). Major Matrix Proteins With Cell Adhesion Motifs Bone sialoprotein and osteopontin are multifunctional molecules associated with cementum formation during development and in repair and regeneration of periodontal tissues. They contain the cell adhesion motif arginine–glycine–aspartic acid and thereby are believed to promote adhesion of selected cells onto the newly forming root. Present data further suggest that both proteins may be implicated in regulating mineral formation on the root surface. The balance between the activities of these two molecules may contribute to establishing and maintaining an unmineralized PDL between cementum and alveolar bone. No major developmental root anomalies have been reported in the osteopontin knockout mouse model. On the other hand, bone sialoprotein expression affects acellular cementum formation and periodontal attachment, possibly by promoting mineralization at the root surface to anchor PDL fibers. Gla Proteins Proteins enriched in γ-carboxyglutamic acid (Gla), a calcium-binding amino acid, are known as Gla proteins. Bone Gla protein (osteocalcin) is a marker for maturation of osteoblasts, odontoblasts, and cementoblasts and is considered to regulate the extent of mineralization. Also, this osteoblast-derived hormone may regulate insulin secretion, insulin sensitivity, and energy expenditure. Matrix Gla protein (MGP) has been identified in periodontal tissues and, based on its suggested role as an inhibitor of mineralization, may act to preserve the PDL width. Mice null for MGP exhibit substantial ectopic calcification. However, periodontal development and tooth formation appear to be normal in MGP-null mice; thus additional studies are required to define the role of MGP within periodontal tissues. Transcription Factors As shown in Chapter 6, Runx-2 (runt-related transcription factor 2), also known as Cbfa1 (core binding factor alpha 1), and osterix, downstream from Runx-2, have been identified as master switches for differentiation of osteoblasts. Runx-2 now has been found to be expressed in dental follicle cells, PDL cells, and cementoblasts. Based on similarities between cementoblasts (at least in cellular cementum) and osteoblasts, it is likely that both factors may be involved in cementoblast differentiation. The exact factors triggering expression or activation of these key transcription factors currently are being investigated; BMPs already have been identified as factors promoting expression of Runx-2. Wnt Signaling Wnt molecules are small secreted glycoproteins that act extracellularly to regulate many different processes such as development, growth, patterning, stemness, and cancer. The Wnt signaling pathway is evolutionarily conserved and extraordinarily complex (see Box 6-1 by Moffatt). Wnt reporter and lineage-tracing strains of mice have allowed researchers to create molecular maps of Wnt responsiveness in the craniofacial tissues, and these patterns of Wnt signaling colocalize with various stem or progenitor populations in alveolar bone, the PDL, and the cementum. Osterix is believed to control cementoblast proliferation by maintaining a low level of Wnt. Inactivation of the Wnt signaling antagonist sclerostin leads to an increase in cementum formation. Phosphate/pyrophosphate levels have also been shown in vitro to influence Wnt signaling. Thus Wnt proteins offer a tremendous potential for promoting periodontal tissue formation and regeneration. (See Yin in Recommended Reading). Other Factors Other molecules that are found within the developing and mature periodontal tissues include alkaline phosphatase, several growth factors (e.g., insulin-like growth factor, transforming growth factor β, and platelet-derived growth factor), metalloproteinases, and proteoglycans. The significance of alkaline phosphatase to cementum formation has long been appreciated and is discussed in a previous section. Proteoglycans accumulate at the dentin-cementum junction, and it has been proposed that, together with noncollagenous matrix proteins such as bone sialoprotein and osteopontin, they may mediate initial mineralization and fiber attachment. Mineralized tissues such as bone are turning over continually and require a delicate balance between formative and resorptive cells. Two key factors that have emerged as critical to this balance are osteoprotegerin and receptor-activated nuclear factor κB ligand (RANKL). Both are produced by osteoblasts and PDL fibroblasts. As discussed in more detail in Chapter 6, RANKL activates osteoclasts by binding to specific cell surface receptors (RANK), whereas osteoprotegerin acts as a decoy interfering with the binding of RANKL to RANK. Growth factors and cytokines in the local region of the periodontium have been shown to modulate expression of osteoprotegerin and RANKL and thus may be important for controlling osteoclastic-mediated bone and root resorption; thus they may be attractive factors in designing therapeutic agents to regulate the behavior of this cell. Equally important to the process of cell maturation and function are the timed expression of specific cell surface receptors and the ability of certain factors to regulate their expression and, subsequently, the signaling pathways mediated by ligand-receptor interactions. Cementum Varieties Table 9-2 lists the various types of cementum along with the origin, location, and function of each. TABLE 9-2 Type, Distribution, and Function of Cementum Type Origin of Location Function Fibers Acellular Extrinsic From cervical Anchorage (primary) (some margin to the intrinsic fibers apical third initially) Cellular Intrinsic Middle to Adaptation and (secondary) apical third repair and furcations Mixed Intrinsic and Apical Adaptation (alternating layers extrinsic portion and of acellular and furcations cellular)  Acellular — Spurs and No known afibrillar patches over function along the enamel and cementoenamel dentin junction Acellular Extrinsic Fiber Cementum (Primary Cementum) Cementoblasts that produce acellular extrinsic fiber cementum differentiate in proximity to the advancing root edge. During root development in human teeth, the first cementoblasts align along the newly formed but not yet mineralized mantle dentin (predentin) surface after disintegration of HERS (Figure 9-8, A and B). These first cementoblasts exhibit fibroblastic characteristics, extend cell processes into the unmineralized dentin, and initially deposit collagen fibrils within it so that the dentin and cementum fibrils intermingle. Mineralization of the mantle dentin starts internally and does not reach the surface until mingling has occurred. Mineralization then spreads across into cementum under the regulatory influence of noncollagenous matrix proteins, thereby establishing the cementodentinal junction. In rodents, initial cementum deposition occurs onto the already mineralized dentin surface, preventing the intermingling of fibers (see Figure 9-3, A).  FIGURE 9-8 Early human acellular extrinsic fiber cementogenesis (AEFC). A, Intermingling of collagen fiber bundles with those at the unmineralized dentin (predentin, PD) surface. Arrowheads indicate the external dentin mineralization front. B, Details of the intermingling. C, The final connection between the collagen fiber bundles of acellular (primary) cementum and dentin (D) surface are shown. D, The fibrous fringe (FF) extending from cementum. Cb, Cementoblast; DCJ, dentinocemental junction; N, nucleus; PDL, periodontal ligament. (Courtesy D.D. Bosshardt.) Initial acellular extrinsic fiber cementum consists of a mineralized layer with a short fringe of collagen fibers implanted perpendicular to the root surface (see Figure 9-8, D). The cells on the root surface then migrate away from the surface but continue to deposit collagen so that the fine fiber bundles lengthen and thicken. These cells also secrete noncollagenous matrix proteins that fill in the spaces between the collagen fibers (Figure 9-9). This activity continues until about 15 to 20 µm of cementum has been formed, at which time the forming PDL fiber bundles become stitched to the fibrous fringe. Thereafter, the surface cells, now clearly defined as cementoblasts, will synthesize and secrete only noncollagenous matrix proteins, and the collagen fibrils that embed in the cementum layer will be formed by PDL fibroblasts. Although this cementum variety is called acellular extrinsic fiber cementum, whether its initial part should be classified instead as having intrinsic fibers is debatable. As described previously, the collagenous matrix of the first-formed cementum results from cementum-associated cells and is elaborated before the PDL forms; therefore the collagen is of local or intrinsic origin. This cementum variety develops slowly as the tooth is erupting and is considered to be acellular because the cells that form it remain on its surface (see Figure 9-8, C).  FIGURE 9-9 Colloidal gold immunocytochemical preparation illustrating the presence and distribution of osteopontin (black dots), a major noncollagenous matrix protein, in rat acellular extrinsic fiber cementum (AEFC). This protein accumulates between the inserted portions of the extrinsic collagen fibers (asterisks) and is more concentrated near dentin, where collagen fibers are sparse and more loosely arranged. Cb, Cementoblast; PDL, periodontal ligament. With the light microscope, acellular extrinsic fiber cementum seems relatively structureless (Figure 9-10, A); however, two sets of striations can be seen with special stains or polarized light. The striations running parallel to the root surface indicate incremental deposition, whereas the short striations at right angles to the root surface indicate the inserted mineralized PDL collagen fiber bundles (Figure 9-11). With the electron microscope, these collagen bundles can be seen clearly to enter cementum, where they become fully mineralized. No well-defined layer of cementoid, akin to osteoid or predentin, can be distinguished on the surface of this cementum. However, the principal PDL fibers, or at least their cementum-related portion, may be regarded as equivalent to the cementoid. The overall degree of mineralization of this cementum is about 45% to 60%, but soft x-ray examination reveals that the innermost layer is less mineralized and that the outer layers are characterized by alternating bands of more and less mineral content that run parallel to the root surface. FIGURE 9-10 Ground sections of human teeth examined by  transmitted light illustrating (A) acellular extrinsic fiber cementum (AEFC) and (B) the transition between the former and cellular intrinsic fiber cementum (CIFC). Both appear as a translucent, structureless layer. Cementocytes (dark, rounded structures) are present in the cellular intrinsic fiber cementum. GLT, Granular layer of Tomes (see Chapter 8). FIGURE 9-11 Histologic section examined by (A) transmitted and (B) polarized light. Polarized microscopy reveals perpendicular striations in the cementum layer and in the surface of alveolar bone. These correspond to the sites of insertion of collagen fiber bundles. C and D, Longitudinal (arrows) and perpendicular lines are also visible  with some histologic stains. The longitudinal layering can appear as thin or thicker lines, essentially denoting the interface between successive layers of cementum. AEFC, Acellular extrinsic fiber cementum; CIFC, cellular intrinsic fiber cementum. (A and B, Courtesy P. Tambasco de Oliveira.) Cellular Intrinsic Fiber Cementum (Secondary Cementum) In some teeth (see the following discussion), after at least half the root is formed, a more rapidly formed and less mineralized variety of cementum, cellular intrinsic fiber cementum, is deposited on the unmineralized dentin surface near the advancing root edge (see Figure 9-3, B) as for acellular cementum. Differentiating cementoblasts deposit the collagen fibrils into the unmineralized dentin so that fibrils from both layers intermingle. These cells also manufacture various noncollagenous matrix proteins that fill in the spaces between collagen fibrils, regulate mineral deposition, and together with the mineral, impart cohesion to the cementum layer (Figure 9-12). A layer of unmineralized matrix, called cementoid, which calcifies gradually, is present at the surface of the mineralized cementum matrix, with a mineralization front between the two layers (Figures 9-12 to 9-14). In contrast to osteoid or predentin, cementoid is not as regular and readily discernible. As cementum deposition progresses, cementoblasts become entrapped in the extracellular matrix they secrete (Figure 9-15; see also Figures 9-12). These entrapped cells, with reduced secretory activity, are called cementocytes, and, similar to osteocytes, reside in a lacuna. Histologic studies suggest that incorporation of cementoblasts within cementum is more haphazard than that of osteoblasts within bone. Cementocytes have processes that lodge in canaliculi that communicate but do not form a syncytium that extends all the way to the surface, as is the case within bone (see Figure 9-12, A). Nourishment of the cells is believed to occur essentially by diffusion, and cementocytes in deeper layers may not be vital. With the electron microscope, cementocytes present a variable picture, depending on the distance of their location from the cement surface and their nutritional supply from the PDL. Loss of intracellular organelles and cell death is progressive in the deeper layers of cellular cementum. Although such features are consistent with loss of cell function, they also may reflect poor tissue preservation in the deeper layers. After a rapid initial phase of matrix formation, the deposition rate slows down and secretion occurs in a more directional manner. This may sometimes lead to the formation of a layer of acellular intrinsic fiber cementum, because the cells are not engulfed in their matrix but remain on its surface. In some species, disaggregating HERS cells get trapped near the cementodentinal junction, and cellular cementum forms around and above them.  FIGURE 9-12 Cellular intrinsic fiber cementum from (A and C) rat and (B) human being. A, Cementoblasts (Cb) line the cementum surface and are apposed against a layer of unmineralized matrix (cementoid). A to C, Cementocytes (Cc) reside within lacunae in cementum and can adopt various shapes. A, The cell processes (cp) of cementocytes generally are directed toward the surface. B and C, Immunocytochemical preparations for bone sialoprotein (BSP). This noncollagenous matrix protein (indicated by the presence of black dots) accumulates among the mineralized collagen in regions that are generally more electron dense. FIGURE 9-13 Electron micrograph illustrating the insertion of periodontal ligament (PDL) fiber bundles into cellular intrinsic fiber cementum. Cementoid is seen at the surface of the mineralized cementum. Cb, Cementoblast.  FIGURE 9-14 Electron micrograph of an oblique section through the periodontal ligament–cementum interface. The distinction between extrinsic and intrinsic fibers within cementum is readily apparent, the intrinsic fibers essentially surrounding the embedded portions of the extrinsic fibers, which constitute Sharpey's fibers. (Courtesy M.A. Listgarten.)  FIGURE 9-15 Cementocyte lacunae in ground section. Most of the canaliculi point toward the tooth surface (arrow). The indistinct dark patches are other cementocyte lacunae deeper within the ground section (and consequently out of focus). Collagen fibrils are deposited haphazardly during the rapid phase; however, subsequently the bulk of fibrils organize as bundles oriented parallel to the root surface (see Figure 9-14). When the PDL becomes organized, cellular cementum continues to be deposited around the ligament fiber bundles, which become incorporated into the cementum and partially mineralized, thereby creating cellular mixed fiber cementum. This constitutes the bulk of secondary cementum, and with the light microscope, this tissue is identified easily because of (1) inclusion of cementocytes within lacunae with processes in canaliculi directed toward the tooth surface (see Figure 9-12, A and 9- 15), (2) its laminated structure, and (3) the presence of cementoid on its surface. The intrinsic fibers are mineralized uniformly, whereas the extrinsic fiber bundles are mineralized variably, with many having a central, unmineralized core.  Cellular (secondary) cementum differs from acellular (primary) cementum in a number of ways. Not only are structural differences obvious in that the cells are incorporated into the matrix, but also the phenotype of the cells producing them may differ. Furthermore, secondary cementum is involved in tooth attachment in a minor and secondary way (this variety of cementum is usually absent from incisor and canine teeth) and is confined to the apical and interradicular regions of the tooth. Acellular Afibrillar Cementum The acellular afibrillar cementum variety consists of an acellular and afibrillar mineralized matrix with a texture similar to the one constituting the bulk of acellular extrinsic fiber cementum or the one found among the collagen fibrils of fibrillar cementum varieties and of bone. This cementum lacks collagen and hence plays no role in tooth attachment. It is deposited over enamel and dentin in proximity to the cementoenamel junction (Figure 9-16, A).  FIGURE 9-16 Three configurations of the cementoenamel junction in ground sections. A, Cementum overlaps the enamel. B, A deficiency of cementum (bracket) leaves root dentin exposed. C, A butt joint is visible (arrow). (B and C, Courtesy P. Tambasco de Oliveira.) The cells responsible for the production of acellular afibrillar cementum still have not been identified with precision. For a long time, this cementum variety was believed to represent a developmental anomaly formed as the result of local disruptions in the reduced enamel epithelium that permit follicular cells to come into contact with the enamel surface and differentiate into cementoblasts. This concept has come under questioning, because the enamel organ itself has been demonstrated to be able to produce mesenchymal proteins found in bone and cementum. Hence the reduced enamel epithelium need not obligatorily retract from the enamel surface to result in deposition of afibrillar cementum.  Researchers also have reported that HERS may produce epithelial products that accumulate on the forming root surface to form a layer, referred to as intermediate cementum. To date, however, no study has demonstrated the consistent presence of a distinct matrix layer between dentin and cementum proper. These may actually correspond to the situation where acellular afibrillar cementum forms on top of enamel (see later discussion). The apparent presence of a layer along the radicular dentin surface in some histologic preparations (see Figure 9-11, C and D) does not consist of enamel proteins and may result from the way dentin and cementum collagen interface and the packing density of noncollagenous matrix proteins among the collagen fibrils. Distribution of Cementum Varieties Along the Root In humans, acellular afibrillar cementum is limited to the cervical enamel surface and occurs as spurs extending from acellular extrinsic fiber cementum or as isolated patches on the enamel surface close to the cementoenamel junction. Acellular extrinsic fiber cementum, which becomes the principal tissue of attachment, extends from the cervical margin of the tooth and covers two thirds of the root and often more. Indeed, in incisors and canines, this form of cementum is often the only one found, and it extends to the apical foramen. At the cervical margin, the cementum is approximately 50 µm thick and increases in thickness as it progresses apically to approximately 200 µm. Cellular cementum is confined to the apical third and interradicular regions of premolar and molar teeth. Cellular cementum is often absent from single-rooted teeth, which indicates that its presence is not essential for tooth support. Both fibrillar cementum varieties can overlap. As mentioned before, the type of cementum formed during periodontal wound healing appears to be cellular in origin. Cementoenamel Junction Classically, in approximately 30% of human teeth the cementum and enamel meet as a butt joint, forming a distinct cementoenamel junction at the cervical margin; 10% have a gap between the cementum and enamel, exposing root dentin; and in about 60% the cementum overlaps the enamel. This information was obtained from the study of ground sections (see Figure 9-16), but studies with a scanning electron microscope indicate that the cementoenamel junction may exhibit all of these forms and shows considerable variation when traced circumferentially. The exposure of root dentin at the cervical margin can lead to sensitivity at this site. It has also been suggested that such morphology may result in increased risk for idiopathic osteoclast-mediated root resorption and root surface caries. Attachment of Cementum Onto Dentin The attachment mechanism of cementum to dentin is of biologic interest and of clinical relevance because pathologic alterations and clinical interventions may influence the nature of the exposed root surface and hence the quality of the new attachment that forms when repair cementum is deposited. The mechanism by which these hard tissues bind together is essentially the same for acellular extrinsic fiber cementum and cellular intrinsic fiber cementum. Mineralization of the mantle dentin starts internally and does not reach the surface until the collagen fibrils of dentin and cementum have had the time to blend together. Mineralization then spreads through the surface layer of dentin, across the dentin-cementum junction and into cementum, essentially resulting in an amalgamated mass of mineral. Although initiation of dentin mineralization occurs in relation to matrix vesicles in the radicular predentin, the subsequent spread of mineral deposition is under the regulatory influence of the various noncollagenous matrix proteins. From a biomechanical perspective, this arrangement appears optimal for a strong union between dentin and cementum. In acellular extrinsic fiber cementum of rodent teeth, cementum is deposited onto mineralized dentin, making amalgamation of dentin and cementum impossible and establishing a weakened interface. Indeed, histologic sections of rodent teeth often show a separation between dentin and cementum in the cervical third of the root. Interestingly, repair cementum adheres well to the root surface if a resorptive phase precedes new matrix deposition, implying that odontoclasts not only remove mineral and matrix but most likely also precondition the root surface. One possibility is that odontoclasts generate an organic matrix fringe with which the matrix of reparative cementum then can blend, thereby recapitulating the developmental sequence. Alveolar Process The alveolar process is the bone of the jaws that contains the sockets (alveoli) for the teeth (Figure 9-17). The alveolar process consists of an outer (buccal and lingual) cortical plate, a central spongiosa, and bone lining the alveolus (alveolar bone). The cortical plate and alveolar bone meet at the alveolar crest (usually 1.5 to 2 mm below the level of the cementoenamel junction on the tooth it surrounds). Alveolar bone comprises inner and outer components; it is perforated by many foramina, which transmit nerves and vessels; thus it sometimes is referred to as the cribriform plate. Radiographically, alveolar bone also is referred to as the lamina dura because of an increased radiopacity (Figure 9-18). This increased radiopacity is a result of the presence of thick bone without trabeculations that x-rays must penetrate and not of increased mineral content.  FIGURE 9-17 A, Trabecular bone is found between the lingual cortical plate and alveolar bone in the region of the apical third of the root and in the body of the mandible. B and C, Histologic sections illustrating (B) a thick alveolar process with an abundant spongiosa (trabecular bone) between the cortical plates and alveolar bone and (C) a thin alveolar process lacking distinct trabecular bone. (A, Courtesy P. Tambasco de Oliveira.)  FIGURE 9-18 The lamina dura (arrows) appears as a thin opaque layer around teeth (A) and around a recent extraction socket (B). (From White SC, Pharoah MJ: Oral radiology: principles and interpretation , ed 6, Mosby, 2009, St. Louis.) The bone directly lining the socket (inner aspect of alveolar bone) specifically is referred to as bundle bone. Embedded within this bone are the extrinsic collagen fiber bundles of the PDL (Figure 9-19), which, as in cellular cementum, are mineralized only at their periphery. Bundle bone thus provides attachment for the PDL fiber bundles that insert into it. Histologically, bundle bone generally is described as containing less intrinsic collagen fibrils than lamellar bone and exhibiting a coarse-fibered texture. Bundle bone is apposed to an outer layer of lamellar bone, but in some cases the alveolar bone can be made up almost completely of bundle bone. This is a simplistic description, however, because the tooth constantly is making minor movements, and therefore the bone of the socket wall constantly must adapt to many forms of stress. Thus practically all histologic forms of bone can be observed lining the alveolus, even in the same field in the same section (Figure 9-20). This considerable variation reflects the functional plasticity of alveolar bone.  FIGURE 9-19 Histologic preparations of alveolar bone examined by (A) transmitted and (B) polarized light microscopy. Periodontal ligament (PDL) fiber bundles (arrows) insert into the bone lining the alveolar socket, giving it the name bundle bone. The inserted fibers are referred to as Sharpey's fibers and appear refringent under polarized light. Bundle bone is apposed to trabecular bone with haversian systems (HS). (Courtesy P. Tambasco de Oliveira.)  FIGURE 9-20 Photomicrographs of the periodontal ligament (PDL) region from a single tooth. The considerable variation in  morphology of the bone lining this alveolus is produced by the resorption and deposition of bone as it responds to functional demands placed on it. The root surface is always on the left and bone on the right. The cortical plate consists of surface layers of lamellar bone supported by compact haversian system bone of variable thickness. The cortical plate is generally thinner in the maxilla and thickest on the buccal aspect of mandibular premolars and molars. The trabecular (or spongy) bone occupying the central part of the alveolar process also consists of lamellae with haversian systems occurring in the larger trabeculae. Yellow marrow, rich in adipose cells, generally fills the intertrabecular spaces, although sometimes there also can be some red or hematopoietic marrow. Trabecular bone is absent in the region of the anterior teeth, and in this case, the cortical plate and alveolar bone are fused together. The important part of this complex in terms of tooth support is the bundle bone. Periodontal Ligament Understanding the cell populations and their function in healthy, mature periodontal tissues is required for developing predictable regenerative therapies. Investigations to date suggest that the PDL region in health contains a heterogeneous population of mesenchymal cells and that some cells within this population, when triggered appropriately, can differentiate toward an osteoblast or cementoblast phenotype, that is, promote formation of bone and cementum. In addition, perivascular and endosteal fibroblasts, again when appropriately induced, have the capacity to form PDL, cementum, and bone. Compelling evidence exists indicating that populations of cells within the PDL, during development and during regeneration, secrete factors that can regulate the extent of mineralization. Thus factors secreted by PDL fibroblasts may inhibit mineralization and prevent the fusion of tooth root with surrounding bone, a situation referred to as ankylosis. Although much research is still to be done, current knowledge has enabled development of improved strategies for attracting and maintaining cells at a regeneration site. The PDL is soft, specialized connective tissue situated between the cementum covering the root of the tooth and the bone forming the socket wall. The PDL ranges in width from 0.15 to 0.38 mm, with its thinnest portion around the middle third of the root (Figures 9-21 and 9-22). The average width is 0.21 mm at 11 to 16 years of age, 0.18 mm at 32 to 52 years of age, and 0.15 mm at 51 to 67 years of age, showing a progressive decrease with age. The PDL is a connective tissue particularly well adapted to its principal function, supporting the teeth in their sockets and at the same time permitting them to withstand the considerable forces of mastication. The PDL also has the important function, in addition to attaching teeth to bone, of acting as a sensory receptor, which is necessary for the proper positioning of the jaws during normal function.  FIGURE 9-21 Longitudinal section along the tooth root. Note the perforation (arrow) in the alveolar bone that transmits neurovascular bundles.  FIGURE 9-22 Periodontal ligament in a cross section between two teeth. Apart from recognition that the PDL is formed within the developing dental follicle region, the exact timing of events associated with the development of an organized PDL varies among species, with individual tooth families, and between deciduous and permanent teeth. What follows is a generalized account from several studies undertaken largely on primates. At the commencement of ligament formation the ligament space consists of unorganized connective tissue with short fiber bundles extending into it from the bone and cemental surfaces (Figure 9-23). Next, ligament mesenchymal cells begin to secrete collagen (mostly type I collagen), which assembles as collagen bundles extending from the bone and cementum surfaces to establish continuity across the ligament space and thereby secure an attachment of the tooth to bone. In addition to collagen, several noncollagenous proteins are secreted that appear to play a role in the maintenance of the PDL space, but this still remains an unresolved question. Eruptive tooth movement and the establishment of occlusion then modify this initial attachment. For example, before the tooth erupts, the crest of the alveolar bone is above the cementoenamel junction, and the developing fiber bundles of the PDL are directed obliquely. Because the tooth moves during eruption, the level of the alveolar crest comes to coincide with the cementoenamel junction, and the oblique fiber bundles just below the free gingival fibers become horizontally aligned. During the process of tooth eruption, osteoclast precursors are activated by a variety of factors secreted by cells within the local environment, including RANKL/osteoprotegerin ligand and macrophage colony–stimulating factor. Functional osteoclasts are critical for the formation of marrow spaces within bone and for tooth eruption. When the tooth finally comes into function, the alveolar crest is positioned nearer the apex. The horizontal fibers, termed the alveolar crest fibers, have become oblique once more, with the difference that now the cemental attachment has reversed its relation to the alveolar attachment and is positioned in a coronal direction, as opposed to its previous apical direction (Figure 9-24). Only after the teeth come into function do the fiber bundles of the PDL thicken appreciably.  FIGURE 9-23 The developing periodontal ligament. Fiber bundles (FB) extend into the unorganized ligament space from the cement and alveolar bone surfaces. HERS, Hertwig's epithelial root sheath; Od, odontoblasts.  FIGURE 9-24 The development of principal fiber groupings in the periodontal ligament. The group of alveolar crest fibers (arrowheads), first forming in A, are initially oblique (B), then horizontal (C), and then oblique again (D). When the periodontium is exposed to increased function, the width of the PDL can increase by as much as 50%, and the principal fiber bundles also increase greatly in thickness. The bony trabeculae supporting the alveoli also increase in number and in thickness, and the alveolar bone itself becomes thicker. Conversely, a reduction in function leads to changes that are the opposite of those described for excess function. The ligament narrows, the fiber bundles decrease in number and thickness, and the trabeculae become fewer. This reduction in width of the PDL is caused mostly by the deposition of additional cementum (Figure 9-25).  FIGURE 9-25 Photomicrographs of the effect of nonfunction on the supporting apparatus of the tooth. A, Normal appearance of tissues supporting the teeth. B, Effect of nonfunction for 6 months. The loss of bone in the area marked by the arrowheads is notable. A narrowing of the ligament also can be distinguished. (Courtesy D.C. Picton.) Similar to all other connective tissues, the PDL consists of cells and an extracellular compartment of collagenous fibers and a noncollagenous extracellular matrix. The cells include osteoblasts and osteoclasts (technically within the ligament but functionally associated with bone), fibroblasts, ERMs, macrophages, undifferentiated mesenchymal cells, stem cells, and cementoblasts (also technically within the ligament but functionally associated with cementum). The extracellular compartment consists of well-defined collagen fiber bundles (Figure 9-26) embedded in an amorphous background material, known as ground substance, consisting of, among others, glycosaminoglycans, glycoproteins, and glycolipids. FIGURE 9-26 Electron micrographs of the periodontal ligament in a pig. A, Elongated fibroblasts can be seen alternating with distinctive collagen fiber bundles. The clear areas are occupied by ground substance. B and C, The periodontal ligament undergoes turnover and remodeling, during which matrix synthesis and breakdown take place. Some collagen degradation takes place intracellularly following its internalization (arrows). G, Golgi complex; m, mitochondria; N, nucleus; rER, rough endoplasmic reticulum. Fibroblasts The principal cells of the PDL are fibroblasts. Although fibroblasts look alike microscopically, heterogeneous cell populations exist between different connective tissues and also within the same connective tissue. In the case of the PDL, its fibroblasts are characterized by an ability to achieve an exceptionally high rate of turnover of proteins within the extracellular compartment, in particular, collagen. PDL fibroblasts are large cells with an extensive cytoplasm containing an abundance of organelles associated with protein synthesis and secretion (i.e., rough endoplasmic reticulum, Golgi complex, and many secretory granules). Ligament fibroblasts also have a well-developed cytoskeleton (see Chapter 4) with a particularly prominent actin network, the presence of which is believed to indicate the functional demands placed on the cells, requiring change in shape and migration. Ligament fibroblasts also show frequent cell-to-cell contacts of the adherens and the gap junction types. Fibroblasts are aligned along the general direction of the fiber bundles and have extensive processes that wrap around the bundles. The collagen fibrils of the fiber bundles are being remodeled continuously. The fibroblast achieves remodeling of collagen; it is capable of simultaneously synthesizing and degrading collagen (see Chapter 4). Because of the exceptionally high rate of turnover of collagen in the ligament, any interference with fibroblast function by disease rapidly produces a loss of the supporting tissue of a tooth. Importantly, in inflammatory situations, such as those associated with periodontal diseases, an increased expression of matrix metalloproteinases occurs that aggressively destroys collagen. Thus attractive therapies for controlling tissue destruction may include host modulators that have the capacity to inhibit matrix metalloproteinases. Fibroblast contractility probably is of greatest significance during posteruptive tooth movements. These include functional movements during mastication, accommodation for growth of the jaws, and compensation for occlusal and interproximal wear. Fibroblasts are associated intimately with the fibrous components of their matrix and respond to changes in tension and compression in the matrix. Integrins, which bind to extracellular matrix components, serve as mechanotransducers to transmit the stimulus to the cell. In addition to contraction, the response of the cell may encompass the pulling of collagen fibrils back toward the cell, the movement of cell processes or individual receptors on the processes, or a combination of all of these events. The fibroblasts and the collagen align parallel to the direction of the principal strain in the matrix, which probably accounts for the highly ordered arrangement of the PDL fiber bundles. Mechanical stress also is a significant stimulus for extracellular matrix production by fibroblasts; the repetitive stress to which the PDL is subjected presumably contributes to the high rates of collagen turnover in this tissue. This rapid turnover of matrix components allows the PDL to adapt to the demands of functional tooth movements. Localized changes in tensile and compressive forces during growth, and the mesial drift resulting from interproximal wear, stimulate bone and cementum formation or resorption. In contrast, the absence of these forces, such as when a tooth has no opponent, results in decreased matrix production, increased collagenase (matrix metalloproteinase 1) secretion, and a thinning of the PDL. Epithelial Cells The epithelial cells in the PDL are remnants of HERS, the ERMs. The epithelial cells occur close to the cementum as clusters or strands of cells and are easily recognized in histologic sections because their nuclei generally stain deeply (see Figure 9-6). Some believe they form a network around roots that possibly interconnect with the junctional epithelium (Figure 9-27). ERMs have been proposed to play a role in periodontal maintenance and to represent a stem cell compartment capable of giving rise to many, if not all, cell types found in the periodontium. It has been shown that under certain circumstances they can be activated and produce epithelial and mesenchymal matrix proteins that are implicated in the mineralization of tooth and bone matrices. When periodontal integrity is compromised, ERMs are activated very early on and dramatically upregulate the expression of the matricellular-like protein odontogenic ameloblast–associated protein (see Chapters 7 and 12 for discussion on this protein).  FIGURE 9-27 A, Epithelial cell rests of Malassez frequently appear as isolated islands (arrows) along the root surface. Three- dimensional reconstruction from serial sections, however, clearly demonstrates that these islands are part of an intricate network that surrounds the tooth roots. B, This network can also be seen in fortuitous tangential sections to the tooth surface. (Reconstruction by C. Rivest.) Undifferentiated Mesenchymal Cells An important cellular constituent of the PDL is the undifferentiated mesenchymal cell or progenitor cell; these cells have a perivascular location. Although they have been demonstrated to be a source of new cells for the PDL, whether a single progenitor cell gives rise to daughter cells that differentiate into fibroblasts, osteoblasts, and cementoblasts or whether separate progenitors exist for each cell line is not known. The fact that new cells are being produced for the PDL while cells of the ligament are in a steady state means that this production of new cells must be balanced by migration of cells out of the ligament or cell death. Selective deletion of ligament cells occurs by apoptosis (see Chapter 7 for descrition of this process), and this process provides cell turnover, which in the rat PDL involves approximately 2% of the population at any time. Stem Cells Pluripotent stem cells are present in the PDL, which represents an easily accessible source of stem cells compared with those found in pulp. These postnatal mesenchymal stem cells have the capacity of self-renewal and have the potential to differentiate into adipogenic, cementogenic, osteogenic, and chondrogenic cells. Some believe that PDL stem cells express distinctive mesenchymal and embryonic markers. Bone and Cementum Cells Although technically situated within the PDL, bone and cementum cells are associated properly with the hard tissues they form and are discussed with these tissues. Fibers The predominant collagens of the PDL are types I, III, and XII, with individual fibrils having a smaller average diameter than tendon collagen fibrils. This difference is believed to reflect the short half-life of ligament collagen, meaning that they have less time for fibrillar assembly. Most collagen fibrils in the PDL are arranged in definite and distinct fiber bundles. Each bundle resembles a spliced rope; individual strands can be remodeled continually, whereas the overall fiber maintains its architecture and function. In this way the fiber bundles are able to adapt to the continual stresses placed on them. These bundles are arranged in groups that can be seen easily in an appropriately stained light microscope section (Figures 9-28 and 9- 29). Those bundles running between the tooth and bone represent the principal fiber bundles of the PDL. These bundles are as follows: FIGURE 9-28 The arrangement of the principal fiber groups within  the periodontium. A, Principal fiber groups. B, Fiber groups of the gingival ligament. C, Gingival ligament fibers as seen interproximally related to the gingival col. FIGURE 9-29 Silver-stained section of some of the fiber groups of the gingival and periodontal ligaments. 1. The alveolar crest group, attached to the cementum just below the cementoenamel junction and running downward and outward to insert into the rim of the alveolus 2. The horizontal group, just apical to the alveolar crest group and running at right angles to the long axis of the tooth from cementum to bone, just below the alveolar crest 3. The oblique group, by far the most numerous in the PDL and running from the cementum in an oblique direction to insert into bone coronally 4. The apical group, radiating from the cementum around the apex of the root to the bone, forming the base of the socket 5. The interradicular group, found only between the roots of multirooted teeth and running from the cementum into the bone, forming the crest of the interradicular septum (see Figure 9-28) At each end, all the principal collagen fiber bundles of the PDL are embedded in cementum or bone (see Figures 9-5, 9-8, 9-11, 9-13, 9- 19, and 9-23). The embedded portion is referred to as Sharpey's fiber. Sharpey's fibers in primary acellular cementum are mineralized fully; those in cellular cementum and bone generally are mineralized only partially at their periphery. Occasionally, Sharpey's fibers pass uninterruptedly through the bone of the alveolar process to continue as principal fibers of an adjacent PDL, or they may mingle buccally and lingually with the fibers of the periosteum that cover the outer cortical plates of the alveolar process. Sharpey's fibers pass through the alveolar process only when the process consists entirely of compact bone and contains no haversian systems, which is not common. Although not strictly part of the PDL, other groups of collagen fibers are associated with maintaining the functional integrity of the periodontium. These groups are found in the lamina propria of the gingiva and collectively form the gingival ligament (see Figures 9-28 and 9-29). Five groups of fiber bundles compose this ligament: 1. Dentogingival group. These are the most numerous fibers, extending from the cervical cementum to the lamina propria of the free and attached gingivae. 2. Alveologingival group. These fibers radiate from the bone of the alveolar crest and extend into the lamina propria of the free and attached gingivae. 3. Circular group. This small group of fibers forms a band around the neck of the tooth, interlacing with other groups of fibers in the free gingiva and helping to bind the free gingiva to the tooth (see Figure 9- 28). 4. Dentoperiosteal group. Running apically from the cementum over the periosteum of the outer cortical plates of the alveolar process, these fibers insert into the alveolar process or the vestibular muscle and floor of the mouth. 5. Transseptal fiber system. These fibers run interdentally from the cementum just apical to the base of the junctional epithelium of one tooth over the alveolar crest and insert into a comparable region of the cementum of the adjacent tooth. Together these fibers constitute the transseptal fiber system, collectively forming an interdental ligament connecting all the teeth of the arch (Figure 9-30). The supracrestal fibers, particularly the transseptal fiber system, have been implicated as a major cause of postretention relapse of orthodontically positioned teeth. The inability of the transseptal fiber system to undergo physiologic rearrangement has led to this conclusion. Although the rate of turnover is not as rapid as in the PDL, studies have shown that the transseptal fiber system is capable of turnover and remodeling under normal physiologic conditions, as well as during therapeutic tooth movement. A sufficiently prolonged retention period after orthodontic tooth movement then would seem reasonable to allow reorganization of the transseptal fiber system to ensure the clinical stability of tooth position.  FIGURE 9-30 Histology of alveolar crest fibers extending from the cementum of the cervical region to the alveolar bone. Periodontal fibers penetrate alveolar bone, and transseptal fibers extend from the tooth on the left to the right. (From Avery JA, Chiego DJ Jr: Essentials of oral histology and embryology , ed 3, Mosby, 2006, St. Louis.) Elastic Fibers The three types of elastic fibers are elastin, oxytalan, and elaunin (see Chapter 4). Only oxytalan fibers are present within the PDL; however, elaunin fibers may be found within fibers of the gingival ligament. Oxytalan fibers (Figure 9-31) are bundles of microfibrils that are distributed extensively in the PDL. The fibers run more or less vertically from the cementum surface of the root apically, forming a three-dimensional branching meshwork that surrounds the root and terminates in the apical complex of arteries, veins, and lymphatic vessels. The fibers also are associated with neural and vascular elements. Oxytalan fibers are numerous and dense in the cervical region of the ligament, where they run parallel to the gingival group of collagen fibers. Although their function has not been determined fully, they are believed to regulate vascular flow in relation to tooth function. Because they are elastic, they can expand in response to tensional variations, with such variations then registered on the walls of the vascular structures. FIGURE 9-31 Oxytalan fibers seen through (A) the light microscope and (B) the electron microscope. These fibers run in an oblique direction, often from the cementum to blood vessels. Ground Substance Ground substance is an amorphous background material that binds tissue and fluids, the latter serving for the diffusion of gases and metabolic substances. Ground substance is a major constituent of the PDL, but few studies have been undertaken to determine its exact composition. What information exists indicates similarity to most other connective tissues in terms of its components, with some variation in ratios, so that in the ligament, dermatan sulfate is the principal glycosaminoglycan. The PDL ground substance has been estimated to be 70% water and is believed to have a significant effect on the ability of the tooth to withstand stress loads. An increase in tissue fluids occurs within the amorphous matrix of the ground substance in areas of injury and inflammation. Blood Supply For a connective tissue, the PDL is exceptionally well vascularized, which reflects the high rate of turnover of its cellular and extracellular constituents. The main blood supply of connective tissue is from the superior and inferior alveolar arteries. These arteries pursue an intraosteal course and give off alveolar branches that ascend within the bone as interalveolar arteries. Numerous branches arise from the interalveolar vessels to run horizontally, penetrate the alveolar bone, and enter the PDL space. Because they enter the ligament, they are called perforating arteries, and they are more abundant in the PDL of posterior teeth than in that of anterior teeth and are in greater numbers in mandibular than in maxillary teeth. In single-rooted teeth, these arteries are found most frequently in the gingival third of the ligament, followed by the apical third. This pattern of distribution has clinical importance. In the healing of extraction wounds, new tissue invades from the perforations, and the formation of a blood clot occupying the socket is more rapid in its gingival and apical areas. Within the ligament, these arteries occupy areas (or bays) of loose connective tissue called interstitial areas between the principal fiber bundles. Vessels course in an apical- occlusal direction with numerous transverse connections (Figure 9- 32). Fenestrated capillaries occur.  FIGURE 9-32 Corrosion cast demonstrating the extensive vasculature of the periodontal ligament. Many transverse connections and the thickened venous network at the apex are visible. (From Selliseth NJ, Selvig KA: J Periodontol 65:1079-87, 1994.) Many arteriovenous anastomoses occur within the PDL, and venous drainage is achieved by axially directed vessels that drain into a system of retia (or networks) in the apical portion of the ligament consisting of large-diameter venules (see Figure 9-32). Lymphatic vessels tend to follow the venous drainage. Nerve Supply The use of radioautographic and immunocytochemical labeling of neural proteins has greatly improved knowledge about the innervation of the PDL over what previously was based on the results of somewhat unpredictable silver staining techniques. Although species differences have been reported, a general pattern of ligament innervation seems to exist (Figure 9-33). First, the general anatomic configuration is applicable to all teeth, with nerve fibers running from the apical region toward the gingival margin and being joined by fibers entering laterally through the foramina of the socket wall (see Figure 9-21). These latter fibers divide into two branches, one extending apically and the other gingivally. Second, regional variation occurs in the termination of neural elements, with the apical region of the ligament containing more nerve endings than elsewhere (except for the upper incisors, where not only is the innervation generally denser than in molars but also further dense distributions of neural elements exist in the coronal half of the labial PDL as well as apically, suggesting that the spatial arrangement of receptors is a factor in determining the response characteristics of the ligament). Third, the manner in which these nerve fibers terminate is being clarified. Four types of neural terminations now have been described (Figure 9-34). The first (and most common) are free nerve endings that ramify in a treelike configuration. These nerve endings are located at regular intervals along the length of the root, suggesting that each termination controls its own territory, and extend to the cementoblast layer. These nerve endings originate largely from unmyelinated fibers but carry with them a Schwann cell envelope with processes that project into the surrounding connective tissue (Figure 9-35). Such endings are believed to be nociceptors and mechanoreceptors. The second type of nerve terminal is found around the root apex and resembles Ruffini's corpuscles. These nerves appear to be dendritic and end in terminal expansions among the PDL fiber bundles. By electron microscopy, such receptors can be seen to have subdivided further into simple and compound forms, the former consisting of a single neurite and the latter of several terminations after branching. Both receptors have ensheathing Schwann cells that are especially close to collagen fiber bundles (Figure 9-36), which provide morphologic evidence of their known physiologic function as mechanoreceptors. An incomplete fibrous capsule sometimes is found associated with the compound receptors. The third type of nerve terminal is a coiled form found in the midregion of the PDL, the function and ultrastructure of which have not been determined yet. The fourth type (the least common) is found associated with the root apex and consists of spindle-like endings surrounded by a fibrous capsule. FIGURE 9-33 Nerve terminals in a human periodontal ligament. (From Maeda T et al: Arch Histol Cytol 53:259-265, 1990.)  FIGURE 9-34 The four types of nerve endings found in a human periodontal ligament. A, Free endings with treelike ramifications. B, Ruffini's ending. C, Coiled ending. D, Encapsulated spindle-type ending. (From Maeda T et al: Arch Histol Cytol 53:259-265, 1990.)  FIGURE 9-35 Electron micrograph of a free nerve ending in a human periodontal ligament with an associated Schwann cell sending finger-like projections into the connective tissue. (From Lambrichts I et al: J Periodontal Res 27:191-196, 1992.)  FIGURE 9-36 Electron micrographs illustrating the close relationship of Ruffini-like endings with collagen fiber bundles. A, Insertion of collagen fibrils into the basal lamina of a Schwann cell. B, Neurite embracing a bundle of collagen fibrils. (From Lambrichts I et al: J Periodontal Res 27:191-196, 1992.) The autonomic supply of the PDL has not been fully determined yet, and the few descriptions available concern sympathetic supply. No evidence indicates the existence of a parasympathetic supply. The many free nerve terminals observed in close association with blood vessels are believed to be sympathetic and to affect regional blood flow. Adaptation to Functional Demand The structural components of the periodontium have been presented. (The gingiva facing teeth are described in Chapter 12.) Together these components form a functional system that provides an attachment for the tooth to the bone of the jaw while permitting the teeth to withstand the considerable forces of mastication. A remarkable capacity of the PDL is that it maintains its width more or less over time. The balance between formation and maintenance of mineralized tissues, bone, and cementum versus soft connective tissues of the PDL requires finely regulated control over cells in the local area. Several situations in which this balance is disrupted result in a variety of abnormal pathologic conditions, for example, (1) lack of tooth eruption because of ankylosis of teeth with surrounding bone, often associated with an osteoclast defect, and (2) lack of cementum formation resulting in exfoliation of teeth, as observed in hypophosphatasia. Compelling evidence exists indicating that populations of cells within the PDL, during development and during regeneration, secrete molecules that can regulate the extent of mineralization and prevent the fusion of the tooth root with surrounding bone. At the cell level, it has been reported that Msx2 prevents the osteogenic differentiation of PDL fibroblasts by repressing Runx2 transcriptional activity. Indeed, Msx2 may play a central role in preventing ligaments and tendons, in general, from mineralizing. At this point, the issue of how the PDL stays uncalcified while it is trapped between two calcified tissues remains unresolved and will require more attention. The PDL also has the capacity to adapt to functional changes. When the functional demand increases, the width of the PDL can increase by as much as 50%, and the fiber bundles also increase greatly in thickness. Conversely, a reduction in function leads to narrowing of the ligament and a decrease in number and thickness of the fiber bundles. These functional modifications of the PDL also implicate corresponding adaptive changes in the bordering cementum and alveolar bone. Another major function of the periodontium is sensory, although the nature of this function of the PDL still is being debated. When teeth move in their sockets, undoubtedly they distort receptors in the PDL and trigger a response. Thus the PDL contributes to the sensations of touch and pressure on the teeth; in addition, the spatial distribution of receptors is significant. What is equally certain, however, is that the ligament receptors are not the only organs from which sensations arise. For example, when teeth are tapped, vibrations are passed through the bone and detected in the middle ear. Debate also exists about the exact physiologic function of these receptors. Stimulation of the teeth causes a reflex jaw opening, and likewise, stimulation of periodontal mechanoreceptors initiates this response. Whether such a reflex is required for the normal masticatory process or is a protective mechanism to prevent forces applied to the teeth from reaching potentially damaging levels is not known.