Periodontology 2000, Vol. 67, 2015, 211–233 Printed in Singapore. All rights reserved

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

PERIODONTOLOGY 2000

Cementum proteins: role in cementogenesis, biomineralization, periodontium formation and regeneration H I G I N I O A R Z A T E , M A R G A R I T A Z E I C H N E R -D A V I D & G A B R I E L A M E R C A D O -C E L I S

Destruction of periodontal tissues is a cardinal sign of periodontal disease, and many factors, such as infections, trauma, orthodontic tooth movement and systemic and genetic diseases, can contribute to this process. In periodontal disease, bacteria on the surface of the teeth produce a chronic inflammation of the gingiva. Cells on the surface of the tooth root and the cementum covering the root are destroyed, and the epithelium from the oral mucosa grows downwards, producing a gingival crevice. Bacteria deposit in this crevice and the ensuing inflammatory process may eventually result in the breakdown of periodontal tissues, including the cementum, periodontal ligament and alveolar bone. The process of periodontal tissue regeneration is initiated at the moment that the damage takes place by the production of growth factors and cytokines by the damaged and inflammatory cells. Periodontal treatment can enhance periodontal healing (60). Root planing or root conditioning is being used as a strategy to increase mesenchymal cell migration and attachment to the exposed root surface. Treatment with acid, in particular citric acid, has been found to widen the orifices of dentinal tubules, thereby accelerating cementogenesis and enhancing cementum apposition and connective tissue attachment (125). However, when periodontal ligament cells are removed from the cementum or are unable to regenerate, bone tissue may invade the periodontal ligament space and establish a direct connection between the tooth and the wall of the alveolar socket, resulting in ankylosis. This nonflexible type of tooth support can lead to loss of function and eventually to resorption of the root (15). Strategies (such as guided

tissue regeneration) have been developed to guide and control regeneration using bioresorbable membranes (3, 138, 142) and bone grafts (175). Although effective to a certain point, these strategies have the problem that they are not predictable and do not completely restore the architecture of the original periodontium. To achieve complete repair and regeneration it is necessary to recapitulate the developmental process with complete formation of cementum, bone and periodontal ligament fibers. The past 20 years of research have seen tremendous advances in our knowledge of the cellular and molecular events involved in the process of developing the periodontium. This knowledge has translated into new therapeutic strategies for periodontal regeneration using molecular approaches (45, 67, 110–112, 116, 129, 146, 158, 210, 223, 240). Amongst these strategies are the use of growth factors, such as platelet-derived growth factor and insulin-like growth factors (32, 54, 90, 120, 163, 181, 222), transforming growth factor-beta1 (127), basic fibroblast growth factor (191), dexamethasone (181) and bone morphogenetic proteins (109, 114, 154, 176–178). It is believed that these molecules are produced during cementum formation and are then stored in the cementum matrix to induce periodontal ligament regeneration when needed (200). However, one of the problems of application of these factors for periodontal repair is the nonspecific activity of some of the factors on different cell lineages and the rapid loss of the topically applied factors over time (15, 120). As our understanding of the structure, function and composition of cementum increases, so does the

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potential for new therapies for periodontal regeneration using molecules formed by these tissues. One such example has been the use of enamel proteins to induce cementum and bone regeneration (50, 51, 72– 74, 84, 85). A preparation of porcine enamel protein has been marketed (Emdogainâ) and is currently being used by practicing dentists (42, 52, 55, 105, 237, 239). Outcomes from studies using Emdogain suggest that its clinical action is the result of contamination with growth factors such as transforming growth factor-beta (107, 143) and bone morphogenetic proteins (100). However, studies using recombinant amelogenin, including results obtained in our laboratory, indicate that amelogenin and ameloblastin also have signaling properties that induce phenotypic changes in cells, and these changes can fluctuate depending on the target cell (20, 219, 237, 239, 240). It is the purpose of this review to focus on the role of cementum and its specific components in the formation, repair and regeneration of the periodontium. As cementum is a matrix rich in growth factors that could influence the activities of various types of periodontal cells, this review will examine the characteristics of cementum, its composition and the role of cementum components, especially the cementum protein-1, during the process of cementogenesis, and their potential usefulness for regeneration of the periodontal structures in a predictable therapeutic manner.

What is cementum? Cementum can be described as the mineralized tissue that covers the roots of teeth and serves to attach the tooth to alveolar bone via collagen fibers of the periodontal ligament. Morphological, histological and functional differences appear to exist along the length of the root, leading cementum to be classified as follows: intermediate cementum (found in the cemento–enamel junction), acellular cementum (found in the coronal and mid-portions of the root) and cellular cementum (present in the apical and inter-radicular portions of the root containing cementoblasts) (21, 33, 71, 75, 183, 187, 207, 240). Studies on dental cementum can be traced back to Malpighi in the 1600s (61). In general, 18th century anatomists regarded human teeth as composed only of enamel and dentin. However, the studies of Tenon on horses’ teeth, of Blake on elephants’ teeth and of Cuvier on the teeth of many species resulted in the recognition that cementum was a constituent part of

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all animal teeth. Initial examination of cementum on the roots of human teeth is attributed to Ringelmann in 1824, followed by physiologists Jan Evangelista Purkinje and his pupils, Fraenkel and Raschkow, in 1835, and later by the classic histologist Anders Adolf Retzius, in 1836, who noticed the presence of ‘striaes’ in cementum (19, 37, 61, 186). In almost all mammalian species the number of incremental structures in the dental cementum (annulations) can be correlated with age and are used as such in archeology and forensics (61). In a very simplistic way we can define cementum as an extracellular matrix composed of calcified collagenous Sharpey’s fibrils, collagen, glycosaminoglycans, proteoglycans and inorganic hydroxyapatite. In the same way we can say that the major functional role of cementum is to serve as the anatomical structural site for the attachment of Sharpey’s fibers of the periodontal ligament. However, the more we study this tissue, the more complex we find its structure and function. Cementum biology goes back to Gottlieb, in 1942, who stated that “the continuous deposition of cementum layers seems to be of great importance and work as a barrier against the downgrowth of the epithelium. Newly deposited cementum seems to have the highest vitality and act as the best barrier. If the ideas about the biology of cementum are correct, it is then our task to find out just how nature provides for continuous cementum deposition and having done so, to imitate the procedure” (62). These studies introduced the notion that not only does cementum act as a barrier to delimit epithelial growth that can impair attachment but also that the presence of a continuous cementum layer is necessary to act as a microbial barrier and that defects in this tissue could result in periodontitis (62, 63). This idea was further supported by observations that the root surfaces of patients with hypophosphatasia contained areas completely devoid of cementum or covered with a hypoplastic form of a cementum-like material. These patients developed early-onset periodontitis, suggesting that abnormalities in the deposition or maintenance of cementum can result in a defective periodontium highly susceptible to microbial invasion and destruction (160). Based on the different functions attributed to cementum it is clear that a thorough understanding of the biological properties of cementum is required to determine its role in periodontal formation and therefore periodontal regeneration. Furthermore, the principles attributed to cementum regeneration might be used in the regeneration of other mineralized tissues.

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Cementum composition In order to understand the process of cementogenesis it is important to know the composition of cementum. As in bone and dentin, the major organic component of cementum is collagen (18). The major type of collagen is type I collagen, which accounts for 90% of all collagens and plays a structural role during the biomineralization process, serving as a reservoir for hydroxyapatite nucleation, which successively develops into intrafibrillar apatite crystals (58). Type III collagen, which coats type I collagen fibrils, is also present, although in considerably lower quantities. In addition to collagens, carboxylated and sulfated mucopolysaccharides (glycosaminoglycans) are present in human cementum (217).

Glycosaminoglycans Glycosaminoglycan-containing proteoglycans are heterogeneous groups of glycoproteins with long repeating disaccharides. The percentage of glycosaminoglycans is high in tissues subjected to compressive forces, such as cementum. Proteoglycans are known to interact specifically with collagens in a variety of tissues. It is postulated that proteoglycans in cementum are integral components of cell substratum attachment matrices and mediate attachment between old and newly formed cementum, thus creating the cementum incremental lines (189, 220). The major glycosaminoglycans present in human cementum are hyaluronic acid, dermatan sulfate and chondroitin sulfate, and their distribution appears to be quite different from that reported for soft tissues such as gingival connective tissue and periodontal ligament, in which dermatan sulfate predominates (12, 165). The proteoglycan content of mineralized tissues is generally relatively low. These differences might reflect differences in function between hard and soft tissue as proteglycans appear to inhibit collagen mineralization by occupying strategic locations normally destined to be filled with hydroxyapatite (189). In bone, dermatan sulfate proteoglycans are oriented parallel to the collagen fiber axis with chondroitin sulfate proteoglycans and hyaluronic acid, occupying the interfibrillar region in a space-filling capacity (190). Amongst other glycosaminoglycans present in cementum, keratan sulfate appears to be one of the major components, which, after digestion with keratanase II and endo-beta-galactosidase, produces two core proteins: lumican and fibromodulin. Interestingly, these proteins are localized predominantly in nonmineralized cementum (precementum and the

pericementocyte area), suggesting that they play major regulatory roles during cementum mineralization (30). In a similar manner, it was also determined that the large chondroitin sulfate glycosaminoglycan, present in cementum, contains the large hyaluronanbinding proteoglycan, versican, and the small interstitial proteoglycans, decorin and biglycan. Versican is localized in lacunae housing cementocytes. Decorin is closely associated with collagen fibers of the periodontal ligament and with biglycan in the cementoblasts/precementum area. The differential tissue distribution suggests that glycosaminoglycans may play distinct roles during the cementogenesis process in addition to regulating the biomineralization of cementum (31). Syndecan-2 has been found to be significantly expressed by cells in close contact with the root surface and within the matrix of reparative cementum, suggesting that it must be associated with cell–matrix interactions during cementum mineralization (224). Biglycan is also associated with the growth of incremental lines in cellular cementum. Furthermore, lumican, decorin, versican and biglycan are associated with the formation of cellular cementum but not of acellular cementum, suggesting different cementocyte subpopulations or a differential response of these cells (1). Osteoadherin, a keratin sulfate-containing proteoglycan, is also associated with the initial phase of cementum formation because Hertwig’s epithelial root sheath cells express this proteoglycan during root development (166), and although acellular cementum does not contain proteoglycans, initial acellular cementum formation requires a dense accumulation of proteoglycans (230).

Ostepontin and bone sialoprotein Cementum contains many noncollagenous proteins, including some major phosphoproteins such as osteopontin and bone sialoprotein. These proteins play a major role in filling spaces created during collagen assembly and imparting cohesion to the mineral-like tissue by allowing mineral deposition to spread across the entire collagen meshwork (22, 148). The role proposed for these proteins is that of regulators of hydroxyapatite crystal nucleation and growth. It has been suggested that osteopontin and sialoprotein are necessary for the initiation of crystal formation at the highly ordered fibrils of type I collagen (179). Both of these proteins are acidic: osteopontin has a poly-Asp and sialoprotein contains two poly-Glu domains, the repetitive sequences of which are known to bind calcium to mineral surfaces. Osteopontin is present within the periodontal ligament in mature teeth.

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Sialoprotein and osteopontin remain bound to collagen matrix and they possess cell-attachment properties through their arginine-glycine-aspartic acid (RGD) sequences (199). In the periodontium, osteopontin is expressed by cells in close contact with acellular cementum as well as by cementocytes (26). It has also been suggested that osteopontin regulates cell migration, differentiation and survival via interactions with avb3 integrin (53). Sialoprotein is also an RGD-containing sialoprotein with cell-attachment properties (156) and has a precise spatial association with early mineral aggregates, binds strongly to hydroxyapatite and acts as a specific and potent nucleator for hydroxyapatite crystal formation in vitro (95). Cementum contains sialoprotein, which, during root formation, is distinctly localized to cells lining the surface of cementum. It has been suggested that sialoprotein modulates the process of cementogenesis and is involved in the process of chemoattraction, adhesion and differentiation of precementoblasts (121–123, 201). Both sialoprotein and osteopontin are believed to play a role in the differentiation of cementoblast progenitor cells to cementoblasts (183).

Gla proteins Matrix gamma-carboxyglutamic acid protein and osteocalcin are the two major Gla-containing proteins associated with calcified hard tissues (80–82, 172, 173). Both proteins have high affinity for Ca2+ and hydroxyapatite through interaction with the Gla residue. The distribution of osteocalcin in the mammalian body is virtually limited to mineralized tissues such as bone, dentin and cementum (34). In the dental root, osteocalcin expression is localized in cells lining cellular cementum and acellular cementum. However, cells at the inter-radicular area also express osteocalcin. In a similar manner, cellular and acellular cementum show expression of matrix gamma-carboxyglutamic acid protein, although acellular cementum expresses those proteins more prominently than does cellular cementum. Matrix gamma-carboxyglutamic acid protein is secreted by cementum-forming cells and is incorporated at the mineralization front (103). One possible explanation for the accumulation of matrix gammacarboxyglutamic acid protein in acellular cementum and the outer surface of cellular cementum could be to prevent hypercalcification of the cementum surface (77, 104). During root development in mice, a high level of osteocalcin mRNA is selectively expressed by cells lining root-surface cementoblasts;

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in contrast, osteocalcin mRNA is not expressed in the periodontal ligament (38). The role of these proteins has been related to regulation of mineralization. Matrix gamma-carboxyglutamic acid protein-deficient mice show abnormal mineralization and a lack of acellular cementum (104). It has been suggested that both osteocalcin and matrix gamma-carboxyglutamic acid protein act as negative regulators of mineralization because matrix gamma-carboxyglutamic acid protein-deficient mice promote calcification of the aortic walls and valves. Thus, both osteocalcin and matrix gamma-carboxyglutamic acid protein seem to regulate mineralization by acting as negative regulators, but to different extents because osteocalcin also inhibits conversion of brushite to hydroxyapatite (81, 173). Other molecules present in the cementum extracellular matrix include osteonectin, which, during cementogenesis, is synthesized by cementum-producing fibroblasts, cementoblasts and cementocytes (174). Osteonectin, synthesized by mineralizing cells, can bind hydroxyapatite and is associated with mineralization (25, 80, 96, 126, 135). Based on the observation that low concentrations of osteonectin delay hydroxyapatiteseeded crystal growth in vitro, it is speculated that osteonectin also acts as a negative regulator by preventing, rather than promoting, matrix mineralization (134, 230).

Alkaline phosphatase Tissue nonspecific alkaline phosphatase (also known simply as alkaline phosphatase) has been studied for more than 80 years and is believed to play an important role in skeletal mineralization. Alkaline phosphatase is a membrane-bound glycoprotein enzyme that hydrolyses phosphate groups at alkaline pH and also inhibits pyrophosphatase, ATPase and protein phosphatase activity at neutral pH (94). Alkaline phosphatase is expressed in most body sites during embryonic development but is confined to bone, kidney, liver and B-lymphocytes during adult life. The fact that it is expressed in nonmineralizing tissues suggests that it has other roles besides those associated with mineralization. Amongst some of these other functions, it has been suggested that alkaline phosphatase can regulate tissue turnover and cell proliferation, differentiation and maturation (94, 226). Alkaline phosphatase is highly expressed in cells of the periodontal ligament (56, 69, 99, 124, 151, 231), where it is thought to play a role in phosphate metabolism and cementum formation (13), particularly formation of acellular cementum (14, 65). Tissue nonspecific alkaline

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phosphatase-deficient mice show defective formation of acellular cementum, which results in very thin and irregular-shaped patches around the bases of the periodontal ligament fibers. No defects were seen in alveolar bone, periodontal ligament and cellular cementum, suggesting that alkaline phosphatase is essential for the formation of acellular cementum (16). However, observations in humans with hypophosphatasia as a result of mutations in the tissue nonspecific alkaline phosphatase gene revealed that cementum formation is almost completely abolished for both acellular and cellular cementum, resulting in premature tooth loss. This phenotype differs from that in tissue nonspecific alkaline phosphatase geneknockout mice, which showed blockage of acellular cementum formation only (213, 214) and might be explained by the different types of mutation possible in the tissue nonspecific alkaline phosphatase gene and the severity of their manifestation in humans. One of the major functions of tissue nonspecific alkaline phosphatase is the hydrolysis of inorganic pyrophosphate, a potent inhibitor of hydroxyapatite formation (206). Cementoblasts are specifically sensitive to the levels of inorganic pyrophosphate/inorganic phosphate within the extracellular matrix (150). Changes in the level of tissue nonspecific alkaline phosphatase protein have a significant effect on the function of osteoblasts, and consequently on matrix mineralization, indicating that tissue nonspecific alkaline phosphatase plays key biological roles in the mineralization of bone and cementum (89). One novel strategy to reduce inorganic pyrophosphate and increase cementum neoformation may center on the modulation of inorganic pyrophosphate/inorganic phosphate in the periodontium, which may result in more predictable regeneration of cementum (180).

Cementum-specific proteins Extracellular matrix from different tissues share many similarities and yet have different functional properties that make them unique. These properties could be the result of quantitative and/or qualitative differences amongst their components. For years it was believed that different mineralized tissues contain specific molecules not present in any other tissue (i.e. amelogenin in enamel, dentin sialophosphoprotein in dentin, etc.) and which could be considered as markers for those tissues. As detection techniques became more sensitive, it was found that many of these molecules were also expressed in other tissues, although at considerably lower concentrations, and

therefore could still be considered as specific markers. Several proteins, some of which are considered to be cementum-specific proteins, have been isolated from cementum and characterized. Cementum-derived growth factor It is now well established that mineralized tissues, such as bone and dentin, are excellent reservoirs of growth factors that, when needed, can be released by demineralization and serve to repair or regenerate tissues. In a similar way, it has been shown that extracts of cementum have the ability to promote a range of biological activities such as cell migration, adhesion, mitogenic activity and differentiation, which are essential for periodontal regeneration (67). Miki et al. (137) were the first to report the presence of mitogenic activity in cementum obtained from human teeth. Later, Nakae et al. (144) isolated and characterized mitogenic factors present in the cementum matrix of bovine teeth. In addition to fibroblast growth factor, which binds strongly to heparin, another mitogenic factor with moderate heparin affinity was present in cementum but not in alveolar bone. This factor was named cementum-derived growth factor and it is the major component in cementum, accounting for 70% of the mitogenic activity extracted from this tissue. The cementumderived growth factor acts synergistically with epidermal growth factor and induces many of the signaling pathways associated with mitogenesis (234). These pathways include an increased concentration of cytosolic Ca2+, activation of the protein kinase C cascade and expression of cellular proto-oncogenes. Additionally, cementum-derived growth factor may promote the migration and growth of progenitor cells, present in the adjacent structures, toward the dentin matrix and participate in their differentiation into cementoblasts (130, 170). Further characterization of cementum-derived growth factor revealed that the mitogenic activity was associated with a 14-kDa protein that showed some homology to insulin-like growth factor-1. Although cementum-derived growth factor activity was inhibited with insulin-like growth factor-1 and insulin-like growth factor-1 receptor antibodies, there were some differences between the canonical insulin-like growth factor-1 and cementum-derived growth factor, thus it was concluded that cementum-derived growth factor is an insulin-like growth factor-1-like molecule (97). The presence of cementum-derived growth factor and other growth factors in cementum indicates that cementum has the potential to regulate the metabolism and turnover of surrounding tissues, that

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cementum could serve as a storage site for those growth-inducing molecules and that the cementum proteins are likely to serve a biological role in promoting periodontal regeneration (67, 149). Cementum attachment protein In addition to cementum-derived growth factor, it has been reported that human and bovine cementum contains potent mediators of cell attachment, and this biological activity is associated with a 55-kDa protein species (128, 157). This protein was named cementum attachment protein and further characterization by amino-acid sequencing showed the presence of four sequences containing Gly-X-Y repeats typical of collagen. A 17-amino-acid peptide had 82% homology with a type XII domain, and another peptide had 95% homology with collagen type Ia1. However, cementum attachment protein did not cross-react with antibodies to type I, type V, type XII and type XIV collagen, and its attachment activity was lost after treatment with bacterial collagenase. These findings, and the fact that a cementum attachment protein monoclonal antibody localizes cementum attachment protein only to cementum (7), suggest that cementum attachment protein might be a collagenous-attachment protein localized exclusively in cementum (225). Characterization of a complementary DNA clone for cementum attachment protein isolated from a human cementifying fibroma-derived cell line k-ZAP expression library, revealed a novel alternatively spliced sequence. This sequence encodes a 140-amino-acid protein that is identical to the first 125 N-terminal amino acids of a truncated isoform of 3-hydroxyacylCoA dehydratase-1/protein-tyrosine phosphatase-like (proline instead of catalytic arginine), known as PTPLA (212). The remainder of the C-terminus of PTPLA/cementum attachment protein is encoded by a read-through of the splice donor site in exon 2, and the truncation eliminates the PTPLA sequence that has the signature phosphatase active site motif. Although PTPLA mRNA is widely expressed in many tissues, the PTPLA/cementum attachment protein mRNA is expressed in cementum cells and only marginally in some periodontal ligament cells in human teeth. PTPLA/cementum attachment protein was not found to be expressed in rat teeth (184), suggesting that there could be some species differences in its distribution. Rat roots are known to overproduce apical cementum after they reach 8 weeks of age, whereas other species maintain a normal layer of cementum. Interestingly, the cementum attachment protein is a 55-kDa collagenous protein, whereas the PTPLA/

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cementum attachment protein codes for a 15-kDa protein that has no collagen sequences. This suggests that perhaps the cementum attachment protein is strongly associated with collagen chains in the cementum matrix, possibly through cross-linking, which increases its molecular weight. This possibility is supported by the observation that immunoprecipitates of cementum extracts obtained with several anti-cementum attachment protein monoclonal antibodies contain two protein species migrating at 55 kDa and ~ 29 kDa, and one monoclonal antibody cross-reacts with both cementum attachment protein and type I collagen (A.S. Narayanan, unpublished data). Expression of PTPLA/cementum attachment protein is limited to cementum and to some cells in the endosteal spaces of bone. This could be explained as a result of the presence of precursors of cementoblasts in the endosteal spaces of alveolar bone (130, 133, 198). These cells are thought to traverse through the periodontal ligament before reaching and differentiating on cementum. It has been shown that cementum attachment protein binds to hydroxyapatite and more strongly to cementum than to the dentin surface (168). Cementum attachment protein also binds to fibronectin, but binds 150 times more strongly to hydroxyapatite than to fibronectin (169). Like the cementum attachment protein obtained from cementum, recombinant PTPLA/cementum attachment protein also binds to hydroxyapatite with high affinity (212). These observations suggest that PTPLA/cementum attachment protein may play a regulatory role during cementum formation (67, 212). It has also been shown that cementum attachment protein promotes the attachment of gingival fibroblasts, endothelial cells and smooth muscle cells, but not oral sulcular epithelial cells (157). Bone cells bind more strongly to cementum attachment protein than do periodontal ligament cells, which, in turn, bind more strongly to cementum attachment protein compared with gingival cells (300% for bone cells, 250% for periodontal ligament cells and 150% for gingival cells). Cementum attachment protein-coated root slices promote preferential adhesion and differentiation of osteoblastic cells (168, 169, 171). Attachment to cementum attachment protein by human gingival fibroblasts is mediated primarily by the integrin a5b1 (98). The a5b1 integrin has been shown to be involved in various aspects of development (131), and it is possible that the a5b1 integrin may also play an important role in cementogenesis and neo-cementogenesis through interactions with cementum attachment protein. Cementum attachment protein has the capacity to

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direct cell migration of alveolar bone cells. Periodontal ligament cell populations differ in their capacity to recognize and respond to cementum attachment protein. Fifteen per cent of clones of periodontal ligament cell cultures bind strongly to cementum attachment protein and this binding capacity is similar to that of alveolar bone cells, suggesting that the origin of cementoblastic precursors could be osteogenic. Therefore, cementum attachment protein may potentially be used to induce preferential repopulation of the root surface by appropriate cells (169). Periodontal ligament cells manifest a higher chemotactic response to cementum attachment protein than do gingival fibroblasts, and this activity is 24-fold greater than that of fibronectin (136). Cementum attachment protein binds selectively to periodontal ligament cells and supports periodontal ligament cell attachment to root surfaces (171). Selective chemotaxis and attachment by cementum attachment protein may represent the natural pathway by which cementoblast progenitors are attracted to the root surface during homeostasis and regeneration. Partially demineralized cementum does not support epithelial outgrowth; however, it enhances migration toward and attachment of periodontal connective tissue cells to dental surfaces in vitro (168, 169). Thus, exposed collagen fibers might improve the capacity of periodontal connective tissue cells to compete with epithelial cells and be the first to attach to and colonize the root surface after periodontal surgery (167). If this is the case, biochemical alterations in cementum may explain the loss of soft-tissue attachment from diseased root surfaces and the failure for its successful restoration. Selective chemotaxis by, and attachment through, cementum attachment protein might represent the natural pathway by which cementoblast progenitors are attracted to the root surface during homeostasis and regeneration. It has been reported that the cementum attachment protein possesses the capacity to bind periodontal ligament progenitor clones and that this is directly related to their alkaline phosphatase expression and mineralized-like tissue formation. These characteristics are found in cementoblastomaderived cells, which produce mineralized nodules (6, 118). A direct correlation between alkaline phosphatase expression and mineralized-like tissue formation was detected in clones with high binding capacity to cementum attachment protein (118), indicating that cementum attachment protein is associated with mineralizing-tissue-forming progenitors in the periodontal ligament. The groups of clones that bind to cementum attachment protein and produce

mineralized-like tissue, similar to cellular cementum, represent 7% of the periodontal ligament population and 15% of the mineralized-tissue forming clones belonging to the cementoblastic lineage (118). The high binding capacity of cementum attachment protein, combined with a low constitutive percentage of alkaline phosphatase expression, is of interest because it supports the view that cementoblastomaderived cells express a low constitutive percentage of alkaline phosphatase-positive cells and that cementoblasts express low levels of alkaline phosphatase compared with alveolar bone-derived osteoblasts (205). Adhesion of human gingival fibroblasts to cementum attachment protein stimulates mitogen-activated protein kinase activity and induces the expression of c-fos mRNA; in contrast, protein-tyrosine phosphorylation and c-fos mRNA were not induced in unattached cells. As mitogen-activated protein kinase and c-fos mRNA were not induced in monolayer cultures, it was presumed that these reactions are induced by adhesion and are not necessary for cell adhesion (182). The kinetics of mitogen-activated protein kinase activation was different for cells attaching to fibronectin or polylysine – c-fos mRNA levels increased only half as much in cells attaching to fibronectin and very little in cells attaching to polylysine. These data demonstrate that cementum attachment protein and other adhesion molecules present in mineralized tissue matrices induce characteristic signaling events during adhesion, which may play a role in the recruitment of specific cell types during wound healing and in mediating their specific biological functions. This differential response is especially important in periodontal regeneration, in which epithelial cells must be excluded and fibroblasts and cementoblasts are to be selected from a pool of various progenitor cells (170). Cementum attachment protein is likely to play a role in the cell selection process. The mitogen-activated protein kinase kinase/mitogenactivated protein kinase pathway participates in cementum attachment protein-mediated fibroblast spreading, but cell attachment and proliferation do not appear to require extracellular signal-regulated kinase-2. Both cementum attachment protein and fibronectin mediate cell attachment through the same a5b1 integrin (98); however, there are differences in the signaling mechanisms induced by cementum attachment protein and fibronectin during cell attachment. There are also differences in attachment and spreading promoted by these substrates. These differences may explain why cells attach, spread and migrate differentially on cementum attachment protein-containing surfaces, and

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such differences can be expected to play a role in the recruitment of cells needed for the regeneration of cementum and other periodontal structures during periodontal regeneration. Cementum attachment protein bound to root surfaces has been shown to enhance the recruitment of putative cementoblastic cells to the root surface in vitro (118). The amount of cementum attachment protein bound might be an indicator of the commitment of a progenitor clone to the mineralized-tissue-forming cell lineage. Cementum attachment protein is instrumental in recruiting putative cementoblastic progenitors to the root surface and is capable of enhancing their differentiation. Also, cells that migrate and attach to root surfaces coated with cementum attachment protein show higher expression of alkaline phosphatase, sialoprotein and cementum attachment protein (118). This indicates that cementum attachment protein plays an important role in promoting the differentiation of putative cementoblast progenitors. Human fibroblasts attached onto surfaces containing cementum attachment protein as the only adhesion substrate can synthesize DNA. However, the synthesis requires the engagement of integrins, presumably the a5b1 to which cementum attachment protein binds (98). This indicates that in vivo, tooth-root surfaces containing cementum attachment protein as the matrix component are conducive for cell proliferation. Cell attachment to cementum attachment protein induces immediate-early G1 phase events, and cyclin D1 levels increase in the cells adhered to cementum attachment protein alone, even without growth factors. The expression of cyclin D1 is regulated by adhesion in the presence of growth factors, and signal reactions generated by binding cementum attachment protein to cementum attachment protein receptors induce expression of cyclin D1 (232). Cementum attachment protein affects cell-cycle progression through mechanisms that are common to other molecules. Nevertheless, differences occur in the type and degree of induction of these events (182). Cells differing in the capacity to bind cementum attachment protein also differ in their ability to form mineralized tissue in culture and to produce cementum attachment protein (11, 118). These observations indicate that substances such as cementum attachment protein present in the local cementum environment could determine which cells are recruited and how they differentiate during normal homeostasis and wound healing, and whether the healing response is repair or regeneration. This is important in periodontal regeneration when regeneration requires new cementum formation and restoration of connective tissue attachment (48, 170).

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Formation of new cementum with inserted Sharpey’s fibers on previously exposed root surfaces is an essential process in the regeneration of periodontal tissues. This process requires the selective repopulation of exposed root surfaces by cementoblastic and fibroblastic cell lineages that originate within the periodontal ligament and possibly bone (132). Selective repopulation invokes that the growth, differentiation, directed migration and attachment of these cell lineages should be specifically regulated in time and space. These actions could be accomplished by cementum components, such as cementum-derived growth factor, cementum attachment protein and cementum protein-1 (67, 171). Cementum protein-1 Cementum protein-1 was first isolated from human cementum and human cementoblastoma-derived conditioned media (8–10). It is expressed from a single-copy gene as a 26-kDa nascent protein that is extensively modified by post-translational events. The human cementum protein-1 gene contains one exon, spans 1.4 kb and maps to the short arm of chromosome 16 (16p13.3). The primary sequence of cementum protein-1 was first deduced from a human complementary DNA sequence that showed 98% homology with a predicted 247-amino-acid sequence present in the Pan troglodytes chromosome 16 (5). No similar sequences have been found so far in other species. Human cementum protein-1 is composed of 247 amino acids with a calculated molecular weight of 26 kDa, and it appears to be an alkaline protein (isoelectric point = 9.73), with no signal peptide. The cementum protein-1 gene product is enriched in proline (11.3%), glycine (10.5%), alanine (10.1%), serine (8.9%), leucine (8.1%), threonine and arginine (each 7.7%) and contains low levels of tryptophan, aspartic acid, isoleucine (each 2.0%) and phenylalanine (1.6%). Tyrosine is not present. The amino-acid sequence indicates that the cementum protein-1 is likely to be a nuclear protein; however, it does not have DNA-binding motifs. Amino acids 30–110 show 48% similarity with the human collagen a I (I) chain, 46% similarity with type XI and 40% similarity with type X. The full-length recombinant cementum protein-1 expressed in human-derived gingival fibroblasts is mainly composed of beta-sheet with 10% alpha-helix, 32.4% anti-parallel, 5.8% parallel, 16.7% beta-turn and 35% random coil. This feature is associated with proteins that have a high percentage of random coil structure, which have been shown to be multifunctional and to have diverse binding properties; such

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proteins include SIBLING and HMGI(Y) (35, 41). This might help to explain why cementum protein-1 regulates crystal growth and composition of apatite crystals (4). According to in silico analysis, cementum protein-1 possesses two N-glycosylation sites, namely asparagine-X-serine in amino acids 20 and 25 – and this is consistent with its shift from a protein size of Mr 50,000 to a protein size of Mr 39,000. The precise role of attached carbohydrates in cementum protein1 is unknown; however, glycosylation may affect the function of cementum protein-1 during the mineralization process because their anionic surface can bind a large number of Ca2+ ions and regulate hydroxyapatite crystal growth (29). Glycans are also implicated in the regulation of endochondral ossification, bone remodeling and fracture healing (66). Cementum protein-1 appears to be a phosphorylated protein because antibodies against phosphor-serine and phosphor-threonine cross-react with cementum protein-1. The presence of phosphate also favors the binding of Ca2+ to the protein (102, 209), and proteins (such as sialoprotein and osteopontin) associated with the mineralization process are highly phosphorylated at threonine and serine residues (236). Thus, cementum protein-1 may play a role at the early stages of mineralization during the formation of octacalcium phosphate (Fig. 1). The cementum protein-1 does not react with sulfhydryl groups; therefore, all cysteine residues in cementum protein-1 might be linked to disulfide bridges, which generally play a role stabilizing protein structure (87, 88, 106). In a steady-state system with the concentrations of calcium and phosphate maintained below the thresh-

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old of spontaneous precipitation, recombinant human-cementum protein-1 is effective in promoting the nucleation of octacalcium phosphate in an agarose gel. It was calculated that as little as 1.0 lg/mL of cementum protein-1 can promote nucleation (218). Human recombinant cementum protein-1 possesses high affinity for hydroxyapatite, even without posttranslational modifications, and it affects the morphology of apatite crystals. Human cementum protein-1 induces the formation of polymorphous crystals, as confirmed by X-ray diffraction. Elemental analysis performed with energy-dispersive X-ray analysis identified a calcium/phosphorus ratio of 1.4, which corresponds to octacalcium phosphate (Fig. 2). These findings indicate that biologically active cementum protein-1 plays a role during the biomineralization process and is required for the synthesis of needle-like shape crystals. Octacalcium phosphate is found to be a transient phase during the growth of biological crystals. In small crystals, octacalcium phosphate is completely transformed into hydroxyapatite by hydrolysis and can only be detected in large crystals because of its slow kinetics of transformation. Octacalcium phosphate has also been presumed to be a necessary precursor of biological apatites. Further characterization of cementum protein-1 using western blots with extracted proteins from human cementoblastoma and antibody to periodontal ligament-derived cells showed the presence of three components: 55-, 50- and 26-kDa species. These products might represent differences in the degree of post-translational modifications, particularly phosphorylation and glycosylation (5). In vitro studies

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Fig. 1. Scanning electron micrograph images of crystal growth. (A) Human recombinant cementum protein-1 (20 lg in a 0.5% agarose gel) induced the formation of a sphere with irradiating prismatic crystals. (B) Enlargement of the box in panel A shows the octacalcium phosphate prismatic crystals. (C) The internal part of the sphere shows a central nucleus with irradiating prismatic crystal. (D) A more detailed view of the box in panel C shows that the irradiating crystals are originating from a central nucleus in a needle-like shape that later acquires the prismatic crystal morphology that grows only in cementum protein-1-containing gels.

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Fig. 2. (A) Representative energy-dispersive X-ray microanalysis spectrum of the crystals formed as a result of the effect of human recombinant cementum protein-1 in a steady-state agarose system. The spectrum shows prominent peaks of calcium (Ca) and phosphorus (P) and the Ca/P ratio indicates that the crystals are octacalcium phosphate. C, carbon; Cl, chlorine; O, oxygen; Si, silicon. (B) Direct visualization of cementum protein-1 nanospheres (39 nm high, 81 nm wide, ovoid morphology), as determined by tapping mode atomic force microscopy in air.

using immunocytochemistry confirmed the expression of cementum protein-1 by cementoblastomaderived cells and periodontal ligament cells. Almost all (95%) of the cementoblastoma-derived cell population was positive for cementum protein-1, whereas only 6% of periodontal ligament cells stained positive for cementum protein-1. Cementum protein-1 was also expressed in a small population (3%) of osteoblastic cells in vitro, whereas cementum protein-1 was not detected in gingival fibroblasts. These small periodontal ligament-positive and osteoblast-positive populations could represent cementoblast precursors, suggesting that cementoblasts and osteoblasts might have a common ancestor and that cementum protein-1 could be a marker for the cementoblastic lineage (10). Immunohistochemistry studies using

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human periodontal tissues showed localization of cementum protein-1 throughout the entire cementum surface, including the cementoid phase of acellular and cellular cementum, cementocytes and cells near the blood vessels in the periodontal ligament (Fig. 3). These cells are considered as cementum progenitor cells (7, 8, 10). Cementum protein-1 is not expressed in any other human tissues, indicating that cementum protein-1 is a tissue-specific protein, restricted to cementoblasts and its progenitor cells, and that it might have a role as a local regulator of cell differentiation and extracellular matrix mineralization (4). An interesting observation was the fact that cementum protein-1 cross-reacts with antibodies to type X collagen. Collagen type X is a product of the hypertrophic chondrocytes and facilitates endochondral ossification (115, 193), which suggests some relationship among cementum protein-1, chondrogenesis and the mineralization processes. This hypothesis is supported by studies showing that human cementum extracts promote attachment of chondrocytes in a dose–dependent manner. Furthermore, mesenchymal bud stem cells grown in the presence of cementum extracts result in the formation of Alcian blue-positive nodules, which synthesize sulfated proteoglycans (8). The expression of proteoglycans rich in chondroitin sulfate is characteristic of cartilage (28, 40, 185, 211, 221). Taken together, these studies suggest that cementum-specific proteins can induce stem cells to express a cartilage phenotype. Furthermore, cementum functions not only as an inducer of cell differentiation but also as an inducer of proliferation (8). The function of cementum protein-1 was further tested by transfection into nonmineralizing cells, such as human gingival fibroblasts. In contrast to normal human gingival fibroblasts, human gingival fibroblasts/cementum protein-1 cells showed increased proliferation, formation of mineralized nodules, increased alkaline phosphatase-specific activity and the de novo expression of osteocalcin, osteopontin, sialoprotein, runt-related transcription factor 2/core-binding factor alpha1 and cementum attachment protein mRNAs and protein. These molecules are all associated with bone/cementum formation (27). The studies strongly support the notion that cementum protein-1 has the ability to change the cell phenotype from nonmineralizing (human gingival fibroblasts) to mineralizing (osteoblast/cementoblast) by regulating proliferation and gene expression, resulting in the differentiation of these cells and the production of a mineralized extracellular matrix resembling cementum.

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Fig. 3. Double immunostaining of human periodontal tissues with cementum proteins. (A) Immunostaining shows that anti-bovine CAP IgG1 (a-CAP) cross-reacts with cementoblasts (CB) paravascular cells (PVC) and cell subpopulations in the periodontal ligament (PL). (B) Cementum protein-1 antibody (a-CEMP1) cross-reacts strongly with CB. (C) Double immunostaining shows that anti-CAP IgG1 and anti-CEMP1 serum are expressed by CB and PVC in the PL that could represent cementum progen-

itor cells. (D) The epithelial cell rests of Malassez (ERM) and CB cross-react strongly with anti-amelogenin monoclonal antibody (a-AMEL). (E) The CEMP1 gene product is expressed by the ERM and CB. (F) Double immunostaining with a-AMEL and a-CEMP1 show that these proteins are expressed by similar structures such as the ERM and CB. AB, alveolar bone; BV, blood vessel; CB, cementoblasts; PVC, paravascular cells.

In-vitro studies have shown that periodontal ligament cells can form alkaline phosphatase-positive and alkaline phosphatase-negative colonies and that the alkaline phosphatase-positive periodontal ligament cells also express higher levels of mineralization-related genes (sialoprotein and osteocalcin) than do the alkaline phosphatase-negative cells. These data suggest that alkaline phosphatase-positive periodontal ligament cells include osteoblast and/or cementoblast subsets (141). It has been found that cementum protein-1 is preferentially expressed in alkaline phosphatase-positive cells and that the expression of cementum protein-1 is reduced when periodontal ligament cells are cultured under osteogenic conditions (i.e. with either bone morphogenetic protein-2 or in osteogenic induction medium) (113). Overexpression of cementum protein-1 increases the expression of cementum attachment protein in periodontal ligament cells at both mRNA and protein levels. The mechanism by which cementum protein-1 regulates the expression of cementum attachment protein in periodontal ligament cells is not clear. However, the expression of cementum protein-1 decreases when cells are committed to osteoblastic/ chondroblastic lineages, which suggests that the expression of cementum protein-1 is being differen-

tially regulated in osteoblasts and cementoblasts and that knockdown of cementum protein-1 expression in periodontal ligament cells only affects sialoprotein expression in cementoblasts, suggesting that cementum protein-1 is associated with the regulation of sialoprotein expression in cementoblasts (113). We have reported the nuclear staining of cementum protein-1 in cementoblasts, whereas periodontal ligament cells exhibit intense staining of cementum protein-1 only in the cytoplasm, indicating that the subcellular location of cementum protein-1 could change during the cementoblastic differentiation of periodontal ligament cells. Overexpression of cementum protein-1 in periodontal ligament cells downregulates periodontal ligament cell markers such as PLAPI/asporin, and increases cementoblast markers such as cementum attachment protein and sialoprotein. These data indicate that cementum protein-1 could select periodontal ligament cells, or progenitor cells present in the periodontal ligament, to differentiate toward the cementoblastic phenotype (113). The ability of cementum protein-1 to induce periodontal ligament cells to differentiate toward cementoblast/osteoblast and/or chondrogenic-like phenotypes was also tested using a three-dimensional cell-culture system. Under those conditions, cemen-

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tum protein-1 stimulated periodontal ligament cells to proliferate in a three-dimensional globular material with morphological features and staining characteristics of cartilage and cementum/bone-like tissues. Cementum protein-1 induced periodontal ligament cells, cultured in a three-dimensional system, to express type II collagen and aggrecan (both markers for pre-hypertrophic chondrocytes) at mRNA and protein levels (36, 91, 140). Expression of type X collagen, a marker of fully differentiated chondrocytes (117), was also increased by the addition of cementum protein-1 to the three-dimensional periodontal ligament cell cultures. The expression of SRY (sex determining region Y)-box 9, a transcription factor that mediates chondrogenic differentiation (2, 17), in cells grown in the presence of human recombinantcementum protein-1 suggests that cementum protein-1 promotes chondrogenic differentiation (91). Cementum protein-1 shows sequence similarity with type X and XI collagens and is immunologically related to type X collagen (5). It is conceivable that cementum protein-1 plays a role during the mineralization of hypertrophic cartilage and facilitates endochondral ossification. Periodontal ligament cells show a multilineage potential to differentiate toward osteogenic, chondrogenic and adipogenic phenotypes by a significant up-regulation of cartilage marker genes and osteoblastic differentiation markers (49, 192). It seems possible that cementum protein-1 exerts a differentiation role on the periodontal ligament cell population by selecting multipotent stem cells, which provide a unique reservoir, to differentiate into various cell phenotypes (229), or as an inducer of the heterogeneous periodontal ligament cell population having a differential effect on cells at various degrees of differentiation. This also explains the expression of cementum protein-1 in periodontal ligament cell subpopulations representing precursors of cementoblasts or osteoblasts, or both (5, 10, 64). Additionally, there is a direct correlation between the capacity of human periodontal ligament-derived cells to bind to a cementum protein and produce mineralized-like tissue in vitro (11). It has been shown that cementum protein-1 increases the activity of alkaline phosphatase. High levels of alkaline phosphatase are also associated with hypertrophic cartilage and bone formation, and alkaline phosphatase is considered to be a marker for chondrocyte differentiation because its activity appears to be increased during chondrocyte hypertrophy (213). The expression of cartilage-formation markers in these cultures suggests that periodontal ligament cells have the potential to differentiate into chondrocytes and then progress rapidly to matu-

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ration and bone formation, as suggested by the expression of collagen type X and sialoprotein. Studies on cementum regeneration, using a dog model for dental pulp necrosis, demonstrated the ability of cementum protein-1 to recruit mesenchymal stem cells from the periodontal ligament and to promote the proliferation and mineralization of these cells. In vivo studies co-localized cementum protein1 and STRO-1 (a marker of mesenchymal stem cells) positive cells adjacent to the root-surface areas where neocementum is deposited, indicating that the cells responsible for reparative cementum deposition are of mesenchymal origin (164). In vitro, cementum protein-1 promotes the proliferation and the migration of periodontal ligament cells, with the migration front comprising STRO-1-positive cells. These studies suggest that cementum protein-1 is a mediator in wound healing and periodontal regeneration because it stimulated the proliferation and migration of periodontal ligament cells. Cementum protein-1 promotes the migration of STRO-1-positive cells and provides a possible mechanism for the recruitment of mesenchymal cells through migration toward the cementum protein-1 signal (164). The role of cementum protein-1 as a chemoattractant and as a promoter of mineralization is further supported by the findings that mineralization is reduced upon blocking cementum protein-1 function in vitro. In cementoblastomaderived cells, blocking cementum protein-1 activity decreases alkaline phosphatase activity and the expression of sialoprotein and osteopontin, but does not alter cell proliferation (4). These studies also showed that extracellular calcium increases the expression of cementum protein-1 and PTPLA/ cementum attachment protein in periodontal ligament stem cells via the mitogen-activated protein kinase signaling pathway. This has been confirmed by blocking extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase-e using small interfering RNA, which down-regulates the expression of PTPLA/cementum attachment protein and cementum protein-1. Furthermore, the use of calcium channel blocker prevented the expression of cementum protein-1 and PTPLA/cementum attachment protein, demonstrating the role for calcium ions in cementogenesis (164). To date, molecules responsible for recruiting mesenchymal cells and inducing their differentiation into cementoblasts have not been identified. These studies suggest that cementum protein-1 could be one of the molecules. In summary, the data presented above strongly indicate that cementum protein-1 is a unique protein that has multiple properties as inducer of mineraliza-

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Fig. 4. Expression of cementum protein-1 (CEMP1) during initial root formation. (A) Inner enamel epithelium (IEE) and outer enamel epithelium (OEE) close to the initial root formation show strong cross-reactivity with anti-cementum protein-1 serum (a-CEMP1) as well as with a few cells in the dental follicle (DF). (B) An enlargement of the area in panel A shows that elongated ameloblasts and flat-like shape cells of the OEE strongly express the CEMP1 gene

product. (C) Cells, possibly cementoblasts (CB), facing the root surface express CEMP1 and CEMP1 is strong expression of at the cemento–enamel junction. (D) Hertwig0 s epithelial root sheath (HERS) cells express CEMP1. (E) Hematoxylin and eosin (H&E) staining for orientation. (F) Cells (CB) facing cementum throughout the length of the root express CEMP1. (G) H&E staining for orientation. AB, alveolar bone; E, enamel; PL, periodontal ligament.

tion, proliferation, differentiation and cell maturation. Additionally, cementum protein-1 might serve to regulate the mesenchymal stem-cell pool present in the periodontal ligament and to induce its differentiation into different pathways. These properties open the possibilities of creating cementum protein-1based therapies for periodontal regeneration.

the root surface forming acellular cementum (43, 44) (Fig. 4). However, the presence of enamel proteins, especially amelogenins, during root development has been the subject of controversy. Some investigators reported the expression of amelogenins by the apical cells of Hertwig’s epithelial root sheath, which secreted small amounts of amelogenins during early differentiation of root development (101), whereas others did not detect the expression of amelogenin in these cells (119), only ameloblastin (238). Nevertheless, a new therapeutic approach, using enamel matrix derivative to achieve periodontal regeneration, was born, based on the assumption that enamel matrix proteins synthesized by cells of the Hertwig’s epithelial root sheath could trigger the differentiation of follicle cells into cementoblasts. Specifically, it has been postulated that amelogenin induces the formation of acellular extrinsic fiber cementum. However, others suggest that the tissue formed by treatment with enamel matrix derivative results in the formation of a cellular cementum-like tissue or bone with the characteristics of cellular intrinsic fiber cementum (23, 24). The bone-like appearance of this tissue is in line with the chondrogenic/osteogenic activity of enamel matrix derivative (24, 215). Numerous studies have suggested that enamel matrix derivative can

Enamel-associated proteins in cementum Many years ago, Slavkin & Boyde (196), proposed a hypothesis that Hertwig’s epithelial root sheathderived extracellular matrix proteins might be related to tooth-crown-derived enamel proteins and that these enamel-related proteins might initiate acellular cementum formation. Several human and mouse cementum proteins were found to be immunologically related to amelogenin and enamelin. These proteins represented species of 72 and 26 kDa that were secreted by Hertwig’s epithelial root sheath cells (197). A few years later, it was demonstrated that ameloblastin, an enamel-associated protein, is expressed by epithelial cells covering the first thin layer of unmineralized root mantle dentin, and a strong signal is expressed in cells enclosed in the cellular cementum known as the epithelial cell rests of Malassez and in cells in a more coronal position at

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have multiple functions, such as the promotion of cell proliferation, differentiation and up-regulation of extracellular matrix production (36, 50, 51, 83, 86, 139, 159, 188, 195, 233). Also, several reports have provided further evidence that enamel matrix proteins may be involved in root formation (20). Furthermore, amelogenin null mice showed defects in cementum and a decreased expression of sialoprotein along the root surface (57, 219). This is of particular importance because sialoprotein acts as a regulator of the mineralization process in cementum (47, 59). Amelogenins are the expression products of X and Y chromosomal genes and give rise to multiple spliced variants (235). One of these alternatively spliced variants is a leucine-rich amelogenin peptide (A-4) which has been demonstrated to increase the expression of osteopontin, sialoprotein and osteoprotegerin, and to decrease the expression of osteocalcin, in cementoblasts (208, 215, 216). Amelogenin and ameloblastin can act as signaling molecules in the periodontal ligament; they have an effect on attachment (237) and proliferation of these cells in vitro. Both proteins have a modulatory role and downregulate the expression of type I collagen, whilst inducing the de novo expression of osteocalcin. Amelogenin also induced the expression of sialoprotein in periodontal ligament cells, indicating that this protein can induce phenotypic changes in these cells (239). Amelogenin has been suggested to have biological effects on cells of mesenchymal origin, such as periodontal ligament and gingival fibroblasts, and enamel matrix derivative enhances the growth of human bone marrow stromal cells (68, 93, 215). In the past, amelogenin has been shown to be expressed by odontoblasts, periodontal ligament cells, Hertwig’s epithelial root sheath cells and cementoblasts (23, 44, 46, 70, 72, 78, 79, 155, 162). Others have described the expression of amelogenin in hematopoietic stem cells, macrophages and megakaryocytes, rat brain and myoepithelial cells, suggesting a regulatory role for amelogenin in the recruitment and differentiation of monocytic cells from the bone marrow toward becoming mineralized tissue-resorbing cells (bone and cementum osteoclasts/cementoclasts). Amelogenin, a major structural protein in mineralizing enamel, is also expressed in brain tissue and cells of the hematopoietic system (39). Progressive deterioration of cementum is observed in amelogenin knockout mice and is characterized by the increased presence of osteoclasts (78, 79). On the other hand, RT-PCR and western blotting results have demonstrated that Hertwig’s epithelial root sheath cells in vitro do not synthesize amelogenin or enamelin, but

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they do synthesize ameloblastin. These studies showed that Hertwig’s epithelial root sheath cells change their morphology and produce Von Kossapositive nodules coincidental with the expression of dentin matrix protein-1, sialoprotein and osteocalcin, and high levels of alkaline phosphatase activity. Transmission electron microscopy comparison of the mineralized extracellular matrix deposited by Hertwig’s epithelial root sheath cells in vitro with acellular cementum deposited in vivo suggests that this extracellular matrix might be cementum (238). Hertwig’s epithelial root sheath cells synthesize cementum attachment protein and cementum protein-1 (Fig. 4), thus supporting the idea that Hertwig’s epithelial root sheath cells are capable of producing cementum along with inducing a high activity of alkaline phosphatase. This finding provides further evidence that the extracellular matrix deposited by these cells is acellular cementum, indicating that alkaline phosphatase is a very important component of acellular cementum. Hertwig’s epithelial root sheath cells express osteocalcin in vitro, thus indicating the possibility that disruption of the basement membrane is caused by Hertwig0 s epithelial root sheath cells when they start depositing the acellular cementum. These studies suggested different cellular origins for acellular (Hertwig’s epithelial root sheath cells) and cellular (mesenchymal cementoblasts) cementum (23, 92). Furthermore, Hertwig’s epithelial root sheath cells/ epithelial cell rests of Malassez are a unique population of epithelial cells in the periodontal ligament and are believed to play a crucial role in cementum repair (203). Hertwig’s epithelial root sheath cells/epithelial cell rests of Malassez could differentiate into cementoblasts through epithelial–mesenchymal transformation (202). Recently it was demonstrated that in vitro Hertwig’s epithelial root sheath/epithelial cell rests of Malassez contain primitive stem cells that express epithelial stem-cell markers such as octamerbinding transcription factor 4, homeobox protein NANOG and stage-specific embryonic antigen 4 (147). These cells might function in creating the border between the ameloblasts and the proliferative region of Hertwig’s epithelial root sheath (145) and might contribute to the formation of cementum and/ or enamel repair (76, 194). More recently it was shown that the epithelial cell rests of Malassez share similar phenotypic and functional characteristics with mesenchymal stem cells and are capable of developing into osteoblasts, adipocytes, chondrocytes and neuron-like cells in vitro (228), similar to that described in vitro for periodontal ligament stem cells (192, 204). These studies suggest that the epithelial

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Fig. 5. (A) Hematoxylin and eosin staining showing the periodontal ligament (PL), cementum (CEM) and alveolar bone (AB). Blood vessels (BV) along with the putative progenitor cells of CEM are located close to the CEM surface. (B) Cementoblasts (CB) are ordered in three to four layers of cells facing the CEM surface. (C) The epithelial cell rests of Malassez (ERM) are located in the vicinity of the CB cell layers and the BV, indicating a possible inter-relationship regulating CEM metabolism and differentiation of CEM

progenitor cells. (D) Ameloblastin (AMBM), an enamel and cementum-related protein, is shown to be expressed by a single layer of CB facing the CEM surface and paravascular progenitor cells (PVC) into the PL. (E) A pan-cytokeratin monoclonal antibody (a-pCK) cross-reacts with CB and subpopulations of PL cells, possibly supporting the statement that CEM is an epithelial product. (F) Cementum attachment protein (CAP) is expressed by subpopulations of ERM and the CB facing the CEM surface.

cell rests of Malassez are epithelial stem cells with the ability to differentiate into epithelial or mesenchymal cells and play a critical function in periodontal repair/regeneration. Dental follicle cells, before the onset of root formation, do not express cementum attachment protein, cementum protein-1 or sialoprotein, which suggests the absence of differentiated cells. However, dental follicle cells are positive for cementum attachment protein and cementum protein-1 when stimulated with enamel matrix derivative or bone morphogenetic protein-2/-7, and their expression was reduced when treated with recombinant human-Noggin, a well-known inhibitor of bone morphogenetic protein activity (108). Some dental follicle cells express STRO1, indicating that populations of the dental follicle have mesenchymal progenitor features (108). Several investigators suggest that the use of enamel matrix derivative enhances the expression of mineralized tissue markers (such as alkaline phosphatase) and nodule mineralization in dental follicle cells along with the expression of bone morphogenetic protein-2 and sialoprotein (108). As enamel matrix derivative also induces expression of cementum attachment protein and cementum protein-1, it is suggested that enamel matrix derivative promotes the differentiation of den-

tal follicle cells to a cementoblast phenotype rather than to an osteoblast phenotype. Recently it was reported that cementum attachment protein and cementum protein-1 are stringently regulated during the cementogenesis process and root formation, and that expression of cementum attachment protein is induced more strongly by runt-related transcription factor 2 than is cementum protein-1 (161). Runtrelated transcription factor 2 is an important transcription factor for osteogenesis and cementogenesis, and is present in the early proliferative osteoblast/cementoblast cell phenotype, a developmental stage at which cell proliferation is still required to obtain a sufficient number of committed cells for matrix formation. Higher expression of cementum attachment protein at this early stage of cementogenesis is therefore consistent with its function of promoting cell proliferation (227). In contrast, cementum protein-1 has a more important role during the mineralization process and its relationship is focused at this early stage to control the mineralization process during cementogenesis (218). Recently we reported that normal human-derived cementoblasts express cementum attachment protein, cementum protein-1 and amelogenin and that they are localized to the cell nucleus. Human cementoblasts express not only

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amelogenin but also other enamel-associated molecules, such as ameloblastin, enamelin and tuftelin (Fig. 5). Furthermore, cementum protein-1 induces the de-novo expression of amelogenin in periodontal ligament cells grown in culture. Perhaps cementum protein-1 promotes the osteoblast/cementobast phenotype in periodontal ligament cells through the expression of amelogenin (91, 152, 153). These data, taken together, suggest that enamel-associated proteins and cementum proteins could act synergistically in the regulation of cementoblast differentiation and cementum deposition. These data offer new approaches to determine how the process of cementogenesis is regulated and point out the role of these proteins during periodontal homeostasis and repair/regeneration of periodontal structures, and could represent new and better therapeutic approaches for the treatment of periodontal disease.

Conclusions Detailed knowledge of the biology of cementum is key for understanding how the periodontium functions, identifying pathological issues and for developing successful therapies for repair and regeneration of damaged periodontal tissue. Of particular importance is the regeneration of cementum, a highly sophisticated and complex system, and the interconnection of this unique tissue with the neoformation of other periodontal tissues. In this review we have highlighted the recent advances in our understanding of the so-called ‘cementum proteins’ – PTPLA/ cementum attachment protein and cementum protein-1 – and their possible role in selecting periodontal stem cells, inducing their differentiation and regulating the biological mineralization process associated with cementum formation. Also, this review points out the synergistic role of enamel-associated proteins and cementum proteins in these processes. Although there have been tremendous advances in our knowledge of the identity and function of ‘cementum proteins’ at the cell and molecular levels, there is still a great deal left to discover about these proteins. We are already in a position to explore new alternatives for regenerative techniques based upon the principles of tissue-engineering methods. These proteins and related peptides with biological activity hold great therapeutic potential for the regeneration of not only periodontal structures but also of other mineralized tissues. Nevertheless, this is only the beginning of our understanding of how these proteins might modulate tissue responses in relation to

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the regeneration process. What may be concluded from the current status of the ‘cementum proteins’ is that they can pave the way to establish effective therapeutic alternatives to achieve the regeneration of the periodontal structures, and that the impact that they could have in the field of periodontology and skeletal tissues looks very promising.

Acknowledgments This work was supported by DGAPA-UNAM IN216711, IT200414 and by CONACYT 130950. The authors are grateful to Professor A. Sampath Narayanan of the University of Washington for critical reading of the manuscript.

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