Cementum Proteins: Role in Cementogenesis, Biomineralization, Periodontium Formation and Regeneration

Cementum proteins: role in cementogenesis, biomineralization, periodontium formation and regeneration

HIGINIO ARZATE,MARGARITA ZEICHNER-DAVID &GABRIELA MERCADO-CELIS

Destruction of periodontal tissues is a cardinal sign of tissue regeneration) have been developed to guide periodontal disease, and many factors, such as infec- and control regeneration using bioresorbable mem- tions, trauma, orthodontic tooth movement and sys- branes (3, 138, 142) and bone grafts (175). Although temic and genetic diseases, can contribute to this effective to a certain point, these strategies have the process. In periodontal disease, bacteria on the sur- problem that they are not predictable and do not face of the teeth produce a chronic inflammation of completely restore the architecture of the original the gingiva. Cells on the surface of the tooth root and periodontium. To achieve complete repair and regen- the cementum covering the root are destroyed, and eration it is necessary to recapitulate the develop- the epithelium from the oral mucosa grows down- mental process with complete formation of wards, producing a gingival crevice. Bacteria deposit cementum, bone and periodontal ligament fibers. in this crevice and the ensuing inflammatory process The past 20 years of research have seen tremen- may eventually result in the breakdown of periodon- dous advances in our knowledge of the cellular and tal tissues, including the cementum, periodontal liga- molecular events involved in the process of devel- ment and alveolar bone. oping the periodontium. This knowledge has trans- The process of periodontal tissue regeneration is lated into new therapeutic strategies for periodontal initiated at the moment that the damage takes place regeneration using molecular approaches (45, 67, by the production of growth factors and cytokines by 110–112, 116, 129, 146, 158, 210, 223, 240). Amongst the damaged and inflammatory cells. Periodontal these strategies are the use of growth factors, such treatment can enhance periodontal healing (60). Root as platelet-derived growth factor and insulin-like planing or root conditioning is being used as a strat- growth factors (32, 54, 90, 120, 163, 181, 222), trans- egy to increase mesenchymal cell migration and forming growth factor-beta1 (127), basic fibroblast attachment to the exposed root surface. Treatment growth factor (191), dexamethasone (181) and bone with acid, in particular citric acid, has been found to morphogenetic proteins (109, 114, 154, 176–178). It widen the orifices of dentinal tubules, thereby accel- is believed that these molecules are produced dur- erating cementogenesis and enhancing cementum ing cementum formation and are then stored in the apposition and connective tissue attachment (125). cementum matrix to induce periodontal ligament However, when periodontal ligament cells are regeneration when needed (200). However, one of removed from the cementum or are unable to regen- the problems of application of these factors for peri- erate, bone tissue may invade the periodontal liga- odontal repair is the nonspecific activity of some of ment space and establish a direct connection the factors on different cell lineages and the rapid between the tooth and the wall of the alveolar socket, loss of the topically applied factors over time (15, resulting in ankylosis. This nonflexible type of tooth 120). support can lead to loss of function and eventually to As our understanding of the structure, function and resorption of the root (15). Strategies (such as guided composition of cementum increases, so does the

211 Arzate et al. potential for new therapies for periodontal regenera- all animal teeth. Initial examination of cementum on tion using molecules formed by these tissues. One the roots of human teeth is attributed to Ringelmann such example has been the use of enamel proteins to in 1824, followed by physiologists Jan Evangelista Pur- induce cementum and bone regeneration (50, 51, 72– kinje and his pupils, Fraenkel and Raschkow, in 1835, 74, 84, 85). A preparation of porcine enamel protein and later by the classic histologist Anders Adolf Retzi- â has been marketed (Emdogain ) and is currently us, in 1836, who noticed the presence of ‘striaes’ in being used by practicing dentists (42, 52, 55, 105, 237, cementum (19, 37, 61, 186). In almost all mammalian 239). Outcomes from studies using Emdogain suggest species the number of incremental structures in the that its clinical action is the result of contamination dental cementum (annulations) can be correlated with growth factors such as transforming growth fac- with age and are used as such in archeology and tor-beta (107, 143) and bone morphogenetic proteins forensics (61). (100). However, studies using recombinant amelo- In a very simplistic way we can define cementum genin, including results obtained in our laboratory, as an extracellular matrix composed of calcified col- indicate that amelogenin and ameloblastin also have lagenous Sharpey’s fibrils, collagen, glycosaminogly- signaling properties that induce phenotypic changes cans, proteoglycans and inorganic hydroxyapatite. In in cells, and these changes can fluctuate depending the same way we can say that the major functional on the target cell (20, 219, 237, 239, 240). role of cementum is to serve as the anatomical struc- It is the purpose of this review to focus on the role tural site for the attachment of Sharpey’s fibers of the of cementum and its specific components in the for- periodontal ligament. However, the more we study mation, repair and regeneration of the periodontium. this tissue, the more complex we find its structure As cementum is a matrix rich in growth factors that and function. Cementum biology goes back to Gott- could influence the activities of various types of peri- lieb, in 1942, who stated that “the continuous deposi- odontal cells, this review will examine the characteris- tion of cementum layers seems to be of great tics of cementum, its composition and the role of importance and work as a barrier against the down- cementum components, especially the cementum growth of the epithelium. Newly deposited cemen- protein-1, during the process of cementogenesis, and tum seems to have the highest vitality and act as the their potential usefulness for regeneration of the best barrier. If the ideas about the biology of cemen- periodontal structures in a predictable therapeutic tum are correct, it is then our task to find out just manner. how nature provides for continuous cementum deposition and having done so, to imitate the procedure” (62). These studies introduced the notion that not What is cementum? only does cementum act as a barrier to delimit epithelial growth that can impair attachment but also Cementum can be described as the mineralized tis- that the presence of a continuous cementum layer is sue that covers the roots of teeth and serves to necessary to act as a microbial barrier and that attach the tooth to alveolar bone via collagen fibers defects in this tissue could result in periodontitis (62, of the periodontal ligament. Morphological, histo- 63). This idea was further supported by observations logical and functional differences appear to exist that the root surfaces of patients with hypophospha- along the length of the root, leading cementum to tasia contained areas completely devoid of cemen- be classified as follows: intermediate cementum tum or covered with a hypoplastic form of a (found in the cemento–enamel junction), acellular cementum-like material. These patients developed cementum (found in the coronal and mid-portions early-onset periodontitis, suggesting that abnormali- of the root) and cellular cementum (present in the ties in the deposition or maintenance of cementum apical and inter-radicular portions of the root con- can result in a defective periodontium highly suscep- taining cementoblasts) (21, 33, 71, 75, 183, 187, 207, tible to microbial invasion and destruction (160). 240). Based on the different functions attributed to Studies on dental cementum can be traced back to cementum it is clear that a thorough understanding Malpighi in the 1600s (61). In general, 18th century of the biological properties of cementum is required anatomists regarded human teeth as composed only to determine its role in periodontal formation and of enamel and dentin. However, the studies of Tenon therefore periodontal regeneration. Furthermore, the on horses’ teeth, of Blake on elephants’ teeth and of principles attributed to cementum regeneration Cuvier on the teeth of many species resulted in the might be used in the regeneration of other mineral- recognition that cementum was a constituent part of ized tissues.

212 Cementum proteins

Cementum composition pericementocyte area), suggesting that they play major regulatory roles during cementum mineraliza- In order to understand the process of cementogenesis tion (30). In a similar manner, it was also determined it is important to know the composition of cemen- that the large chondroitin sulfate glycosaminoglycan, tum. As in bone and dentin, the major organic com- present in cementum, contains the large hyaluronan- ponent of cementum is collagen (18). The major type binding proteoglycan, versican, and the small intersti- of collagen is type I collagen, which accounts for 90% tial proteoglycans, decorin and biglycan. Versican is of all collagens and plays a structural role during the localized in lacunae housing cementocytes. Decorin fi biomineralization process, serving as a reservoir for is closely associated with collagen bers of the peri- hydroxyapatite nucleation, which successively devel- odontal ligament and with biglycan in the cemento- ops into intrafibrillar apatite crystals (58). Type III col- blasts/precementum area. The differential tissue lagen, which coats type I collagen fibrils, is also distribution suggests that glycosaminoglycans may present, although in considerably lower quantities. In play distinct roles during the cementogenesis process addition to collagens, carboxylated and sulfated mu- in addition to regulating the biomineralization of copolysaccharides (glycosaminoglycans) are present cementum (31). Syndecan-2 has been found to be sig- fi in human cementum (217). ni cantly expressed by cells in close contact with the root surface and within the matrix of reparative Glycosaminoglycans cementum, suggesting that it must be associated with cell–matrix interactions during cementum mineraliza- Glycosaminoglycan-containing proteoglycans are tion (224). Biglycan is also associated with the growth heterogeneous groups of glycoproteins with long of incremental lines in cellular cementum. Further- repeating disaccharides. The percentage of glycosa- more, lumican, decorin, versican and biglycan are minoglycans is high in tissues subjected to compres- associated with the formation of cellular cementum sive forces, such as cementum. Proteoglycans are but not of acellular cementum, suggesting different known to interact specifically with collagens in a vari- cementocyte subpopulations or a differential ety of tissues. It is postulated that proteoglycans in response of these cells (1). Osteoadherin, a keratin sul- cementum are integral components of cell substra- fate-containing proteoglycan, is also associated with tum attachment matrices and mediate attachment the initial phase of cementum formation because Her- between old and newly formed cementum, thus cre- twig’s epithelial root sheath cells express this proteo- ating the cementum incremental lines (189, 220). glycan during root development (166), and although The major glycosaminoglycans present in human acellular cementum does not contain proteoglycans, cementum are hyaluronic acid, dermatan sulfate and initial acellular cementum formation requires a dense chondroitin sulfate, and their distribution appears to accumulation of proteoglycans (230). be quite different from that reported for soft tissues such as gingival connective tissue and periodontal lig- Ostepontin and bone sialoprotein ament, in which dermatan sulfate predominates (12, 165). The proteoglycan content of mineralized tissues Cementum contains many noncollagenous proteins, is generally relatively low. These differences might including some major phosphoproteins such as os- reflect differences in function between hard and soft teopontin and bone sialoprotein. These proteins play tissue as proteglycans appear to inhibit collagen min- a major role in filling spaces created during collagen eralization by occupying strategic locations normally assembly and imparting cohesion to the mineral-like destined to be filled with hydroxyapatite (189). In tissue by allowing mineral deposition to spread across bone, dermatan sulfate proteoglycans are oriented the entire collagen meshwork (22, 148). The role pro- parallel to the collagen fiber axis with chondroitin sul- posed for these proteins is that of regulators of fate proteoglycans and hyaluronic acid, occupying the hydroxyapatite crystal nucleation and growth. It has interfibrillar region in a space-filling capacity (190). been suggested that osteopontin and sialoprotein are Amongst other glycosaminoglycans present in necessary for the initiation of crystal formation at the cementum, keratan sulfate appears to be one of the highly ordered fibrils of type I collagen (179). Both of major components, which, after digestion with kera- these proteins are acidic: osteopontin has a poly-Asp tanase II and endo-beta-galactosidase, produces two and sialoprotein contains two poly-Glu domains, the core proteins: lumican and fibromodulin. Interest- repetitive sequences of which are known to bind cal- ingly, these proteins are localized predominantly in cium to mineral surfaces. Osteopontin is present nonmineralized cementum (precementum and the within the periodontal ligament in mature teeth.

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Sialoprotein and osteopontin remain bound to colla- in contrast, osteocalcin mRNA is not expressed in gen matrix and they possess cell-attachment proper- the periodontal ligament (38). ties through their arginine-glycine-aspartic acid The role of these proteins has been related to regu- (RGD) sequences (199). In the periodontium, osteo- lation of mineralization. Matrix gamma-carboxyglu- pontin is expressed by cells in close contact with acel- tamic acid protein-deficient mice show abnormal lular cementum as well as by cementocytes (26). It mineralization and a lack of acellular cementum has also been suggested that osteopontin regulates (104). It has been suggested that both osteocalcin and cell migration, differentiation and survival via interac- matrix gamma-carboxyglutamic acid protein act as tions with avb3 integrin (53). Sialoprotein is also an negative regulators of mineralization because matrix RGD-containing sialoprotein with cell-attachment gamma-carboxyglutamic acid protein-deficient mice properties (156) and has a precise spatial association promote calcification of the aortic walls and valves. with early mineral aggregates, binds strongly to Thus, both osteocalcin and matrix gamma-carboxy- hydroxyapatite and acts as a specific and potent glutamic acid protein seem to regulate mineralization nucleator for hydroxyapatite crystal formation in vitro by acting as negative regulators, but to different (95). Cementum contains sialoprotein, which, during extents because osteocalcin also inhibits conversion root formation, is distinctly localized to cells lining of brushite to hydroxyapatite (81, 173). Other mole- the surface of cementum. It has been suggested cules present in the cementum extracellular matrix that sialoprotein modulates the process of ceme- include osteonectin, which, during cementogenesis, ntogenesis and is involved in the process of chem- is synthesized by cementum-producing fibroblasts, oattraction, adhesion and differentiation of cementoblasts and cementocytes (174). Osteonectin, precementoblasts (121–123, 201). Both sialoprotein synthesized by mineralizing cells, can bind hydroxy- and osteopontin are believed to play a role in the dif- apatite and is associated with mineralization (25, 80, ferentiation of cementoblast progenitor cells to ce- 96, 126, 135). Based on the observation that low con- mentoblasts (183). centrations of osteonectin delay hydroxyapatite- seeded crystal growth in vitro, it is speculated that Gla proteins osteonectin also acts as a negative regulator by pre- venting, rather than promoting, matrix mineralization Matrix gamma-carboxyglutamic acid protein and (134, 230). osteocalcin are the two major Gla-containing pro- fi – teins associated with calci ed hard tissues (80 82, Alkaline phosphatase 172, 173). Both proteins have high affinity for Ca2+ and hydroxyapatite through interaction with the Gla Tissue nonspecific alkaline phosphatase (also known residue. The distribution of osteocalcin in the mam- simply as alkaline phosphatase) has been studied for malian body is virtually limited to mineralized tis- more than 80 years and is believed to play an impor- sues such as bone, dentin and cementum (34). In tant role in skeletal mineralization. Alkaline phospha- the dental root, osteocalcin expression is localized tase is a membrane-bound glycoprotein enzyme that in cells lining cellular cementum and acellular hydrolyses phosphate groups at alkaline pH and also cementum. However, cells at the inter-radicular inhibits pyrophosphatase, ATPase and protein phos- area also express osteocalcin. In a similar manner, phatase activity at neutral pH (94). Alkaline phospha- cellular and acellular cementum show expression tase is expressed in most body sites during embryonic of matrix gamma-carboxyglutamic acid protein, development but is confined to bone, kidney, liver although acellular cementum expresses those pro- and B-lymphocytes during adult life. The fact that it is teins more prominently than does cellular cemen- expressed in nonmineralizing tissues suggests that it tum. Matrix gamma-carboxyglutamic acid protein is has other roles besides those associated with mineral- secreted by cementum-forming cells and is incorpo- ization. Amongst some of these other functions, it has rated at the mineralization front (103). One possible been suggested that alkaline phosphatase can regu- explanation for the accumulation of matrix gamma- late tissue turnover and cell proliferation, differentia- carboxyglutamic acid protein in acellular cementum tion and maturation (94, 226). Alkaline phosphatase is and the outer surface of cellular cementum could highly expressed in cells of the periodontal ligament be to prevent hypercalcification of the cementum (56, 69, 99, 124, 151, 231), where it is thought to play surface (77, 104). During root development in mice, a role in phosphate metabolism and cementum for- a high level of osteocalcin mRNA is selectively mation (13), particularly formation of acellular expressed by cells lining root-surface cementoblasts; cementum (14, 65). Tissue nonspecific alkaline

214 Cementum proteins phosphatase-deficient mice show defective formation therefore could still be considered as specific mark- of acellular cementum, which results in very thin and ers. Several proteins, some of which are considered to irregular-shaped patches around the bases of the be cementum-specific proteins, have been isolated periodontal ligament fibers. No defects were seen in from cementum and characterized. alveolar bone, periodontal ligament and cellular Cementum-derived growth factor cementum, suggesting that alkaline phosphatase is essential for the formation of acellular cementum It is now well established that mineralized tissues, (16). However, observations in humans with hypo- such as bone and dentin, are excellent reservoirs of phosphatasia as a result of mutations in the tissue growth factors that, when needed, can be released by nonspecific alkaline phosphatase gene revealed that demineralization and serve to repair or regenerate tis- cementum formation is almost completely abolished sues. In a similar way, it has been shown that extracts for both acellular and cellular cementum, resulting in of cementum have the ability to promote a range of premature tooth loss. This phenotype differs from biological activities such as cell migration, adhesion, that in tissue nonspecific alkaline phosphatase gene- mitogenic activity and differentiation, which are knockout mice, which showed blockage of acellular essential for periodontal regeneration (67). Miki et al. cementum formation only (213, 214) and might be (137) were the first to report the presence of mito- explained by the different types of mutation possible genic activity in cementum obtained from human in the tissue nonspecific alkaline phosphatase gene teeth. Later, Nakae et al. (144) isolated and character- and the severity of their manifestation in humans. ized mitogenic factors present in the cementum One of the major functions of tissue nonspecific alka- matrix of bovine teeth. In addition to fibroblast line phosphatase is the hydrolysis of inorganic pyro- growth factor, which binds strongly to heparin, phosphate, a potent inhibitor of hydroxyapatite another mitogenic factor with moderate heparin formation (206). Cementoblasts are specifically sensi- affinity was present in cementum but not in alveolar tive to the levels of inorganic pyrophosphate/inor- bone. This factor was named cementum-derived ganic phosphate within the extracellular matrix (150). growth factor and it is the major component in Changes in the level of tissue nonspecific alkaline cementum, accounting for 70% of the mitogenic phosphatase protein have a significant effect on the activity extracted from this tissue. The cementum- function of osteoblasts, and consequently on matrix derived growth factor acts synergistically with epider- mineralization, indicating that tissue nonspecific mal growth factor and induces many of the signaling alkaline phosphatase plays key biological roles in the pathways associated with mitogenesis (234). These mineralization of bone and cementum (89). One pathways include an increased concentration of cyto- novel strategy to reduce inorganic pyrophosphate solic Ca2+, activation of the protein kinase C cascade and increase cementum neoformation may center on and expression of cellular proto-oncogenes. Addition- the modulation of inorganic pyrophosphate/inor- ally, cementum-derived growth factor may promote ganic phosphate in the periodontium, which may the migration and growth of progenitor cells, present result in more predictable regeneration of cementum in the adjacent structures, toward the dentin matrix (180). and participate in their differentiation into cementoblasts (130, 170). Further characterization of cemen- Cementum-specific proteins tum-derived growth factor revealed that the mitogenic activity was associated with a 14-kDa pro- Extracellular matrix from different tissues share many tein that showed some homology to insulin-like similarities and yet have different functional proper- growth factor-1. Although cementum-derived growth ties that make them unique. These properties could factor activity was inhibited with insulin-like growth be the result of quantitative and/or qualitative differ- factor-1 and insulin-like growth factor-1 receptor ences amongst their components. For years it was antibodies, there were some differences between the believed that different mineralized tissues contain canonical insulin-like growth factor-1 and cemen- specific molecules not present in any other tissue (i.e. tum-derived growth factor, thus it was concluded that amelogenin in enamel, dentin sialophosphoprotein cementum-derived growth factor is an insulin-like in dentin, etc.) and which could be considered as growth factor-1-like molecule (97). markers for those tissues. As detection techniques The presence of cementum-derived growth factor became more sensitive, it was found that many of and other growth factors in cementum indicates that these molecules were also expressed in other tissues, cementum has the potential to regulate the although at considerably lower concentrations, and metabolism and turnover of surrounding tissues, that

215 Arzate et al. cementum could serve as a storage site for those cementum attachment protein codes for a 15-kDa growth-inducing molecules and that the cementum protein that has no collagen sequences. This suggests proteins are likely to serve a biological role in promot- that perhaps the cementum attachment protein is ing periodontal regeneration (67, 149). strongly associated with collagen chains in the cementum matrix, possibly through cross-linking, Cementum attachment protein which increases its molecular weight. This possibility In addition to cementum-derived growth factor, it is supported by the observation that immunoprecipi- has been reported that human and bovine cementum tates of cementum extracts obtained with several contains potent mediators of cell attachment, and anti-cementum attachment protein monoclonal anti- this biological activity is associated with a 55-kDa bodies contain two protein species migrating at protein species (128, 157). This protein was named 55 kDa and ~ 29 kDa, and one monoclonal antibody cementum attachment protein and further character- cross-reacts with both cementum attachment protein ization by amino-acid sequencing showed the pres- and type I collagen (A.S. Narayanan, unpublished ence of four sequences containing Gly-X-Y repeats data). Expression of PTPLA/cementum attachment typical of collagen. A 17-amino-acid peptide had 82% protein is limited to cementum and to some cells in homology with a type XII domain, and another the endosteal spaces of bone. This could be explained peptide had 95% homology with collagen type Ia1. as a result of the presence of precursors of cemento- However, cementum attachment protein did not blasts in the endosteal spaces of alveolar bone (130, cross-react with antibodies to type I, type V, type XII 133, 198). These cells are thought to traverse through and type XIV collagen, and its attachment activity the periodontal ligament before reaching and differ- was lost after treatment with bacterial collagenase. entiating on cementum. These findings, and the fact that a cementum It has been shown that cementum attachment pro- attachment protein monoclonal antibody localizes tein binds to hydroxyapatite and more strongly to cementum attachment protein only to cementum (7), cementum than to the dentin surface (168). Cemen- suggest that cementum attachment protein might tum attachment protein also binds to fibronectin, but be a collagenous-attachment protein localized exclu- binds 150 times more strongly to hydroxyapatite than sively in cementum (225). to fibronectin (169). Like the cementum attachment Characterization of a complementary DNA clone for protein obtained from cementum, recombinant cementum attachment protein isolated from a human PTPLA/cementum attachment protein also binds to cementifying fibroma-derived cell line k-ZAP expres- hydroxyapatite with high affinity (212). These obser- sion library, revealed a novel alternatively spliced vations suggest that PTPLA/cementum attachment sequence. This sequence encodes a 140-amino-acid protein may play a regulatory role during cementum protein that is identical to the first 125 N-terminal formation (67, 212). It has also been shown that amino acids of a truncated isoform of 3-hydroxyacyl- cementum attachment protein promotes the attach- CoA dehydratase-1/protein-tyrosine phosphatase-like ment of gingival fibroblasts, endothelial cells and (proline instead of catalytic arginine), known as smooth muscle cells, but not oral sulcular epithelial PTPLA (212). The remainder of the C-terminus of cells (157). Bone cells bind more strongly to cemen- PTPLA/cementum attachment protein is encoded by tum attachment protein than do periodontal liga- a read-through of the splice donor site in exon 2, and ment cells, which, in turn, bind more strongly to the truncation eliminates the PTPLA sequence that cementum attachment protein compared with gingi- has the signature phosphatase active site motif. val cells (300% for bone cells, 250% for periodontal Although PTPLA mRNA is widely expressed in many ligament cells and 150% for gingival cells). Cementum tissues, the PTPLA/cementum attachment protein attachment protein-coated root slices promote pref- mRNA is expressed in cementum cells and only mar- erential adhesion and differentiation of osteoblastic ginally in some periodontal ligament cells in human cells (168, 169, 171). Attachment to cementum attach- teeth. PTPLA/cementum attachment protein was not ment protein by human gingival fibroblasts is medi- found to be expressed in rat teeth (184), suggesting ated primarily by the integrin a5b1 (98). The a5b1 that there could be some species differences in its dis- integrin has been shown to be involved in various tribution. Rat roots are known to overproduce apical aspects of development (131), and it is possible that cementum after they reach 8 weeks of age, whereas the a5b1 integrin may also play an important role in other species maintain a normal layer of cementum. cementogenesis and neo-cementogenesis through Interestingly, the cementum attachment protein is interactions with cementum attachment protein. a 55-kDa collagenous protein, whereas the PTPLA/ Cementum attachment protein has the capacity to

216 Cementum proteins direct cell migration of alveolar bone cells. Periodon- mineralized-like tissue, similar to cellular cementum, tal ligament cell populations differ in their capacity to represent 7% of the periodontal ligament population recognize and respond to cementum attachment pro- and 15% of the mineralized-tissue forming clones tein. Fifteen per cent of clones of periodontal liga- belonging to the cementoblastic lineage (118). The ment cell cultures bind strongly to cementum high binding capacity of cementum attachment pro- attachment protein and this binding capacity is simi- tein, combined with a low constitutive percentage of lar to that of alveolar bone cells, suggesting that the alkaline phosphatase expression, is of interest origin of cementoblastic precursors could be osteo- because it supports the view that cementoblastoma- genic. Therefore, cementum attachment protein may derived cells express a low constitutive percentage of potentially be used to induce preferential repopula- alkaline phosphatase-positive cells and that cemento- tion of the root surface by appropriate cells (169). blasts express low levels of alkaline phosphatase com- Periodontal ligament cells manifest a higher chemo- pared with alveolar bone-derived osteoblasts (205). tactic response to cementum attachment protein Adhesion of human gingival fibroblasts to cemen- than do gingival fibroblasts, and this activity is 24-fold tum attachment protein stimulates mitogen-activated greater than that of fibronectin (136). Cementum protein kinase activity and induces the expression of attachment protein binds selectively to periodontal c-fos mRNA; in contrast, protein-tyrosine phosphory- ligament cells and supports periodontal ligament cell lation and c-fos mRNA were not induced in unat- attachment to root surfaces (171). Selective chemo- tached cells. As mitogen-activated protein kinase and taxis and attachment by cementum attachment pro- c-fos mRNA were not induced in monolayer cultures, tein may represent the natural pathway by which it was presumed that these reactions are induced by cementoblast progenitors are attracted to the root adhesion and are not necessary for cell adhesion surface during homeostasis and regeneration. Par- (182). The kinetics of mitogen-activated protein tially demineralized cementum does not support epi- kinase activation was different for cells attaching to thelial outgrowth; however, it enhances migration fibronectin or polylysine – c-fos mRNA levels toward and attachment of periodontal connective tis- increased only half as much in cells attaching to sue cells to dental surfaces in vitro (168, 169). Thus, fibronectin and very little in cells attaching to polyly- exposed collagen fibers might improve the capacity of sine. These data demonstrate that cementum attach- periodontal connective tissue cells to compete with ment protein and other adhesion molecules present epithelial cells and be the first to attach to and colo- in mineralized tissue matrices induce characteristic nize the root surface after periodontal surgery (167). signaling events during adhesion, which may play a If this is the case, biochemical alterations in cemen- role in the recruitment of specific cell types during tum may explain the loss of soft-tissue attachment wound healing and in mediating their specific biolog- from diseased root surfaces and the failure for its suc- ical functions. This differential response is especially cessful restoration. Selective chemotaxis by, and important in periodontal regeneration, in which epi- attachment through, cementum attachment protein thelial cells must be excluded and fibroblasts and ce- might represent the natural pathway by which cementoblasts are to be selected from a pool of various mentoblast progenitors are attracted to the root sur- progenitor cells (170). Cementum attachment protein face during homeostasis and regeneration. is likely to play a role in the cell selection process. The It has been reported that the cementum attach- mitogen-activated protein kinase kinase/mitogen- ment protein possesses the capacity to bind peri- activated protein kinase pathway participates in odontal ligament progenitor clones and that this is cementum attachment protein-mediated fibroblast directly related to their alkaline phosphatase expres- spreading, but cell attachment and proliferation do sion and mineralized-like tissue formation. These not appear to require extracellular signal-regulated characteristics are found in cementoblastoma- kinase-2. Both cementum attachment protein and derived cells, which produce mineralized nodules (6, fibronectin mediate cell attachment through the

118). A direct correlation between alkaline phospha- same a5b1 integrin (98); however, there are differ- tase expression and mineralized-like tissue formation ences in the signaling mechanisms induced by was detected in clones with high binding capacity to cementum attachment protein and ﬁbronectin dur- cementum attachment protein (118), indicating that ing cell attachment. There are also differences in cementum attachment protein is associated with attachment and spreading promoted by these sub- mineralizing-tissue-forming progenitors in the peri- strates. These differences may explain why cells odontal ligament. The groups of clones that bind to attach, spread and migrate differentially on cemen- cementum attachment protein and produce tum attachment protein-containing surfaces, and

217 Arzate et al. such differences can be expected to play a role in the Formation of new cementum with inserted Shar- recruitment of cells needed for the regeneration of pey’s fibers on previously exposed root surfaces is an cementum and other periodontal structures during essential process in the regeneration of periodontal periodontal regeneration. Cementum attachment tissues. This process requires the selective repopula- protein bound to root surfaces has been shown to tion of exposed root surfaces by cementoblastic and enhance the recruitment of putative cementoblastic fibroblastic cell lineages that originate within the cells to the root surface in vitro (118). The amount of periodontal ligament and possibly bone (132). Selec- cementum attachment protein bound might be an tive repopulation invokes that the growth, differentia- indicator of the commitment of a progenitor clone to tion, directed migration and attachment of these cell the mineralized-tissue-forming cell lineage. Cemen- lineages should be specifically regulated in time and tum attachment protein is instrumental in recruiting space. These actions could be accomplished by putative cementoblastic progenitors to the root sur- cementum components, such as cementum-derived face and is capable of enhancing their differentiation. growth factor, cementum attachment protein and Also, cells that migrate and attach to root surfaces cementum protein-1 (67, 171). coated with cementum attachment protein show Cementum protein-1 higher expression of alkaline phosphatase, sialoprotein and cementum attachment protein (118). This Cementum protein-1 was first isolated from human indicates that cementum attachment protein plays an cementum and human cementoblastoma-derived important role in promoting the differentiation of conditioned media (8–10). It is expressed from a sin- putative cementoblast progenitors. Human fibro- gle-copy gene as a 26-kDa nascent protein that is blasts attached onto surfaces containing cementum extensively modified by post-translational events. attachment protein as the only adhesion substrate The human cementum protein-1 gene contains one can synthesize DNA. However, the synthesis requires exon, spans 1.4 kb and maps to the short arm of chro- the engagement of integrins, presumably the a5b1 to mosome 16 (16p13.3). The primary sequence of which cementum attachment protein binds (98). This cementum protein-1 was first deduced from a human indicates that in vivo, tooth-root surfaces containing complementary DNA sequence that showed 98% cementum attachment protein as the matrix compo- homology with a predicted 247-amino-acid sequence nent are conducive for cell proliferation. Cell attach- present in the Pan troglodytes chromosome 16 (5). No ment to cementum attachment protein induces similar sequences have been found so far in other immediate-early G1 phase events, and cyclin D1 levels species. Human cementum protein-1 is composed of increase in the cells adhered to cementum attach- 247 amino acids with a calculated molecular weight ment protein alone, even without growth factors. The of 26 kDa, and it appears to be an alkaline protein expression of cyclin D1 is regulated by adhesion in (isoelectric point = 9.73), with no signal peptide. The the presence of growth factors, and signal reactions cementum protein-1 gene product is enriched in pro- generated by binding cementum attachment protein line (11.3%), glycine (10.5%), alanine (10.1%), serine to cementum attachment protein receptors induce (8.9%), leucine (8.1%), threonine and arginine (each expression of cyclin D1 (232). Cementum attachment 7.7%) and contains low levels of tryptophan, aspartic protein affects cell-cycle progression through mecha- acid, isoleucine (each 2.0%) and phenylalanine nisms that are common to other molecules. Never- (1.6%). Tyrosine is not present. The amino-acid theless, differences occur in the type and degree of sequence indicates that the cementum protein-1 is induction of these events (182). Cells differing in the likely to be a nuclear protein; however, it does not capacity to bind cementum attachment protein also have DNA-binding motifs. Amino acids 30–110 show differ in their ability to form mineralized tissue in cul- 48% similarity with the human collagen a I (I) chain, ture and to produce cementum attachment protein 46% similarity with type XI and 40% similarity with (11, 118). These observations indicate that substances type X. such as cementum attachment protein present in the The full-length recombinant cementum protein-1 local cementum environment could determine which expressed in human-derived gingival fibroblasts is cells are recruited and how they differentiate during mainly composed of beta-sheet with 10% alpha-helix, normal homeostasis and wound healing, and whether 32.4% anti-parallel, 5.8% parallel, 16.7% beta-turn the healing response is repair or regeneration. This is and 35% random coil. This feature is associated with important in periodontal regeneration when regener- proteins that have a high percentage of random coil ation requires new cementum formation and restora- structure, which have been shown to be multifunc- tion of connective tissue attachment (48, 170). tional and to have diverse binding properties; such

218 Cementum proteins proteins include SIBLING and HMGI(Y) (35, 41). This old of spontaneous precipitation, recombinant might help to explain why cementum protein-1 regu- human-cementum protein-1 is effective in promoting lates crystal growth and composition of apatite crys- the nucleation of octacalcium phosphate in an aga- tals (4). According to in silico analysis, cementum rose gel. It was calculated that as little as 1.0 lg/mL of protein-1 possesses two N-glycosylation sites, namely cementum protein-1 can promote nucleation (218). asparagine-X-serine in amino acids 20 and 25 – and Human recombinant cementum protein-1 possesses this is consistent with its shift from a protein size of high afﬁnity for hydroxyapatite, even without post-

Mr 50,000 to a protein size of Mr 39,000. The precise translational modifications, and it affects the mor- role of attached carbohydrates in cementum protein- phology of apatite crystals. Human cementum pro- 1 is unknown; however, glycosylation may affect the tein-1 induces the formation of polymorphous function of cementum protein-1 during the minerali- crystals, as confirmed by X-ray diffraction. Elemental zation process because their anionic surface can bind analysis performed with energy-dispersive X-ray a large number of Ca2+ ions and regulate hydroxyapa- analysis identified a calcium/phosphorus ratio of 1.4, tite crystal growth (29). Glycans are also implicated in which corresponds to octacalcium phosphate (Fig. 2). the regulation of endochondral ossification, bone These findings indicate that biologically active remodeling and fracture healing (66). Cementum pro- cementum protein-1 plays a role during the biomin- tein-1 appears to be a phosphorylated protein eralization process and is required for the synthesis of because antibodies against phosphor-serine and needle-like shape crystals. Octacalcium phosphate is phosphor-threonine cross-react with cementum pro- found to be a transient phase during the growth of tein-1. The presence of phosphate also favors the biological crystals. In small crystals, octacalcium binding of Ca2+ to the protein (102, 209), and proteins phosphate is completely transformed into hydroxyap- (such as sialoprotein and osteopontin) associated atite by hydrolysis and can only be detected in large with the mineralization process are highly phosphor- crystals because of its slow kinetics of transformation. ylated at threonine and serine residues (236). Thus, Octacalcium phosphate has also been presumed to cementum protein-1 may play a role at the early be a necessary precursor of biological apatites. stages of mineralization during the formation of octa- Further characterization of cementum protein-1 calcium phosphate (Fig. 1). The cementum protein-1 using western blots with extracted proteins from does not react with sulfhydryl groups; therefore, all human cementoblastoma and antibody to periodon- cysteine residues in cementum protein-1 might be tal ligament-derived cells showed the presence of linked to disulfide bridges, which generally play a role three components: 55-, 50- and 26-kDa species. These stabilizing protein structure (87, 88, 106). products might represent differences in the degree of In a steady-state system with the concentrations of post-translational modifications, particularly phos- calcium and phosphate maintained below the thresh- phorylation and glycosylation (5). In vitro studies

A B

Fig. 1. Scanning electron micro- graph 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) C D 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.

219 Arzate et al.

A 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 B 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. Further- more, 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 Fig. 2. (A) Representative energy-dispersive X-ray micro- together, these studies suggest that cementum-spe- analysis spectrum of the crystals formed as a result of the cific proteins can induce stem cells to express a carti- effect of human recombinant cementum protein-1 in a steady-state agarose system. The spectrum shows promi- lage phenotype. Furthermore, cementum functions nent peaks of calcium (Ca) and phosphorus (P) and the not only as an inducer of cell differentiation but also Ca/P ratio indicates that the crystals are octacalcium phos- as an inducer of proliferation (8). phate. C, carbon; Cl, chlorine; O, oxygen; Si, silicon. (B) The function of cementum protein-1 was further Direct visualization of cementum protein-1 nanospheres tested by transfection into nonmineralizing cells, (39 nm high, 81 nm wide, ovoid morphology), as deter- fi mined by tapping mode atomic force microscopy in air. such as human gingival broblasts. In contrast to normal human gingival fibroblasts, human gingival fi- using immunocytochemistry confirmed the expres- broblasts/cementum protein-1 cells showed sion of cementum protein-1 by cementoblastoma- increased proliferation, formation of mineralized derived cells and periodontal ligament cells. Almost nodules, increased alkaline phosphatase-specific all (95%) of the cementoblastoma-derived cell popu- activity and the de novo expression of osteocalcin, os- lation was positive for cementum protein-1, whereas teopontin, sialoprotein, runt-related transcription only 6% of periodontal ligament cells stained positive factor 2/core-binding factor alpha1 and cementum for cementum protein-1. Cementum protein-1 was attachment protein mRNAs and protein. These mole- also expressed in a small population (3%) of osteo- cules are all associated with bone/cementum forma- blastic cells in vitro, whereas cementum protein-1 tion (27). The studies strongly support the notion that was not detected in gingival fibroblasts. These small cementum protein-1 has the ability to change the cell periodontal ligament-positive and osteoblast-positive phenotype from nonmineralizing (human gingival fi- populations could represent cementoblast precur- broblasts) to mineralizing (osteoblast/cementoblast) sors, suggesting that cementoblasts and osteoblasts by regulating proliferation and gene expression, might have a common ancestor and that cementum resulting in the differentiation of these cells and the protein-1 could be a marker for the cementoblastic production of a mineralized extracellular matrix lineage (10). Immunohistochemistry studies using resembling cementum.

220 Cementum proteins

α-CAP α-CEMP1 MERGE

PL PL PL

CB CB CB

BV BV BV CEM CEM CEM

AB AB AB

PVC PVC PVC ABC

α-AMEL α-CEMP1 MERGE

CB CB CB

CEM CEM CEM

ERM ERM ERM

DEF Fig. 3. Double immunostaining of human periodontal tis- itor cells. (D) The epithelial cell rests of Malassez (ERM) sues with cementum proteins. (A) Immunostaining shows and CB cross-react strongly with anti-amelogenin mono- that anti-bovine CAP IgG1 (a-CAP) cross-reacts with ce- clonal antibody (a-AMEL). (E) The CEMP1 gene product is mentoblasts (CB) paravascular cells (PVC) and cell sub- expressed by the ERM and CB. (F) Double immunostaining populations in the periodontal ligament (PL). (B) with a-AMEL and a-CEMP1 show that these proteins are Cementum protein-1 antibody (a-CEMP1) cross-reacts expressed by similar structures such as the ERM and CB. strongly with CB. (C) Double immunostaining shows that AB, alveolar bone; BV, blood vessel; CB, cementoblasts; anti-CAP IgG1 and anti-CEMP1 serum are expressed by CB PVC, paravascular cells. and PVC in the PL that could represent cementum progen- In-vitro studies have shown that periodontal liga- tially regulated in osteoblasts and cementoblasts and ment cells can form alkaline phosphatase-positive that knockdown of cementum protein-1 expression and alkaline phosphatase-negative colonies and that in periodontal ligament cells only affects sialoprotein the alkaline phosphatase-positive periodontal liga- expression in cementoblasts, suggesting that cemen- ment cells also express higher levels of mineraliza- tum protein-1 is associated with the regulation of tion-related genes (sialoprotein and osteocalcin) than sialoprotein expression in cementoblasts (113). We do the alkaline phosphatase-negative cells. These have reported the nuclear staining of cementum pro- data suggest that alkaline phosphatase-positive peri- tein-1 in cementoblasts, whereas periodontal liga- odontal ligament cells include osteoblast and/or ce- ment cells exhibit intense staining of cementum mentoblast subsets (141). It has been found that protein-1 only in the cytoplasm, indicating that the cementum protein-1 is preferentially expressed in subcellular location of cementum protein-1 could alkaline phosphatase-positive cells and that the change during the cementoblastic differentiation of expression of cementum protein-1 is reduced when periodontal ligament cells. Overexpression of cemen- periodontal ligament cells are cultured under osteo- tum protein-1 in periodontal ligament cells down- genic conditions (i.e. with either bone morphogenetic regulates periodontal ligament cell markers such as protein-2 or in osteogenic induction medium) (113). PLAPI/asporin, and increases cementoblast markers Overexpression of cementum protein-1 increases the such as cementum attachment protein and sialopro- expression of cementum attachment protein in peri- tein. These data indicate that cementum protein-1 odontal ligament cells at both mRNA and protein lev- could select periodontal ligament cells, or progenitor els. The mechanism by which cementum protein-1 cells present in the periodontal ligament, to differen- regulates the expression of cementum attachment tiate toward the cementoblastic phenotype (113). protein in periodontal ligament cells is not clear. The ability of cementum protein-1 to induce peri- However, the expression of cementum protein-1 odontal ligament cells to differentiate toward ce- decreases when cells are committed to osteoblastic/ mentoblast/osteoblast and/or chondrogenic-like chondroblastic lineages, which suggests that the phenotypes was also tested using a three-dimensional expression of cementum protein-1 is being differen- cell-culture system. Under those conditions, cemen-

221 Arzate et al. tum protein-1 stimulated periodontal ligament cells ration and bone formation, as suggested by the to proliferate in a three-dimensional globular mate- expression of collagen type X and sialoprotein. rial with morphological features and staining charac- Studies on cementum regeneration, using a dog teristics of cartilage and cementum/bone-like tissues. model for dental pulp necrosis, demonstrated the Cementum protein-1 induced periodontal ligament ability of cementum protein-1 to recruit mesenchy- cells, cultured in a three-dimensional system, to mal stem cells from the periodontal ligament and to express type II collagen and aggrecan (both markers promote the proliferation and mineralization of these for pre-hypertrophic chondrocytes) at mRNA and cells. In vivo studies co-localized cementum protein- protein levels (36, 91, 140). Expression of type X colla- 1 and STRO-1 (a marker of mesenchymal stem cells) gen, a marker of fully differentiated chondrocytes positive cells adjacent to the root-surface areas where (117), was also increased by the addition of cemen- neocementum is deposited, indicating that the cells tum protein-1 to the three-dimensional periodontal responsible for reparative cementum deposition are ligament cell cultures. The expression of SRY (sex of mesenchymal origin (164). In vitro, cementum pro- determining region Y)-box 9, a transcription factor tein-1 promotes the proliferation and the migration that mediates chondrogenic differentiation (2, 17), in of periodontal ligament cells, with the migration front cells grown in the presence of human recombinant- comprising STRO-1-positive cells. These studies sug- cementum protein-1 suggests that cementum pro- gest that cementum protein-1 is a mediator in wound tein-1 promotes chondrogenic differentiation (91). healing and periodontal regeneration because it stim- Cementum protein-1 shows sequence similarity with ulated the proliferation and migration of periodontal type X and XI collagens and is immunologically ligament cells. Cementum protein-1 promotes the related to type X collagen (5). It is conceivable that migration of STRO-1-positive cells and provides a cementum protein-1 plays a role during the minerali- possible mechanism for the recruitment of mesen- zation of hypertrophic cartilage and facilitates endo- chymal cells through migration toward the cemen- chondral ossification. Periodontal ligament cells show tum protein-1 signal (164). The role of cementum a multilineage potential to differentiate toward osteo- protein-1 as a chemoattractant and as a promoter of genic, chondrogenic and adipogenic phenotypes by a mineralization is further supported by the findings significant up-regulation of cartilage marker genes that mineralization is reduced upon blocking cemen- and osteoblastic differentiation markers (49, 192). It tum protein-1 function in vitro. In cementoblastoma- seems possible that cementum protein-1 exerts a dif- derived cells, blocking cementum protein-1 activity ferentiation role on the periodontal ligament cell decreases alkaline phosphatase activity and the population by selecting multipotent stem cells, which expression of sialoprotein and osteopontin, but does provide a unique reservoir, to differentiate into vari- not alter cell proliferation (4). These studies also ous cell phenotypes (229), or as an inducer of the het- showed that extracellular calcium increases the erogeneous periodontal ligament cell population expression of cementum protein-1 and PTPLA/ having a differential effect on cells at various degrees cementum attachment protein in periodontal liga- of differentiation. This also explains the expression of ment stem cells via the mitogen-activated protein cementum protein-1 in periodontal ligament cell sub- kinase signaling pathway. This has been confirmed by populations representing precursors of cemento- blocking extracellular signal-regulated kinase-1 and blasts or osteoblasts, or both (5, 10, 64). Additionally, extracellular signal-regulated kinase-e using small there is a direct correlation between the capacity of interfering RNA, which down-regulates the expression human periodontal ligament-derived cells to bind to of PTPLA/cementum attachment protein and cemen- a cementum protein and produce mineralized-like tum protein-1. Furthermore, the use of calcium chan- tissue in vitro (11). It has been shown that cementum nel blocker prevented the expression of cementum protein-1 increases the activity of alkaline phospha- protein-1 and PTPLA/cementum attachment protein, tase. High levels of alkaline phosphatase are also demonstrating the role for calcium ions in cemento- associated with hypertrophic cartilage and bone for- genesis (164). To date, molecules responsible for mation, and alkaline phosphatase is considered to be recruiting mesenchymal cells and inducing their dif- a marker for chondrocyte differentiation because its ferentiation into cementoblasts have not been identi- activity appears to be increased during chondrocyte fied. These studies suggest that cementum protein-1 hypertrophy (213). The expression of cartilage-forma- could be one of the molecules. tion markers in these cultures suggests that periodon- In summary, the data presented above strongly tal ligament cells have the potential to differentiate indicate that cementum protein-1 is a unique protein into chondrocytes and then progress rapidly to matu- that has multiple properties as inducer of mineraliza-

222 Cementum proteins

α-CEMP1 α-CEMP1 α-CEMP1 E E

IEE IEE C CB OEE OEE DF DF A B C α-CEMP1 H&E α-CEMP1 H&E

PL PL AB

AB CEM

CB HERS CB HERS D E F G Fig. 4. Expression of cementum protein-1 (CEMP1) during product. (C) Cells, possibly cementoblasts (CB), facing the initial root formation. (A) Inner enamel epithelium (IEE) root surface express CEMP1 and CEMP1 is strong expres- and outer enamel epithelium (OEE) close to the initial root sion of at the cemento–enamel junction. (D) Hertwig0s epi- formation show strong cross-reactivity with anti-cemen- thelial root sheath (HERS) cells express CEMP1. (E) tum protein-1 serum (a-CEMP1) as well as with a few cells Hematoxylin and eosin (H&E) staining for orientation. (F) in the dental follicle (DF). (B) An enlargement of the area Cells (CB) facing cementum throughout the length of the in panel A shows that elongated ameloblasts and flat-like root express CEMP1. (G) H&E staining for orientation. AB, shape cells of the OEE strongly express the CEMP1 gene alveolar bone; E, enamel; PL, periodontal ligament. tion, proliferation, differentiation and cell matura- the root surface forming acellular cementum (43, 44) tion. Additionally, cementum protein-1 might serve (Fig. 4). However, the presence of enamel proteins, to regulate the mesenchymal stem-cell pool present especially amelogenins, during root development has in the periodontal ligament and to induce its differen- been the subject of controversy. Some investigators tiation into different pathways. These properties open reported the expression of amelogenins by the apical the possibilities of creating cementum protein-1- cells of Hertwig’s epithelial root sheath, which based therapies for periodontal regeneration. secreted small amounts of amelogenins during early differentiation of root development (101), whereas Enamel-associated proteins in cementum others did not detect the expression of amelogenin in Many years ago, Slavkin & Boyde (196), proposed a these cells (119), only ameloblastin (238). Neverthe- hypothesis that Hertwig’s epithelial root sheath- less, a new therapeutic approach, using enamel derived extracellular matrix proteins might be related matrix derivative to achieve periodontal regeneration, to tooth-crown-derived enamel proteins and that was born, based on the assumption that enamel these enamel-related proteins might initiate acellular matrix proteins synthesized by cells of the Hertwig’s cementum formation. Several human and mouse epithelial root sheath could trigger the differentiation cementum proteins were found to be immunologi- of follicle cells into cementoblasts. Specifically, it has cally related to amelogenin and enamelin. These pro- been postulated that amelogenin induces the forma- teins represented species of 72 and 26 kDa that were tion of acellular extrinsic fiber cementum. However, secreted by Hertwig’s epithelial root sheath cells others suggest that the tissue formed by treatment (197). A few years later, it was demonstrated that a- with enamel matrix derivative results in the formation meloblastin, an enamel-associated protein, is of a cellular cementum-like tissue or bone with the expressed by epithelial cells covering the first thin characteristics of cellular intrinsic fiber cementum layer of unmineralized root mantle dentin, and a (23, 24). The bone-like appearance of this tissue is in strong signal is expressed in cells enclosed in the cel- line with the chondrogenic/osteogenic activity of lular cementum known as the epithelial cell rests of enamel matrix derivative (24, 215). Numerous studies Malassez and in cells in a more coronal position at have suggested that enamel matrix derivative can

223 Arzate et al. have multiple functions, such as the promotion of cell they do synthesize ameloblastin. These studies proliferation, differentiation and up-regulation of showed that Hertwig’s epithelial root sheath cells extracellular matrix production (36, 50, 51, 83, 86, 139, change their morphology and produce Von Kossa- 159, 188, 195, 233). Also, several reports have provided positive nodules coincidental with the expression of further evidence that enamel matrix proteins may be dentin matrix protein-1, sialoprotein and osteocalcin, involved in root formation (20). Furthermore, amelo- and high levels of alkaline phosphatase activity. genin null mice showed defects in cementum and a Transmission electron microscopy comparison of the decreased expression of sialoprotein along the root mineralized extracellular matrix deposited by Her- surface (57, 219). This is of particular importance twig’s epithelial root sheath cells in vitro with acellu- because sialoprotein acts as a regulator of the miner- lar cementum deposited in vivo suggests that this alization process in cementum (47, 59). extracellular matrix might be cementum (238). Amelogenins are the expression products of X and Hertwig’s epithelial root sheath cells synthesize Y chromosomal genes and give rise to multiple cementum attachment protein and cementum pro- spliced variants (235). One of these alternatively tein-1 (Fig. 4), thus supporting the idea that Hertwig’s spliced variants is a leucine-rich amelogenin peptide epithelial root sheath cells are capable of producing (A-4) which has been demonstrated to increase the cementum along with inducing a high activity of alka- expression of osteopontin, sialoprotein and osteopro- line phosphatase. This finding provides further evi- tegerin, and to decrease the expression of osteocalcin, dence that the extracellular matrix deposited by these in cementoblasts (208, 215, 216). Amelogenin and a- cells is acellular cementum, indicating that alkaline meloblastin can act as signaling molecules in the phosphatase is a very important component of acel- periodontal ligament; they have an effect on attach- lular cementum. Hertwig’s epithelial root sheath cells ment (237) and proliferation of these cells in vitro. express osteocalcin in vitro, thus indicating the possi- Both proteins have a modulatory role and down- bility that disruption of the basement membrane is regulate the expression of type I collagen, whilst caused by Hertwig0s epithelial root sheath cells when inducing the de novo expression of osteocalcin. they start depositing the acellular cementum. These Amelogenin also induced the expression of sialopro- studies suggested different cellular origins for acellu- tein in periodontal ligament cells, indicating that this lar (Hertwig’s epithelial root sheath cells) and cellular protein can induce phenotypic changes in these cells (mesenchymal cementoblasts) cementum (23, 92). (239). Amelogenin has been suggested to have biolog- Furthermore, Hertwig’s epithelial root sheath cells/ ical effects on cells of mesenchymal origin, such as epithelial cell rests of Malassez are a unique popula- periodontal ligament and gingival fibroblasts, and tion of epithelial cells in the periodontal ligament and enamel matrix derivative enhances the growth of are believed to play a crucial role in cementum repair human bone marrow stromal cells (68, 93, 215). In (203). Hertwig’s epithelial root sheath cells/epithelial the past, amelogenin has been shown to be expressed cell rests of Malassez could differentiate into ce- by odontoblasts, periodontal ligament cells, Hertwig’s mentoblasts through epithelial–mesenchymal trans- epithelial root sheath cells and cementoblasts (23, 44, formation (202). Recently it was demonstrated that in 46, 70, 72, 78, 79, 155, 162). Others have described the vitro Hertwig’s epithelial root sheath/epithelial cell expression of amelogenin in hematopoietic stem rests of Malassez contain primitive stem cells that cells, macrophages and megakaryocytes, rat brain express epithelial stem-cell markers such as octamer- and myoepithelial cells, suggesting a regulatory role binding transcription factor 4, homeobox protein for amelogenin in the recruitment and differentiation NANOG and stage-specific embryonic antigen 4 of monocytic cells from the bone marrow toward (147). These cells might function in creating the bor- becoming mineralized tissue-resorbing cells (bone der between the ameloblasts and the proliferative and cementum osteoclasts/cementoclasts). Amelo- region of Hertwig’s epithelial root sheath (145) and genin, a major structural protein in mineralizing might contribute to the formation of cementum and/ enamel, is also expressed in brain tissue and cells of or enamel repair (76, 194). More recently it was the hematopoietic system (39). Progressive deteriora- shown that the epithelial cell rests of Malassez share tion of cementum is observed in amelogenin knock- similar phenotypic and functional characteristics with out mice and is characterized by the increased mesenchymal stem cells and are capable of develop- presence of osteoclasts (78, 79). On the other hand, ing into osteoblasts, adipocytes, chondrocytes and RT-PCR and western blotting results have demon- neuron-like cells in vitro (228), similar to that strated that Hertwig’s epithelial root sheath cells in described in vitro for periodontal ligament stem cells vitro do not synthesize amelogenin or enamelin, but (192, 204). These studies suggest that the epithelial

224 Cementum proteins

BV PL PL PL CB BV CB ERM CEM AB CEM CEM

BV A B C

α-AMBN α-pCK α-CAP PL PL PL

BV PVC CB ERM CEM CB CEM CEM

DFE Fig. 5. (A) Hematoxylin and eosin staining showing the progenitor cells. (D) Ameloblastin (AMBM), an enamel and periodontal ligament (PL), cementum (CEM) and alveolar cementum-related protein, is shown to be expressed by a bone (AB). Blood vessels (BV) along with the putative pro- single layer of CB facing the CEM surface and paravascular genitor cells of CEM are located close to the CEM surface. progenitor cells (PVC) into the PL. (E) A pan-cytokeratin (B) Cementoblasts (CB) are ordered in three to four layers monoclonal antibody (a-pCK) cross-reacts with CB and of cells facing the CEM surface. (C) The epithelial cell rests subpopulations of PL cells, possibly supporting the state- of Malassez (ERM) are located in the vicinity of the CB cell ment that CEM is an epithelial product. (F) Cementum layers and the BV, indicating a possible inter-relationship attachment protein (CAP) is expressed by subpopulations regulating CEM metabolism and differentiation of CEM of ERM and the CB facing the CEM surface. cell rests of Malassez are epithelial stem cells with the tal follicle cells to a cementoblast phenotype rather ability to differentiate into epithelial or mesenchymal than to an osteoblast phenotype. Recently it was cells and play a critical function in periodontal reported that cementum attachment protein and repair/regeneration. cementum protein-1 are stringently regulated during Dental follicle cells, before the onset of root forma- the cementogenesis process and root formation, and tion, do not express cementum attachment protein, that expression of cementum attachment protein is cementum protein-1 or sialoprotein, which suggests induced more strongly by runt-related transcription the absence of differentiated cells. However, dental factor 2 than is cementum protein-1 (161). Runt- follicle cells are positive for cementum attachment related transcription factor 2 is an important tran- protein and cementum protein-1 when stimulated scription factor for osteogenesis and cementogenesis, with enamel matrix derivative or bone morphoge- and is present in the early proliferative osteoblast/ce- netic protein-2/-7, and their expression was reduced mentoblast cell phenotype, a developmental stage at when treated with recombinant human-Noggin, a which cell proliferation is still required to obtain a well-known inhibitor of bone morphogenetic protein sufﬁcient number of committed cells for matrix for- activity (108). Some dental follicle cells express STRO- mation. Higher expression of cementum attachment 1, indicating that populations of the dental follicle protein at this early stage of cementogenesis is there- have mesenchymal progenitor features (108). Several fore consistent with its function of promoting cell investigators suggest that the use of enamel matrix proliferation (227). In contrast, cementum protein-1 derivative enhances the expression of mineralized tis- has a more important role during the mineralization sue markers (such as alkaline phosphatase) and nod- process and its relationship is focused at this early ule mineralization in dental follicle cells along with stage to control the mineralization process during ce- the expression of bone morphogenetic protein-2 and mentogenesis (218). Recently we reported that nor- sialoprotein (108). As enamel matrix derivative also mal human-derived cementoblasts express induces expression of cementum attachment protein cementum attachment protein, cementum protein-1 and cementum protein-1, it is suggested that enamel and amelogenin and that they are localized to the cell matrix derivative promotes the differentiation of den- nucleus. Human cementoblasts express not only

225 Arzate et al. amelogenin but also other enamel-associated mole- the regeneration process. What may be concluded cules, such as ameloblastin, enamelin and tuftelin from the current status of the ‘cementum proteins’ (Fig. 5). Furthermore, cementum protein-1 induces is that they can pave the way to establish effective the de-novo expression of amelogenin in periodontal therapeutic alternatives to achieve the regeneration ligament cells grown in culture. Perhaps cementum of the periodontal structures, and that the impact protein-1 promotes the osteoblast/cementobast phe- that they could have in the ﬁeld of periodontology notype in periodontal ligament cells through the and skeletal tissues looks very promising. expression of amelogenin (91, 152, 153). These data, taken together, suggest that enamel-associated proteins and cementum proteins could act synergistically Acknowledgments in the regulation of cementoblast differentiation and cementum deposition. These data offer new This work was supported by DGAPA-UNAM approaches to determine how the process of ce- IN216711, IT200414 and by CONACYT 130950. The mentogenesis is regulated and point out the role of authors are grateful to Professor A. Sampath Naraya- these proteins during periodontal homeostasis and nan of the University of Washington for critical read- repair/regeneration of periodontal structures, and ing of the manuscript. could represent new and better therapeutic approaches for the treatment of periodontal disease. References

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