Cementum proteins: role incementogenesis, biomineralization,periodontium formation andregenerationHIGINIO ARZATE, MARGARITA ZEICHNER-DAVID & GABRIELA MERCADO-CELIS

Destruction of periodontal tissues is a cardinal sign ofperiodontal disease, and many factors, such as infec-tions, trauma, orthodontic tooth movement and sys-temic and genetic diseases, can contribute to thisprocess. In periodontal disease, bacteria on the sur-face of the teeth produce a chronic inflammation ofthe gingiva. Cells on the surface of the tooth root andthe cementum covering the root are destroyed, andthe epithelium from the oral mucosa grows down-wards, producing a gingival crevice. Bacteria depositin this crevice and the ensuing inflammatory processmay eventually result in the breakdown of periodon-tal tissues, including the cementum, periodontal liga-ment and alveolar bone.

The process of periodontal tissue regeneration isinitiated at the moment that the damage takes placeby the production of growth factors and cytokines bythe damaged and inflammatory cells. Periodontaltreatment can enhance periodontal healing (60). Rootplaning or root conditioning is being used as a strat-egy to increase mesenchymal cell migration andattachment to the exposed root surface. Treatmentwith acid, in particular citric acid, has been found towiden the orifices of dentinal tubules, thereby accel-erating cementogenesis and enhancing cementumapposition and connective tissue attachment (125).However, when periodontal ligament cells areremoved from the cementum or are unable to regen-erate, bone tissue may invade the periodontal liga-ment space and establish a direct connectionbetween the tooth and the wall of the alveolar socket,resulting in ankylosis. This nonflexible type of toothsupport can lead to loss of function and eventually toresorption of the root (15). Strategies (such as guided

tissue regeneration) have been developed to guideand control regeneration using bioresorbable mem-branes (3, 138, 142) and bone grafts (175). Althougheffective to a certain point, these strategies have theproblem that they are not predictable and do notcompletely restore the architecture of the originalperiodontium. To achieve complete repair and regen-eration it is necessary to recapitulate the develop-mental process with complete formation ofcementum, bone and periodontal ligament fibers.

The past 20 years of research have seen tremen-dous advances in our knowledge of the cellular andmolecular events involved in the process of devel-oping the periodontium. This knowledge has trans-lated into new therapeutic strategies for periodontalregeneration using molecular approaches (45, 67,110–112, 116, 129, 146, 158, 210, 223, 240). Amongstthese strategies are the use of growth factors, suchas platelet-derived growth factor and insulin-likegrowth factors (32, 54, 90, 120, 163, 181, 222), trans-forming growth factor-beta1 (127), basic fibroblastgrowth factor (191), dexamethasone (181) and bonemorphogenetic proteins (109, 114, 154, 176–178). Itis believed that these molecules are produced dur-ing cementum formation and are then stored in thecementum matrix to induce periodontal ligamentregeneration when needed (200). However, one ofthe problems of application of these factors for peri-odontal repair is the nonspecific activity of some ofthe factors on different cell lineages and the rapidloss of the topically applied factors over time (15,120).

As our understanding of the structure, function andcomposition of cementum increases, so does the

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Periodontology 2000, Vol. 67, 2015, 211–233 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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potential for new therapies for periodontal regenera-tion using molecules formed by these tissues. Onesuch example has been the use of enamel proteins toinduce cementum and bone regeneration (50, 51, 72–74, 84, 85). A preparation of porcine enamel proteinhas been marketed (Emdogain�) and is currentlybeing used by practicing dentists (42, 52, 55, 105, 237,239). Outcomes from studies using Emdogain suggestthat its clinical action is the result of contaminationwith growth factors such as transforming growth fac-tor-beta (107, 143) and bone morphogenetic proteins(100). However, studies using recombinant amelo-genin, including results obtained in our laboratory,indicate that amelogenin and ameloblastin also havesignaling properties that induce phenotypic changesin cells, and these changes can fluctuate dependingon the target cell (20, 219, 237, 239, 240).

It is the purpose of this review to focus on the roleof cementum and its specific components in the for-mation, repair and regeneration of the periodontium.As cementum is a matrix rich in growth factors thatcould influence the activities of various types of peri-odontal cells, this review will examine the characteris-tics of cementum, its composition and the role ofcementum components, especially the cementumprotein-1, during the process of cementogenesis, andtheir potential usefulness for regeneration of theperiodontal structures in a predictable therapeuticmanner.

What is cementum?

Cementum can be described as the mineralized tis-sue that covers the roots of teeth and serves toattach the tooth to alveolar bone via collagen fibersof the periodontal ligament. Morphological, histo-logical and functional differences appear to existalong the length of the root, leading cementum tobe classified as follows: intermediate cementum(found in the cemento–enamel junction), acellularcementum (found in the coronal and mid-portionsof the root) and cellular cementum (present in theapical and inter-radicular portions of the root con-taining cementoblasts) (21, 33, 71, 75, 183, 187, 207,240).

Studies on dental cementum can be traced back toMalpighi in the 1600s (61). In general, 18th centuryanatomists regarded human teeth as composed onlyof enamel and dentin. However, the studies of Tenonon horses’ teeth, of Blake on elephants’ teeth and ofCuvier on the teeth of many species resulted in therecognition that cementum was a constituent part of

all animal teeth. Initial examination of cementum onthe roots of human teeth is attributed to Ringelmannin 1824, followed by physiologists Jan Evangelista Pur-kinje and his pupils, Fraenkel and Raschkow, in 1835,and later by the classic histologist Anders Adolf Retzi-us, in 1836, who noticed the presence of ‘striaes’ incementum (19, 37, 61, 186). In almost all mammalianspecies the number of incremental structures in thedental cementum (annulations) can be correlatedwith age and are used as such in archeology andforensics (61).

In a very simplistic way we can define cementumas an extracellular matrix composed of calcified col-lagenous Sharpey’s fibrils, collagen, glycosaminogly-cans, proteoglycans and inorganic hydroxyapatite. Inthe same way we can say that the major functionalrole of cementum is to serve as the anatomical struc-tural site for the attachment of Sharpey’s fibers of theperiodontal ligament. However, the more we studythis tissue, the more complex we find its structureand function. Cementum biology goes back to Gott-lieb, in 1942, who stated that “the continuous deposi-tion of cementum layers seems to be of greatimportance and work as a barrier against the down-growth of the epithelium. Newly deposited cemen-tum seems to have the highest vitality and act as thebest barrier. If the ideas about the biology of cemen-tum are correct, it is then our task to find out justhow nature provides for continuous cementum depo-sition and having done so, to imitate the procedure”(62). These studies introduced the notion that notonly does cementum act as a barrier to delimit epi-thelial growth that can impair attachment but alsothat the presence of a continuous cementum layer isnecessary to act as a microbial barrier and thatdefects in this tissue could result in periodontitis (62,63). This idea was further supported by observationsthat the root surfaces of patients with hypophospha-tasia contained areas completely devoid of cemen-tum or covered with a hypoplastic form of acementum-like material. These patients developedearly-onset periodontitis, suggesting that abnormali-ties in the deposition or maintenance of cementumcan result in a defective periodontium highly suscep-tible to microbial invasion and destruction (160).

Based on the different functions attributed tocementum it is clear that a thorough understandingof the biological properties of cementum is requiredto determine its role in periodontal formation andtherefore periodontal regeneration. Furthermore, theprinciples attributed to cementum regenerationmight be used in the regeneration of other mineral-ized tissues.

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Cementum composition

In order to understand the process of cementogenesisit is important to know the composition of cemen-tum. As in bone and dentin, the major organic com-ponent of cementum is collagen (18). The major typeof collagen is type I collagen, which accounts for 90%of all collagens and plays a structural role during thebiomineralization process, serving as a reservoir forhydroxyapatite nucleation, which successively devel-ops into intrafibrillar apatite crystals (58). Type III col-lagen, which coats type I collagen fibrils, is alsopresent, although in considerably lower quantities. Inaddition to collagens, carboxylated and sulfated mu-copolysaccharides (glycosaminoglycans) are presentin human cementum (217).

Glycosaminoglycans

Glycosaminoglycan-containing proteoglycans areheterogeneous groups of glycoproteins with longrepeating disaccharides. The percentage of glycosa-minoglycans is high in tissues subjected to compres-sive forces, such as cementum. Proteoglycans areknown to interact specifically with collagens in a vari-ety of tissues. It is postulated that proteoglycans incementum are integral components of cell substra-tum attachment matrices and mediate attachmentbetween old and newly formed cementum, thus cre-ating the cementum incremental lines (189, 220).

The major glycosaminoglycans present in humancementum are hyaluronic acid, dermatan sulfate andchondroitin sulfate, and their distribution appears tobe quite different from that reported for soft tissuessuch as gingival connective tissue and periodontal lig-ament, in which dermatan sulfate predominates (12,165). The proteoglycan content of mineralized tissuesis generally relatively low. These differences mightreflect differences in function between hard and softtissue as proteglycans appear to inhibit collagen min-eralization by occupying strategic locations normallydestined to be filled with hydroxyapatite (189). Inbone, dermatan sulfate proteoglycans are orientedparallel to the collagen fiber axis with chondroitin sul-fate proteoglycans and hyaluronic acid, occupying theinterfibrillar region in a space-filling capacity (190).

Amongst other glycosaminoglycans present incementum, keratan sulfate appears to be one of themajor components, which, after digestion with kera-tanase II and endo-beta-galactosidase, produces twocore proteins: lumican and fibromodulin. Interest-ingly, these proteins are localized predominantly innonmineralized cementum (precementum and the

pericementocyte area), suggesting that they playmajor regulatory roles during cementum mineraliza-tion (30). In a similar manner, it was also determinedthat the large chondroitin sulfate glycosaminoglycan,present in cementum, contains the large hyaluronan-binding proteoglycan, versican, and the small intersti-tial proteoglycans, decorin and biglycan. Versican islocalized in lacunae housing cementocytes. Decorinis closely associated with collagen fibers of the peri-odontal ligament and with biglycan in the cemento-blasts/precementum area. The differential tissuedistribution suggests that glycosaminoglycans mayplay distinct roles during the cementogenesis processin addition to regulating the biomineralization ofcementum (31). Syndecan-2 has been found to be sig-nificantly expressed by cells in close contact with theroot surface and within the matrix of reparativecementum, suggesting that it must be associated withcell–matrix interactions during cementummineraliza-tion (224). Biglycan is also associated with the growthof incremental lines in cellular cementum. Further-more, lumican, decorin, versican and biglycan areassociated with the formation of cellular cementumbut not of acellular cementum, suggesting differentcementocyte subpopulations or a differentialresponse of these cells (1). Osteoadherin, a keratin sul-fate-containing proteoglycan, is also associated withthe initial phase of cementum formation because Her-twig’s epithelial root sheath cells express this proteo-glycan during root development (166), and althoughacellular cementum does not contain proteoglycans,initial acellular cementum formation requires a denseaccumulation of proteoglycans (230).

Ostepontin and bone sialoprotein

Cementum contains many noncollagenous proteins,including some major phosphoproteins such as os-teopontin and bone sialoprotein. These proteins playa major role in filling spaces created during collagenassembly and imparting cohesion to the mineral-liketissue by allowing mineral deposition to spread acrossthe entire collagen meshwork (22, 148). The role pro-posed for these proteins is that of regulators ofhydroxyapatite crystal nucleation and growth. It hasbeen suggested that osteopontin and sialoprotein arenecessary for the initiation of crystal formation at thehighly ordered fibrils of type I collagen (179). Both ofthese proteins are acidic: osteopontin has a poly-Aspand sialoprotein contains two poly-Glu domains, therepetitive sequences of which are known to bind cal-cium to mineral surfaces. Osteopontin is presentwithin the periodontal ligament in mature teeth.

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Sialoprotein and osteopontin remain bound to colla-gen matrix and they possess cell-attachment proper-ties through their arginine-glycine-aspartic acid(RGD) sequences (199). In the periodontium, osteo-pontin is expressed by cells in close contact with acel-lular cementum as well as by cementocytes (26). Ithas also been suggested that osteopontin regulatescell migration, differentiation and survival via interac-tions with avb3 integrin (53). Sialoprotein is also anRGD-containing sialoprotein with cell-attachmentproperties (156) and has a precise spatial associationwith early mineral aggregates, binds strongly tohydroxyapatite and acts as a specific and potentnucleator for hydroxyapatite crystal formation in vitro(95). Cementum contains sialoprotein, which, duringroot formation, is distinctly localized to cells liningthe surface of cementum. It has been suggestedthat sialoprotein modulates the process of ceme-ntogenesis and is involved in the process of chem-oattraction, adhesion and differentiation ofprecementoblasts (121–123, 201). Both sialoproteinand osteopontin are believed to play a role in the dif-ferentiation of cementoblast progenitor cells to ce-mentoblasts (183).

Gla proteins

Matrix gamma-carboxyglutamic acid protein andosteocalcin are the two major Gla-containing pro-teins associated with calcified hard tissues (80–82,172, 173). Both proteins have high affinity for Ca2+

and hydroxyapatite through interaction with the Glaresidue. The distribution of osteocalcin in the mam-malian body is virtually limited to mineralized tis-sues such as bone, dentin and cementum (34). Inthe dental root, osteocalcin expression is localizedin cells lining cellular cementum and acellularcementum. However, cells at the inter-radiculararea also express osteocalcin. In a similar manner,cellular and acellular cementum show expressionof matrix gamma-carboxyglutamic acid protein,although acellular cementum expresses those pro-teins more prominently than does cellular cemen-tum. Matrix gamma-carboxyglutamic acid protein issecreted by cementum-forming cells and is incorpo-rated at the mineralization front (103). One possibleexplanation for the accumulation of matrix gamma-carboxyglutamic acid protein in acellular cementumand the outer surface of cellular cementum couldbe to prevent hypercalcification of the cementumsurface (77, 104). During root development in mice,a high level of osteocalcin mRNA is selectivelyexpressed by cells lining root-surface cementoblasts;

in contrast, osteocalcin mRNA is not expressed inthe periodontal ligament (38).

The role of these proteins has been related to regu-lation of mineralization. Matrix gamma-carboxyglu-tamic acid protein-deficient mice show abnormalmineralization and a lack of acellular cementum(104). It has been suggested that both osteocalcin andmatrix gamma-carboxyglutamic acid protein act asnegative regulators of mineralization because matrixgamma-carboxyglutamic acid protein-deficient micepromote calcification of the aortic walls and valves.Thus, both osteocalcin and matrix gamma-carboxy-glutamic acid protein seem to regulate mineralizationby acting as negative regulators, but to differentextents because osteocalcin also inhibits conversionof brushite to hydroxyapatite (81, 173). Other mole-cules present in the cementum extracellular matrixinclude osteonectin, which, during cementogenesis,is synthesized by cementum-producing fibroblasts,cementoblasts and cementocytes (174). Osteonectin,synthesized by mineralizing cells, can bind hydroxy-apatite and is associated with mineralization (25, 80,96, 126, 135). Based on the observation that low con-centrations of osteonectin delay hydroxyapatite-seeded crystal growth in vitro, it is speculated thatosteonectin also acts as a negative regulator by pre-venting, rather than promoting, matrix mineralization(134, 230).

Alkaline phosphatase

Tissue nonspecific alkaline phosphatase (also knownsimply as alkaline phosphatase) has been studied formore than 80 years and is believed to play an impor-tant role in skeletal mineralization. Alkaline phospha-tase is a membrane-bound glycoprotein enzyme thathydrolyses phosphate groups at alkaline pH and alsoinhibits pyrophosphatase, ATPase and protein phos-phatase activity at neutral pH (94). Alkaline phospha-tase is expressed in most body sites during embryonicdevelopment but is confined to bone, kidney, liverand B-lymphocytes during adult life. The fact that it isexpressed in nonmineralizing tissues suggests that ithas other roles besides those associated with mineral-ization. Amongst some of these other functions, it hasbeen suggested that alkaline phosphatase can regu-late tissue turnover and cell proliferation, differentia-tion and maturation (94, 226). Alkaline phosphatase ishighly expressed in cells of the periodontal ligament(56, 69, 99, 124, 151, 231), where it is thought to playa role in phosphate metabolism and cementum for-mation (13), particularly formation of acellularcementum (14, 65). Tissue nonspecific alkaline

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phosphatase-deficient mice show defective formationof acellular cementum, which results in very thin andirregular-shaped patches around the bases of theperiodontal ligament fibers. No defects were seen inalveolar bone, periodontal ligament and cellularcementum, suggesting that alkaline phosphatase isessential for the formation of acellular cementum(16). However, observations in humans with hypo-phosphatasia as a result of mutations in the tissuenonspecific alkaline phosphatase gene revealed thatcementum formation is almost completely abolishedfor both acellular and cellular cementum, resulting inpremature tooth loss. This phenotype differs fromthat in tissue nonspecific alkaline phosphatase gene-knockout mice, which showed blockage of acellularcementum formation only (213, 214) and might beexplained by the different types of mutation possiblein the tissue nonspecific alkaline phosphatase geneand the severity of their manifestation in humans.One of the major functions of tissue nonspecific alka-line phosphatase is the hydrolysis of inorganic pyro-phosphate, a potent inhibitor of hydroxyapatiteformation (206). Cementoblasts are specifically sensi-tive to the levels of inorganic pyrophosphate/inor-ganic phosphate within the extracellular matrix (150).Changes in the level of tissue nonspecific alkalinephosphatase protein have a significant effect on thefunction of osteoblasts, and consequently on matrixmineralization, indicating that tissue nonspecificalkaline phosphatase plays key biological roles in themineralization of bone and cementum (89). Onenovel strategy to reduce inorganic pyrophosphateand increase cementum neoformation may center onthe modulation of inorganic pyrophosphate/inor-ganic phosphate in the periodontium, which mayresult in more predictable regeneration of cementum(180).

Cementum-specific proteins

Extracellular matrix from different tissues share manysimilarities and yet have different functional proper-ties that make them unique. These properties couldbe the result of quantitative and/or qualitative differ-ences amongst their components. For years it wasbelieved that different mineralized tissues containspecific molecules not present in any other tissue (i.e.amelogenin in enamel, dentin sialophosphoproteinin dentin, etc.) and which could be considered asmarkers for those tissues. As detection techniquesbecame more sensitive, it was found that many ofthese molecules were also expressed in other tissues,although at considerably lower concentrations, and

therefore could still be considered as specific mark-ers. Several proteins, some of which are considered tobe cementum-specific proteins, have been isolatedfrom cementum and characterized.

Cementum-derived growth factor

It is now well established that mineralized tissues,such as bone and dentin, are excellent reservoirs ofgrowth factors that, when needed, can be released bydemineralization and serve to repair or regenerate tis-sues. In a similar way, it has been shown that extractsof cementum have the ability to promote a range ofbiological activities such as cell migration, adhesion,mitogenic activity and differentiation, which areessential for periodontal regeneration (67). Miki et al.(137) were the first to report the presence of mito-genic activity in cementum obtained from humanteeth. Later, Nakae et al. (144) isolated and character-ized mitogenic factors present in the cementummatrix of bovine teeth. In addition to fibroblastgrowth factor, which binds strongly to heparin,another mitogenic factor with moderate heparinaffinity was present in cementum but not in alveolarbone. This factor was named cementum-derivedgrowth factor and it is the major component incementum, accounting for 70% of the mitogenicactivity extracted from this tissue. The cementum-derived growth factor acts synergistically with epider-mal growth factor and induces many of the signalingpathways associated with mitogenesis (234). Thesepathways include an increased concentration of cyto-solic Ca2+, activation of the protein kinase C cascadeand expression of cellular proto-oncogenes. Addition-ally, cementum-derived growth factor may promotethe migration and growth of progenitor cells, presentin the adjacent structures, toward the dentin matrixand participate in their differentiation into cemento-blasts (130, 170). Further characterization of cemen-tum-derived growth factor revealed that themitogenic activity was associated with a 14-kDa pro-tein that showed some homology to insulin-likegrowth factor-1. Although cementum-derived growthfactor activity was inhibited with insulin-like growthfactor-1 and insulin-like growth factor-1 receptorantibodies, there were some differences between thecanonical insulin-like growth factor-1 and cemen-tum-derived growth factor, thus it was concluded thatcementum-derived growth factor is an insulin-likegrowth factor-1-like molecule (97).

The presence of cementum-derived growth factorand other growth factors in cementum indicates thatcementum has the potential to regulate themetabolism and turnover of surrounding tissues, that

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cementum could serve as a storage site for thosegrowth-inducing molecules and that the cementumproteins are likely to serve a biological role in promot-ing periodontal regeneration (67, 149).

Cementum attachment protein

In addition to cementum-derived growth factor, ithas been reported that human and bovine cementumcontains potent mediators of cell attachment, andthis biological activity is associated with a 55-kDaprotein species (128, 157). This protein was namedcementum attachment protein and further character-ization by amino-acid sequencing showed the pres-ence of four sequences containing Gly-X-Y repeatstypical of collagen. A 17-amino-acid peptide had 82%homology with a type XII domain, and anotherpeptide had 95% homology with collagen type Ia1.However, cementum attachment protein did notcross-react with antibodies to type I, type V, type XIIand type XIV collagen, and its attachment activitywas lost after treatment with bacterial collagenase.These findings, and the fact that a cementumattachment protein monoclonal antibody localizescementum attachment protein only to cementum (7),suggest that cementum attachment protein mightbe a collagenous-attachment protein localized exclu-sively in cementum (225).

Characterization of a complementary DNA clone forcementum attachment protein isolated from a humancementifying fibroma-derived cell line k-ZAP expres-sion library, revealed a novel alternatively splicedsequence. This sequence encodes a 140-amino-acidprotein that is identical to the first 125 N-terminalamino acids of a truncated isoform of 3-hydroxyacyl-CoA dehydratase-1/protein-tyrosine phosphatase-like(proline instead of catalytic arginine), known asPTPLA (212). The remainder of the C-terminus ofPTPLA/cementum attachment protein is encoded bya read-through of the splice donor site in exon 2, andthe truncation eliminates the PTPLA sequence thathas the signature phosphatase active site motif.Although PTPLA mRNA is widely expressed in manytissues, the PTPLA/cementum attachment proteinmRNA is expressed in cementum cells and only mar-ginally in some periodontal ligament cells in humanteeth. PTPLA/cementum attachment protein was notfound to be expressed in rat teeth (184), suggestingthat there could be some species differences in its dis-tribution. Rat roots are known to overproduce apicalcementum after they reach 8 weeks of age, whereasother species maintain a normal layer of cementum.

Interestingly, the cementum attachment protein isa 55-kDa collagenous protein, whereas the PTPLA/

cementum attachment protein codes for a 15-kDaprotein that has no collagen sequences. This suggeststhat perhaps the cementum attachment protein isstrongly associated with collagen chains in thecementum matrix, possibly through cross-linking,which increases its molecular weight. This possibilityis supported by the observation that immunoprecipi-tates of cementum extracts obtained with severalanti-cementum attachment protein monoclonal anti-bodies contain two protein species migrating at55 kDa and ~ 29 kDa, and one monoclonal antibodycross-reacts with both cementum attachment proteinand type I collagen (A.S. Narayanan, unpublisheddata). Expression of PTPLA/cementum attachmentprotein is limited to cementum and to some cells inthe endosteal spaces of bone. This could be explainedas a result of the presence of precursors of cemento-blasts in the endosteal spaces of alveolar bone (130,133, 198). These cells are thought to traverse throughthe periodontal ligament before reaching and differ-entiating on cementum.

It has been shown that cementum attachment pro-tein binds to hydroxyapatite and more strongly tocementum than to the dentin surface (168). Cemen-tum attachment protein also binds to fibronectin, butbinds 150 times more strongly to hydroxyapatite thanto fibronectin (169). Like the cementum attachmentprotein obtained from cementum, recombinantPTPLA/cementum attachment protein also binds tohydroxyapatite with high affinity (212). These obser-vations suggest that PTPLA/cementum attachmentprotein may play a regulatory role during cementumformation (67, 212). It has also been shown thatcementum attachment protein promotes the attach-ment of gingival fibroblasts, endothelial cells andsmooth muscle cells, but not oral sulcular epithelialcells (157). Bone cells bind more strongly to cemen-tum attachment protein than do periodontal liga-ment cells, which, in turn, bind more strongly tocementum attachment protein compared with gingi-val cells (300% for bone cells, 250% for periodontalligament cells and 150% for gingival cells). Cementumattachment protein-coated root slices promote pref-erential adhesion and differentiation of osteoblasticcells (168, 169, 171). Attachment to cementum attach-ment protein by human gingival fibroblasts is medi-ated primarily by the integrin a5b1 (98). The a5b1integrin has been shown to be involved in variousaspects of development (131), and it is possible thatthe a5b1 integrin may also play an important role incementogenesis and neo-cementogenesis throughinteractions with cementum attachment protein.Cementum attachment protein has the capacity to

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direct cell migration of alveolar bone cells. Periodon-tal ligament cell populations differ in their capacity torecognize and respond to cementum attachment pro-tein. Fifteen per cent of clones of periodontal liga-ment cell cultures bind strongly to cementumattachment protein and this binding capacity is simi-lar to that of alveolar bone cells, suggesting that theorigin of cementoblastic precursors could be osteo-genic. Therefore, cementum attachment protein maypotentially be used to induce preferential repopula-tion of the root surface by appropriate cells (169).Periodontal ligament cells manifest a higher chemo-tactic response to cementum attachment proteinthan do gingival fibroblasts, and this activity is 24-foldgreater than that of fibronectin (136). Cementumattachment protein binds selectively to periodontalligament cells and supports periodontal ligament cellattachment to root surfaces (171). Selective chemo-taxis and attachment by cementum attachment pro-tein may represent the natural pathway by whichcementoblast progenitors are attracted to the rootsurface during homeostasis and regeneration. Par-tially demineralized cementum does not support epi-thelial outgrowth; however, it enhances migrationtoward and attachment of periodontal connective tis-sue cells to dental surfaces in vitro (168, 169). Thus,exposed collagen fibers might improve the capacity ofperiodontal connective tissue cells to compete withepithelial cells and be the first to attach to and colo-nize the root surface after periodontal surgery (167).If this is the case, biochemical alterations in cemen-tum may explain the loss of soft-tissue attachmentfrom diseased root surfaces and the failure for its suc-cessful restoration. Selective chemotaxis by, andattachment through, cementum attachment proteinmight represent the natural pathway by which ce-mentoblast progenitors are attracted to the root sur-face during homeostasis and regeneration.

It has been reported that the cementum attach-ment protein possesses the capacity to bind peri-odontal ligament progenitor clones and that this isdirectly related to their alkaline phosphatase expres-sion and mineralized-like tissue formation. Thesecharacteristics are found in cementoblastoma-derived cells, which produce mineralized nodules (6,118). A direct correlation between alkaline phospha-tase expression and mineralized-like tissue formationwas detected in clones with high binding capacity tocementum attachment protein (118), indicating thatcementum attachment protein is associated withmineralizing-tissue-forming progenitors in the peri-odontal ligament. The groups of clones that bind tocementum attachment protein and produce

mineralized-like tissue, similar to cellular cementum,represent 7% of the periodontal ligament populationand 15% of the mineralized-tissue forming clonesbelonging to the cementoblastic lineage (118). Thehigh binding capacity of cementum attachment pro-tein, combined with a low constitutive percentage ofalkaline phosphatase expression, is of interestbecause it supports the view that cementoblastoma-derived cells express a low constitutive percentage ofalkaline phosphatase-positive cells and that cemento-blasts express low levels of alkaline phosphatase com-pared with alveolar bone-derived osteoblasts (205).

Adhesion of human gingival fibroblasts to cemen-tum attachment protein stimulates mitogen-activatedprotein kinase activity and induces the expression ofc-fos mRNA; in contrast, protein-tyrosine phosphory-lation and c-fos mRNA were not induced in unat-tached cells. As mitogen-activated protein kinase andc-fos mRNA were not induced in monolayer cultures,it was presumed that these reactions are induced byadhesion and are not necessary for cell adhesion(182). The kinetics of mitogen-activated proteinkinase activation was different for cells attaching tofibronectin or polylysine – c-fos mRNA levelsincreased only half as much in cells attaching tofibronectin and very little in cells attaching to polyly-sine. These data demonstrate that cementum attach-ment protein and other adhesion molecules presentin mineralized tissue matrices induce characteristicsignaling events during adhesion, which may play arole in the recruitment of specific cell types duringwound healing and in mediating their specific biolog-ical functions. This differential response is especiallyimportant in periodontal regeneration, in which epi-thelial cells must be excluded and fibroblasts and ce-mentoblasts are to be selected from a pool of variousprogenitor cells (170). Cementum attachment proteinis likely to play a role in the cell selection process. Themitogen-activated protein kinase kinase/mitogen-activated protein kinase pathway participates incementum attachment protein-mediated fibroblastspreading, but cell attachment and proliferation donot appear to require extracellular signal-regulatedkinase-2. Both cementum attachment protein andfibronectin mediate cell attachment through thesame a5b1 integrin (98); however, there are differ-ences in the signaling mechanisms induced bycementum attachment protein and fibronectin dur-ing cell attachment. There are also differences inattachment and spreading promoted by these sub-strates. These differences may explain why cellsattach, spread and migrate differentially on cemen-tum attachment protein-containing surfaces, and

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such differences can be expected to play a role in therecruitment of cells needed for the regeneration ofcementum and other periodontal structures duringperiodontal regeneration. Cementum attachmentprotein bound to root surfaces has been shown toenhance the recruitment of putative cementoblasticcells to the root surface in vitro (118). The amount ofcementum attachment protein bound might be anindicator of the commitment of a progenitor clone tothe mineralized-tissue-forming cell lineage. Cemen-tum attachment protein is instrumental in recruitingputative cementoblastic progenitors to the root sur-face and is capable of enhancing their differentiation.Also, cells that migrate and attach to root surfacescoated with cementum attachment protein showhigher expression of alkaline phosphatase, sialopro-tein and cementum attachment protein (118). Thisindicates that cementum attachment protein plays animportant role in promoting the differentiation ofputative cementoblast progenitors. Human fibro-blasts attached onto surfaces containing cementumattachment protein as the only adhesion substratecan synthesize DNA. However, the synthesis requiresthe engagement of integrins, presumably the a5b1 towhich cementum attachment protein binds (98). Thisindicates that in vivo, tooth-root surfaces containingcementum attachment protein as the matrix compo-nent are conducive for cell proliferation. Cell attach-ment to cementum attachment protein inducesimmediate-early G1 phase events, and cyclin D1 levelsincrease in the cells adhered to cementum attach-ment protein alone, even without growth factors. Theexpression of cyclin D1 is regulated by adhesion inthe presence of growth factors, and signal reactionsgenerated by binding cementum attachment proteinto cementum attachment protein receptors induceexpression of cyclin D1 (232). Cementum attachmentprotein affects cell-cycle progression through mecha-nisms that are common to other molecules. Never-theless, differences occur in the type and degree ofinduction of these events (182). Cells differing in thecapacity to bind cementum attachment protein alsodiffer in their ability to form mineralized tissue in cul-ture and to produce cementum attachment protein(11, 118). These observations indicate that substancessuch as cementum attachment protein present in thelocal cementum environment could determine whichcells are recruited and how they differentiate duringnormal homeostasis and wound healing, and whetherthe healing response is repair or regeneration. This isimportant in periodontal regeneration when regener-ation requires new cementum formation and restora-tion of connective tissue attachment (48, 170).

Formation of new cementum with inserted Shar-pey’s fibers on previously exposed root surfaces is anessential process in the regeneration of periodontaltissues. This process requires the selective repopula-tion of exposed root surfaces by cementoblastic andfibroblastic cell lineages that originate within theperiodontal ligament and possibly bone (132). Selec-tive repopulation invokes that the growth, differentia-tion, directed migration and attachment of these celllineages should be specifically regulated in time andspace. These actions could be accomplished bycementum components, such as cementum-derivedgrowth factor, cementum attachment protein andcementum protein-1 (67, 171).

Cementum protein-1

Cementum protein-1 was first isolated from humancementum and human cementoblastoma-derivedconditioned media (8–10). It is expressed from a sin-gle-copy gene as a 26-kDa nascent protein that isextensively modified by post-translational events.The human cementum protein-1 gene contains oneexon, spans 1.4 kb and maps to the short arm of chro-mosome 16 (16p13.3). The primary sequence ofcementum protein-1 was first deduced from a humancomplementary DNA sequence that showed 98%homology with a predicted 247-amino-acid sequencepresent in the Pan troglodytes chromosome 16 (5). Nosimilar sequences have been found so far in otherspecies. Human cementum protein-1 is composed of247 amino acids with a calculated molecular weightof 26 kDa, and it appears to be an alkaline protein(isoelectric point = 9.73), with no signal peptide. Thecementum protein-1 gene product is enriched in pro-line (11.3%), glycine (10.5%), alanine (10.1%), serine(8.9%), leucine (8.1%), threonine and arginine (each7.7%) and contains low levels of tryptophan, asparticacid, isoleucine (each 2.0%) and phenylalanine(1.6%). Tyrosine is not present. The amino-acidsequence indicates that the cementum protein-1 islikely to be a nuclear protein; however, it does nothave DNA-binding motifs. Amino acids 30–110 show48% similarity with the human collagen a I (I) chain,46% similarity with type XI and 40% similarity withtype X.

The full-length recombinant cementum protein-1expressed in human-derived gingival fibroblasts ismainly composed of beta-sheet with 10% alpha-helix,32.4% anti-parallel, 5.8% parallel, 16.7% beta-turnand 35% random coil. This feature is associated withproteins that have a high percentage of random coilstructure, which have been shown to be multifunc-tional and to have diverse binding properties; such

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proteins include SIBLING and HMGI(Y) (35, 41). Thismight help to explain why cementum protein-1 regu-lates crystal growth and composition of apatite crys-tals (4). According to in silico analysis, cementumprotein-1 possesses two N-glycosylation sites, namelyasparagine-X-serine in amino acids 20 and 25 – andthis is consistent with its shift from a protein size ofMr 50,000 to a protein size of Mr 39,000. The preciserole of attached carbohydrates in cementum protein-1 is unknown; however, glycosylation may affect thefunction of cementum protein-1 during the minerali-zation process because their anionic surface can binda large number of Ca2+ ions and regulate hydroxyapa-tite crystal growth (29). Glycans are also implicated inthe regulation of endochondral ossification, boneremodeling and fracture healing (66). Cementum pro-tein-1 appears to be a phosphorylated proteinbecause antibodies against phosphor-serine andphosphor-threonine cross-react with cementum pro-tein-1. The presence of phosphate also favors thebinding of Ca2+ to the protein (102, 209), and proteins(such as sialoprotein and osteopontin) associatedwith the mineralization process are highly phosphor-ylated at threonine and serine residues (236). Thus,cementum protein-1 may play a role at the earlystages of mineralization during the formation of octa-calcium phosphate (Fig. 1). The cementum protein-1does not react with sulfhydryl groups; therefore, allcysteine residues in cementum protein-1 might belinked to disulfide bridges, which generally play a rolestabilizing protein structure (87, 88, 106).

In a steady-state system with the concentrations ofcalcium and phosphate maintained below the thresh-

old of spontaneous precipitation, recombinanthuman-cementum protein-1 is effective in promotingthe nucleation of octacalcium phosphate in an aga-rose gel. It was calculated that as little as 1.0 lg/mL ofcementum protein-1 can promote nucleation (218).Human recombinant cementum protein-1 possesseshigh affinity for hydroxyapatite, even without post-translational modifications, and it affects the mor-phology of apatite crystals. Human cementum pro-tein-1 induces the formation of polymorphouscrystals, as confirmed by X-ray diffraction. Elementalanalysis performed with energy-dispersive X-rayanalysis identified a calcium/phosphorus ratio of 1.4,which corresponds to octacalcium phosphate (Fig. 2).These findings indicate that biologically activecementum protein-1 plays a role during the biomin-eralization process and is required for the synthesis ofneedle-like shape crystals. Octacalcium phosphate isfound to be a transient phase during the growth ofbiological crystals. In small crystals, octacalciumphosphate is completely transformed into hydroxyap-atite by hydrolysis and can only be detected in largecrystals because of its slow kinetics of transformation.Octacalcium phosphate has also been presumed tobe a necessary precursor of biological apatites.

Further characterization of cementum protein-1using western blots with extracted proteins fromhuman cementoblastoma and antibody to periodon-tal ligament-derived cells showed the presence ofthree components: 55-, 50- and 26-kDa species. Theseproducts might represent differences in the degree ofpost-translational modifications, particularly phos-phorylation and glycosylation (5). In vitro studies

A B

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Fig. 1. Scanning electron micro-graph images of crystal growth. (A)Human recombinant cementumprotein-1 (20 lg in a 0.5% agarosegel) induced the formation of asphere with irradiating prismaticcrystals. (B) Enlargement of the boxin panel A shows the octacalciumphosphate prismatic crystals. (C)The internal part of the sphereshows a central nucleus with irradi-ating prismatic crystal. (D) A moredetailed view of the box in panel Cshows that the irradiating crystalsare originating from a centralnucleus in a needle-like shape thatlater acquires the prismatic crystalmorphology that grows only incementum protein-1-containinggels.

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using immunocytochemistry confirmed the expres-sion of cementum protein-1 by cementoblastoma-derived cells and periodontal ligament cells. Almostall (95%) of the cementoblastoma-derived cell popu-lation was positive for cementum protein-1, whereasonly 6% of periodontal ligament cells stained positivefor cementum protein-1. Cementum protein-1 wasalso expressed in a small population (3%) of osteo-blastic cells in vitro, whereas cementum protein-1was not detected in gingival fibroblasts. These smallperiodontal ligament-positive and osteoblast-positivepopulations could represent cementoblast precur-sors, suggesting that cementoblasts and osteoblastsmight have a common ancestor and that cementumprotein-1 could be a marker for the cementoblasticlineage (10). Immunohistochemistry studies using

human periodontal tissues showed localization ofcementum protein-1 throughout the entire cementumsurface, including the cementoid phase of acellularand cellular cementum, cementocytes and cells nearthe blood vessels in the periodontal ligament (Fig. 3).These cells are considered as cementum progenitorcells (7, 8, 10). Cementum protein-1 is not expressed inany other human tissues, indicating that cementumprotein-1 is a tissue-specific protein, restricted to ce-mentoblasts and its progenitor cells, and that it mighthave a role as a local regulator of cell differentiationand extracellular matrix mineralization (4).

An interesting observation was the fact thatcementum protein-1 cross-reacts with antibodies totype X collagen. Collagen type X is a product of thehypertrophic chondrocytes and facilitates endochon-dral ossification (115, 193), which suggests some rela-tionship among cementum protein-1,chondrogenesis and the mineralization processes.This hypothesis is supported by studies showing thathuman cementum extracts promote attachment ofchondrocytes in a dose–dependent manner. Further-more, mesenchymal bud stem cells grown in thepresence of cementum extracts result in the forma-tion of Alcian blue-positive nodules, which synthe-size sulfated proteoglycans (8). The expression ofproteoglycans rich in chondroitin sulfate is charac-teristic of cartilage (28, 40, 185, 211, 221). Takentogether, these studies suggest that cementum-spe-cific proteins can induce stem cells to express a carti-lage phenotype. Furthermore, cementum functionsnot only as an inducer of cell differentiation but alsoas an inducer of proliferation (8).

The function of cementum protein-1 was furthertested by transfection into nonmineralizing cells,such as human gingival fibroblasts. In contrast tonormal human gingival fibroblasts, human gingival fi-broblasts/cementum protein-1 cells showedincreased proliferation, formation of mineralizednodules, increased alkaline phosphatase-specificactivity and the de novo expression of osteocalcin, os-teopontin, sialoprotein, runt-related transcriptionfactor 2/core-binding factor alpha1 and cementumattachment protein mRNAs and protein. These mole-cules are all associated with bone/cementum forma-tion (27). The studies strongly support the notion thatcementum protein-1 has the ability to change the cellphenotype from nonmineralizing (human gingival fi-broblasts) to mineralizing (osteoblast/cementoblast)by regulating proliferation and gene expression,resulting in the differentiation of these cells and theproduction of a mineralized extracellular matrixresembling cementum.

A

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Fig. 2. (A) Representative energy-dispersive X-ray micro-analysis spectrum of the crystals formed as a result of theeffect of human recombinant cementum protein-1 in asteady-state agarose system. The spectrum shows promi-nent peaks of calcium (Ca) and phosphorus (P) and theCa/P ratio indicates that the crystals are octacalcium phos-phate. 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 deter-mined by tapping mode atomic force microscopy in air.

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In-vitro studies have shown that periodontal liga-ment cells can form alkaline phosphatase-positiveand alkaline phosphatase-negative colonies and thatthe alkaline phosphatase-positive periodontal liga-ment cells also express higher levels of mineraliza-tion-related genes (sialoprotein and osteocalcin) thando the alkaline phosphatase-negative cells. Thesedata suggest that alkaline phosphatase-positive peri-odontal ligament cells include osteoblast and/or ce-mentoblast subsets (141). It has been found thatcementum protein-1 is preferentially expressed inalkaline phosphatase-positive cells and that theexpression of cementum protein-1 is reduced whenperiodontal ligament cells are cultured under osteo-genic conditions (i.e. with either bone morphogeneticprotein-2 or in osteogenic induction medium) (113).Overexpression of cementum protein-1 increases theexpression of cementum attachment protein in peri-odontal ligament cells at both mRNA and protein lev-els. The mechanism by which cementum protein-1regulates the expression of cementum attachmentprotein in periodontal ligament cells is not clear.However, the expression of cementum protein-1decreases when cells are committed to osteoblastic/chondroblastic lineages, which suggests that theexpression of cementum protein-1 is being differen-

tially regulated in osteoblasts and cementoblasts andthat knockdown of cementum protein-1 expressionin periodontal ligament cells only affects sialoproteinexpression in cementoblasts, suggesting that cemen-tum protein-1 is associated with the regulation ofsialoprotein expression in cementoblasts (113). Wehave reported the nuclear staining of cementum pro-tein-1 in cementoblasts, whereas periodontal liga-ment cells exhibit intense staining of cementumprotein-1 only in the cytoplasm, indicating that thesubcellular location of cementum protein-1 couldchange during the cementoblastic differentiation ofperiodontal ligament cells. Overexpression of cemen-tum protein-1 in periodontal ligament cells down-regulates periodontal ligament cell markers such asPLAPI/asporin, and increases cementoblast markerssuch as cementum attachment protein and sialopro-tein. These data indicate that cementum protein-1could select periodontal ligament cells, or progenitorcells present in the periodontal ligament, to differen-tiate toward the cementoblastic phenotype (113).

The ability of cementum protein-1 to induce peri-odontal ligament cells to differentiate toward ce-mentoblast/osteoblast and/or chondrogenic-likephenotypes was also tested using a three-dimensionalcell-culture system. Under those conditions, cemen-

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Fig. 3. Double immunostaining of human periodontal tis-sues with cementum proteins. (A) Immunostaining showsthat anti-bovine CAP IgG1 (a-CAP) cross-reacts with ce-mentoblasts (CB) paravascular cells (PVC) and cell sub-populations in the periodontal ligament (PL). (B)Cementum protein-1 antibody (a-CEMP1) cross-reactsstrongly with CB. (C) Double immunostaining shows thatanti-CAP IgG1 and anti-CEMP1 serum are expressed by CBand 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 mono-clonal antibody (a-AMEL). (E) The CEMP1 gene product isexpressed by the ERM and CB. (F) Double immunostainingwith a-AMEL and a-CEMP1 show that these proteins areexpressed by similar structures such as the ERM and CB.AB, alveolar bone; BV, blood vessel; CB, cementoblasts;PVC, paravascular cells.

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tum protein-1 stimulated periodontal ligament cellsto proliferate in a three-dimensional globular mate-rial with morphological features and staining charac-teristics of cartilage and cementum/bone-like tissues.Cementum protein-1 induced periodontal ligamentcells, cultured in a three-dimensional system, toexpress type II collagen and aggrecan (both markersfor pre-hypertrophic chondrocytes) at mRNA andprotein levels (36, 91, 140). Expression of type X colla-gen, a marker of fully differentiated chondrocytes(117), was also increased by the addition of cemen-tum protein-1 to the three-dimensional periodontalligament cell cultures. The expression of SRY (sexdetermining region Y)-box 9, a transcription factorthat mediates chondrogenic differentiation (2, 17), incells grown in the presence of human recombinant-cementum protein-1 suggests that cementum pro-tein-1 promotes chondrogenic differentiation (91).Cementum protein-1 shows sequence similarity withtype X and XI collagens and is immunologicallyrelated to type X collagen (5). It is conceivable thatcementum protein-1 plays a role during the minerali-zation of hypertrophic cartilage and facilitates endo-chondral ossification. Periodontal ligament cells showa multilineage potential to differentiate toward osteo-genic, chondrogenic and adipogenic phenotypes by asignificant up-regulation of cartilage marker genesand osteoblastic differentiation markers (49, 192). Itseems possible that cementum protein-1 exerts a dif-ferentiation role on the periodontal ligament cellpopulation by selecting multipotent stem cells, whichprovide a unique reservoir, to differentiate into vari-ous cell phenotypes (229), or as an inducer of the het-erogeneous periodontal ligament cell populationhaving a differential effect on cells at various degreesof differentiation. This also explains the expression ofcementum protein-1 in periodontal ligament cell sub-populations representing precursors of cemento-blasts or osteoblasts, or both (5, 10, 64). Additionally,there is a direct correlation between the capacity ofhuman periodontal ligament-derived cells to bind toa cementum protein and produce mineralized-liketissue in vitro (11). It has been shown that cementumprotein-1 increases the activity of alkaline phospha-tase. High levels of alkaline phosphatase are alsoassociated with hypertrophic cartilage and bone for-mation, and alkaline phosphatase is considered to bea marker for chondrocyte differentiation because itsactivity appears to be increased during chondrocytehypertrophy (213). The expression of cartilage-forma-tion markers in these cultures suggests that periodon-tal ligament cells have the potential to differentiateinto chondrocytes and then progress rapidly to matu-

ration and bone formation, as suggested by theexpression of collagen type X and sialoprotein.

Studies on cementum regeneration, using a dogmodel for dental pulp necrosis, demonstrated theability of cementum protein-1 to recruit mesenchy-mal stem cells from the periodontal ligament and topromote the proliferation and mineralization of thesecells. In vivo studies co-localized cementum protein-1 and STRO-1 (a marker of mesenchymal stem cells)positive cells adjacent to the root-surface areas whereneocementum is deposited, indicating that the cellsresponsible for reparative cementum deposition areof mesenchymal origin (164). In vitro, cementum pro-tein-1 promotes the proliferation and the migrationof periodontal ligament cells, with the migration frontcomprising STRO-1-positive cells. These studies sug-gest that cementum protein-1 is a mediator in woundhealing and periodontal regeneration because it stim-ulated the proliferation and migration of periodontalligament cells. Cementum protein-1 promotes themigration of STRO-1-positive cells and provides apossible mechanism for the recruitment of mesen-chymal cells through migration toward the cemen-tum protein-1 signal (164). The role of cementumprotein-1 as a chemoattractant and as a promoter ofmineralization is further supported by the findingsthat mineralization is reduced upon blocking cemen-tum protein-1 function in vitro. In cementoblastoma-derived cells, blocking cementum protein-1 activitydecreases alkaline phosphatase activity and theexpression of sialoprotein and osteopontin, but doesnot alter cell proliferation (4). These studies alsoshowed that extracellular calcium increases theexpression of cementum protein-1 and PTPLA/cementum attachment protein in periodontal liga-ment stem cells via the mitogen-activated proteinkinase signaling pathway. This has been confirmed byblocking extracellular signal-regulated kinase-1 andextracellular signal-regulated kinase-e using smallinterfering RNA, which down-regulates the expressionof PTPLA/cementum attachment protein and cemen-tum protein-1. Furthermore, the use of calcium chan-nel blocker prevented the expression of cementumprotein-1 and PTPLA/cementum attachment protein,demonstrating the role for calcium ions in cemento-genesis (164). To date, molecules responsible forrecruiting mesenchymal cells and inducing their dif-ferentiation into cementoblasts have not been identi-fied. These studies suggest that cementum protein-1could be one of the molecules.

In summary, the data presented above stronglyindicate that cementum protein-1 is a unique proteinthat has multiple properties as inducer of mineraliza-

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tion, proliferation, differentiation and cell matura-tion. Additionally, cementum protein-1 might serveto regulate the mesenchymal stem-cell pool presentin the periodontal ligament and to induce its differen-tiation into different pathways. These properties openthe possibilities of creating cementum protein-1-based therapies for periodontal regeneration.

Enamel-associated proteins in cementum

Many years ago, Slavkin & Boyde (196), proposed ahypothesis that Hertwig’s epithelial root sheath-derived extracellular matrix proteins might be relatedto tooth-crown-derived enamel proteins and thatthese enamel-related proteins might initiate acellularcementum formation. Several human and mousecementum proteins were found to be immunologi-cally related to amelogenin and enamelin. These pro-teins represented species of 72 and 26 kDa that weresecreted by Hertwig’s epithelial root sheath cells(197). A few years later, it was demonstrated that a-meloblastin, an enamel-associated protein, isexpressed by epithelial cells covering the first thinlayer of unmineralized root mantle dentin, and astrong signal is expressed in cells enclosed in the cel-lular cementum known as the epithelial cell rests ofMalassez and in cells in a more coronal position at

the root surface forming acellular cementum (43, 44)(Fig. 4). However, the presence of enamel proteins,especially amelogenins, during root development hasbeen the subject of controversy. Some investigatorsreported the expression of amelogenins by the apicalcells of Hertwig’s epithelial root sheath, whichsecreted small amounts of amelogenins during earlydifferentiation of root development (101), whereasothers did not detect the expression of amelogenin inthese cells (119), only ameloblastin (238). Neverthe-less, a new therapeutic approach, using enamelmatrix derivative to achieve periodontal regeneration,was born, based on the assumption that enamelmatrix proteins synthesized by cells of the Hertwig’sepithelial root sheath could trigger the differentiationof follicle cells into cementoblasts. Specifically, it hasbeen postulated that amelogenin induces the forma-tion of acellular extrinsic fiber cementum. However,others suggest that the tissue formed by treatmentwith enamel matrix derivative results in the formationof a cellular cementum-like tissue or bone with thecharacteristics of cellular intrinsic fiber cementum(23, 24). The bone-like appearance of this tissue is inline with the chondrogenic/osteogenic activity ofenamel matrix derivative (24, 215). Numerous studieshave suggested that enamel matrix derivative can

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

product. (C) Cells, possibly cementoblasts (CB), facing theroot surface express CEMP1 and CEMP1 is strong expres-sion of at the cemento–enamel junction. (D) Hertwig0s epi-thelial root sheath (HERS) cells express CEMP1. (E)Hematoxylin and eosin (H&E) staining for orientation. (F)Cells (CB) facing cementum throughout the length of theroot express CEMP1. (G) H&E staining for orientation. AB,alveolar bone; E, enamel; PL, periodontal ligament.

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have multiple functions, such as the promotion of cellproliferation, differentiation and up-regulation ofextracellular matrix production (36, 50, 51, 83, 86, 139,159, 188, 195, 233). Also, several reports have providedfurther evidence that enamel matrix proteins may beinvolved in root formation (20). Furthermore, amelo-genin null mice showed defects in cementum and adecreased expression of sialoprotein along the rootsurface (57, 219). This is of particular importancebecause sialoprotein acts as a regulator of the miner-alization process in cementum (47, 59).

Amelogenins are the expression products of X andY chromosomal genes and give rise to multiplespliced variants (235). One of these alternativelyspliced variants is a leucine-rich amelogenin peptide(A-4) which has been demonstrated to increase theexpression of osteopontin, sialoprotein and osteopro-tegerin, and to decrease the expression of osteocalcin,in cementoblasts (208, 215, 216). Amelogenin and a-meloblastin can act as signaling molecules in theperiodontal ligament; they have an effect on attach-ment (237) and proliferation of these cells in vitro.Both proteins have a modulatory role and down-regulate the expression of type I collagen, whilstinducing the de novo expression of osteocalcin.Amelogenin also induced the expression of sialopro-tein in periodontal ligament cells, indicating that thisprotein can induce phenotypic changes in these cells(239). Amelogenin has been suggested to have biolog-ical effects on cells of mesenchymal origin, such asperiodontal ligament and gingival fibroblasts, andenamel matrix derivative enhances the growth ofhuman bone marrow stromal cells (68, 93, 215). Inthe past, amelogenin has been shown to be expressedby odontoblasts, periodontal ligament cells, Hertwig’sepithelial root sheath cells and cementoblasts (23, 44,46, 70, 72, 78, 79, 155, 162). Others have described theexpression of amelogenin in hematopoietic stemcells, macrophages and megakaryocytes, rat brainand myoepithelial cells, suggesting a regulatory rolefor amelogenin in the recruitment and differentiationof monocytic cells from the bone marrow towardbecoming mineralized tissue-resorbing cells (boneand cementum osteoclasts/cementoclasts). Amelo-genin, a major structural protein in mineralizingenamel, is also expressed in brain tissue and cells ofthe hematopoietic system (39). Progressive deteriora-tion of cementum is observed in amelogenin knock-out mice and is characterized by the increasedpresence of osteoclasts (78, 79). On the other hand,RT-PCR and western blotting results have demon-strated that Hertwig’s epithelial root sheath cells invitro do not synthesize amelogenin or enamelin, but

they do synthesize ameloblastin. These studiesshowed that Hertwig’s epithelial root sheath cellschange their morphology and produce Von Kossa-positive nodules coincidental with the expression ofdentin matrix protein-1, sialoprotein and osteocalcin,and high levels of alkaline phosphatase activity.Transmission electron microscopy comparison of themineralized extracellular matrix deposited by Her-twig’s epithelial root sheath cells in vitro with acellu-lar cementum deposited in vivo suggests that thisextracellular matrix might be cementum (238).

Hertwig’s epithelial root sheath cells synthesizecementum attachment protein and cementum pro-tein-1 (Fig. 4), thus supporting the idea that Hertwig’sepithelial root sheath cells are capable of producingcementum along with inducing a high activity of alka-line phosphatase. This finding provides further evi-dence that the extracellular matrix deposited by thesecells is acellular cementum, indicating that alkalinephosphatase is a very important component of acel-lular cementum. Hertwig’s epithelial root sheath cellsexpress osteocalcin in vitro, thus indicating the possi-bility that disruption of the basement membrane iscaused by Hertwig0s epithelial root sheath cells whenthey start depositing the acellular cementum. Thesestudies suggested different cellular origins for acellu-lar (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 popula-tion of epithelial cells in the periodontal ligament andare believed to play a crucial role in cementum repair(203). Hertwig’s epithelial root sheath cells/epithelialcell rests of Malassez could differentiate into ce-mentoblasts through epithelial–mesenchymal trans-formation (202). Recently it was demonstrated that invitro Hertwig’s epithelial root sheath/epithelial cellrests of Malassez contain primitive stem cells thatexpress epithelial stem-cell markers such as octamer-binding transcription factor 4, homeobox proteinNANOG and stage-specific embryonic antigen 4(147). These cells might function in creating the bor-der between the ameloblasts and the proliferativeregion of Hertwig’s epithelial root sheath (145) andmight contribute to the formation of cementum and/or enamel repair (76, 194). More recently it wasshown that the epithelial cell rests of Malassez sharesimilar phenotypic and functional characteristics withmesenchymal stem cells and are capable of develop-ing into osteoblasts, adipocytes, chondrocytes andneuron-like cells in vitro (228), similar to thatdescribed in vitro for periodontal ligament stem cells(192, 204). These studies suggest that the epithelial

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cell rests of Malassez are epithelial stem cells with theability to differentiate into epithelial or mesenchymalcells and play a critical function in periodontalrepair/regeneration.

Dental follicle cells, before the onset of root forma-tion, do not express cementum attachment protein,cementum protein-1 or sialoprotein, which suggeststhe absence of differentiated cells. However, dentalfollicle cells are positive for cementum attachmentprotein and cementum protein-1 when stimulatedwith enamel matrix derivative or bone morphoge-netic protein-2/-7, and their expression was reducedwhen treated with recombinant human-Noggin, awell-known inhibitor of bone morphogenetic proteinactivity (108). Some dental follicle cells express STRO-1, indicating that populations of the dental folliclehave mesenchymal progenitor features (108). Severalinvestigators suggest that the use of enamel matrixderivative enhances the expression of mineralized tis-sue markers (such as alkaline phosphatase) and nod-ule mineralization in dental follicle cells along withthe expression of bone morphogenetic protein-2 andsialoprotein (108). As enamel matrix derivative alsoinduces expression of cementum attachment proteinand cementum protein-1, it is suggested that enamelmatrix derivative promotes the differentiation of den-

tal follicle cells to a cementoblast phenotype ratherthan to an osteoblast phenotype. Recently it wasreported that cementum attachment protein andcementum protein-1 are stringently regulated duringthe cementogenesis process and root formation, andthat expression of cementum attachment protein isinduced more strongly by runt-related transcriptionfactor 2 than is cementum protein-1 (161). Runt-related transcription factor 2 is an important tran-scription factor for osteogenesis and cementogenesis,and is present in the early proliferative osteoblast/ce-mentoblast cell phenotype, a developmental stage atwhich cell proliferation is still required to obtain asufficient number of committed cells for matrix for-mation. Higher expression of cementum attachmentprotein at this early stage of cementogenesis is there-fore consistent with its function of promoting cellproliferation (227). In contrast, cementum protein-1has a more important role during the mineralizationprocess and its relationship is focused at this earlystage to control the mineralization process during ce-mentogenesis (218). Recently we reported that nor-mal human-derived cementoblasts expresscementum attachment protein, cementum protein-1and amelogenin and that they are localized to the cellnucleus. Human cementoblasts express not only

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Fig. 5. (A) Hematoxylin and eosin staining showing theperiodontal ligament (PL), cementum (CEM) and alveolarbone (AB). Blood vessels (BV) along with the putative pro-genitor cells of CEM are located close to the CEM surface.(B) Cementoblasts (CB) are ordered in three to four layersof cells facing the CEM surface. (C) The epithelial cell restsof Malassez (ERM) are located in the vicinity of the CB celllayers and the BV, indicating a possible inter-relationshipregulating CEM metabolism and differentiation of CEM

progenitor cells. (D) Ameloblastin (AMBM), an enamel andcementum-related protein, is shown to be expressed by asingle layer of CB facing the CEM surface and paravascularprogenitor cells (PVC) into the PL. (E) A pan-cytokeratinmonoclonal antibody (a-pCK) cross-reacts with CB andsubpopulations of PL cells, possibly supporting the state-ment that CEM is an epithelial product. (F) Cementumattachment protein (CAP) is expressed by subpopulationsof ERM and the CB facing the CEM surface.

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amelogenin but also other enamel-associated mole-cules, such as ameloblastin, enamelin and tuftelin(Fig. 5). Furthermore, cementum protein-1 inducesthe de-novo expression of amelogenin in periodontalligament cells grown in culture. Perhaps cementumprotein-1 promotes the osteoblast/cementobast phe-notype in periodontal ligament cells through theexpression of amelogenin (91, 152, 153). These data,taken together, suggest that enamel-associated pro-teins and cementum proteins could act synergisticallyin the regulation of cementoblast differentiation andcementum deposition. These data offer newapproaches to determine how the process of ce-mentogenesis is regulated and point out the role ofthese proteins during periodontal homeostasis andrepair/regeneration of periodontal structures, andcould represent new and better therapeuticapproaches for the treatment of periodontal disease.

Conclusions

Detailed knowledge of the biology of cementum iskey for understanding how the periodontium func-tions, identifying pathological issues and for develop-ing successful therapies for repair and regenerationof damaged periodontal tissue. Of particular impor-tance is the regeneration of cementum, a highlysophisticated and complex system, and the intercon-nection of this unique tissue with the neoformationof other periodontal tissues. In this review we havehighlighted the recent advances in our understand-ing of the so-called ‘cementum proteins’ – PTPLA/cementum attachment protein and cementum pro-tein-1 – and their possible role in selecting periodon-tal stem cells, inducing their differentiation andregulating the biological mineralization process asso-ciated with cementum formation. Also, this reviewpoints out the synergistic role of enamel-associatedproteins and cementum proteins in these processes.Although there have been tremendous advances inour 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 theseproteins. We are already in a position to explore newalternatives for regenerative techniques based uponthe principles of tissue-engineering methods. Theseproteins and related peptides with biological activityhold great therapeutic potential for the regenerationof not only periodontal structures but also of othermineralized tissues. Nevertheless, this is only thebeginning of our understanding of how these pro-teins might modulate tissue responses in relation to

the regeneration process. What may be concludedfrom the current status of the ‘cementum proteins’is that they can pave the way to establish effectivetherapeutic alternatives to achieve the regenerationof the periodontal structures, and that the impactthat they could have in the field of periodontologyand skeletal tissues looks very promising.

Acknowledgments

This work was supported by DGAPA-UNAMIN216711, IT200414 and by CONACYT 130950. Theauthors are grateful to Professor A. Sampath Naraya-nan of the University of Washington for critical read-ing of the manuscript.

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