Braz J Oral Sci. January/March 2005 - Vol.4 - Number 12

Dental reviewed: development, structure, composition, regeneration and potential functions Patricia Furtado Gonçalves 1 Enilson Antonio Sallum 1 Abstract Antonio Wilson Sallum 1 This article reviews developmental and structural characteristics of Márcio Zaffalon Casati 1 cementum, a unique avascular mineralized tissue covering the root Sérgio de Toledo 1 surface that forms the interface between root and periodontal Francisco Humberto Nociti Junior 1 . Besides describing the types of cementum and 1 Dept. of Prosthodontics and Periodontics, , attention is given to recent advances in scientific Division of Periodontics, School of understanding of the molecular and cellular aspects of the formation at Piracicaba - UNICAMP, Piracicaba, São and regeneration of cementum. The understanding of the mechanisms Paulo, Brazil. involved in the dynamic of this tissue should allow for the development of new treatment strategies concerning the approach of the root surface affected by and periodontal regeneration techniques.

Received for publication: October 01, 2004 Key Words: Accepted: December 17, 2004 dental cementum, review

Correspondence to: Francisco H. Nociti Jr. Av. Limeira 901 - Caixa Postal: 052 - CEP: 13414-903 - Piracicaba - S.P. - Brazil Tel: ++ 55 19 34125298 Fax: ++ 55 19 3412 5218 E-mail: [email protected]

651 Braz J Oral Sci. 4(12): 651-658 Dental cementum reviewed: development, structure, composition, regeneration and potential functions

Introduction junction (Figure 1). The areas and location of acellular Cementum is an avascular mineralized tissue covering the afibrillar cementum vary from to tooth and along the entire root surface. Due to its intermediary position, forming of the same tooth6-9. Its major the interface between root dentin and periodontal ligament, structural organic components are glycosaminoglycans6, cementum is a component of the tooth itself, but belongs and its functional significance is unknown. The lack of functionally to the dental attachment apparatus, that is, fibrils indicates that this cementum variety has no the . One of the main functions of cementum function in tooth attachment. is to anchor the principal collagen fibers of the periodontal ligament to the root surface, but it also has important adaptative and reparative functions, playing a crucial role to maintain occlusal relationship and to protect the integrity of the root surface. Until recently, knowledge of the development and the general structure of the periodontium was limited to morphological information, although, during the last decade, rapid progress has been made in understanding the molecular and cellular biology of periodontal tissue development and regeneration, including cementum. Dental cementum is unique in various aspects: it is avascular and not innervated, does not undergo continuous remodelling like , but continues to grow in thickness 1 throughout life . In contrast with these specific histological Fig. 1. Acellular cementum (arrow). Notice that acellular afibrillar characteristics, it appears not to be specific at the cellular cementum overlaps the cervical end of the enamel. This is the most and molecular level2. Unlike dentine and enamel, where there common relationship between the two tissues. Unstained, 100x are clear differences in the proteins present in these tissues and the factors regulating their functions when compared Cellular intrinsic fiber cementum contains cementocytes with bone, cementum has not demonstrated to express embedded in a collagenous matrix of intrinsic collagen specific proteins, appearing to contain factors in common fibers (Figures 3 and 4). These collagen fibers are oriented with bone and to be developmentally controlled by similar mostly parallel to the root surface and course in a circular factors2-3. fashion around the root6. A fast matrix deposition by the There is accumulating histological evidence that cementum , which occurs in the space between is critical for appropriate maturation of the periodontium, deviating epithelial cells of Hertwig´s root sheath and the both during development and as well as that associated dentinal surface appears to be the reason for the with regeneration of periodontal tissues3. Recent studies incorporation of some of the cementoblasts10-11. Cellular have contributed to an understanding of the possible intrinsic fiber cementum is found in the furcation, on the involvement of some of the molecular factors in cementum apical root portion, in old resorption lacunae and in root regeneration4, but cementogenesis, on a biological fracture sites, playing an important role as an adaptative basis, continues to be poorly understood. The aim of this tissue that brings and maintains the tooth in its proper chapter is to give a comprehensive review about some position and also participating in the repair process, important aspects of this unique tissue, including its although it has no immediate function in tooth varieties, development, structure, composition, attachment1,6. Only cellular intrinsic fiber cementum can regeneration and potential functions. repair a resorptive defect of the root in a reasonable time, due to its capacity to grow much faster than any other Types of cementum type of cementum10. Traditionally, cementum has been classified as cellular and Acellular extrinsic fiber cementum is mainly found on acellular, depending on the presence or absence of cervical and middle root portions, covering 40% to 70% of cementocytes in its structure. Another classification the root surface. On anterior teeth, it may also cover part of includes intrinsic or extrinsic fiber cementum, depending the apical root portion, since its apical extension increases on the presence of collagen fibers formed by cementoblasts from posterior to anterior teeth. It serves the exclusive or by , respectively5-6. Mainly three cementum function of anchoring the root to periodontal ligament. The types differing in these aspects are distinguished in humans. acellular extrinsic fiber cementum matrix consists of a dense Acellular afibrillar cementum covers minor areas of the fringe of short collagenous fibers that are implanted into enamel, particularly at and along the cementoenamel the dentinal matrix (glycosaminoglycans) and are oriented 652 Braz J Oral Sci. 4(12): 651-658 Dental cementum reviewed: development, structure, composition, regeneration and potential functions

about perpendicularly to the root surface6. When they become elongated and eventually continuous with the principal periodontal ligament fibers they are called Sharpey´s fibers. Although acellular extrinsic fiber cementum is a quite constantly growing tissue, appositional lines (Figure 2) represent the periodical deposition of cementum layers in frequent association with an abrupt change in the direction of Sharpey´s fibers12. Moreover, as can be deducted from faster growth rates on distal (4.3mm/year) than on mesial (1.4mm/year) root surfaces13, acellular extrinsic fiber cementum has the potential to adapt to functional alterations such as mesial tooth drift. In humans, the extrinsic fibers traverse and intermingles the intrinsic fiber cementum variety either sporadically or densely arrayed in parallel; this mixed Fig. 3b: Higher magnification of previous image. Notice that the cementum is referred to as cellular mixed stratified canaliculi of cementocytes (*) have a tendency to be polarized and extend toward the periodontal ligament side of the root. Unstained, 400x cementum6.

Origin and development of cementum Based on the observation that populations of cementoblasts are phenotipically distinct, some authors defended the possibility that acellular and cellular cementum have different developmental origin14. Some studies15-16 suggested that cementoprogenitor cells arise from the proper, which is of ectomesenchymal origin (that is, a derivative of the cranial ). However, as pointed out by Thomas & Kollar17, labeled cementoblasts could also be of epithelial origin, since cells of the , which give rise to HERS, also incorporate 3H- thimidine prior to transplantation in mice. More recent ultrastructural and immunohistochemical studies support, Fig. 2: Acellular cementum. At a higher magnification, notice indeed, the hypothesis that cementoblasts originate from that this type of cementum has incremental lines of growth (arrow) which are oriented parallel to the root surface. Tomes’ granular epithelial cells of HERS when they undergo an epithelial- layer (*)and dentinal tubules (thin arrow) are also visible in the mesenchymal transformation18-20. There is increasing field. evidence that Hertwig’s is actively involved in the formation of both acellular and cellular cementum. Diekwisch21 (2001), published an extensive literature review on early cementogenesis and performed a detailed morphological and molecular analysis to illustrate and verify key issues in the current debate about epithelial and mesenchymal contributions to root cementum. The author demonstrated that prior to cementogenesis, Hertwig’s epithelial root sheath disintegrates and dental follicle cells penetrate the epithelial layer to invade the root surface. Other studies confirmed that HERS became disrupted or disintegrated prior to cementum deposition, and visualized how mesenchymal cells from the dental follicle penetrated the HERS bilayer and deposited initial cementum, while immediately adjacent epithelial cells were separated from Fig. 3a: Cellular cementum (arrow). It is characterized by the presence of lacunae with canaliculi in which reside cementocytes. the root surface by a basal lamina and did not secrete any However, the cells are absent in ground section preparations. cementum22-23. Human specimen from the Gottlieb collection Unstained, 40x indicated that HERS was removed from the root surface 653 Braz J Oral Sci. 4(12): 651-658 Dental cementum reviewed: development, structure, composition, regeneration and potential functions

prior to cementum deposition. In situ hybridization and from the same teeth30-31, although not without exception32. immunolocalization data revealed that both amelogenin Acellular extrinsic fiber cementum appears more highly mRNAs and enamel proteins were restricted to the mineralized than cellular intrinsic fiber cementum and enamel and were absent from the root surface and from the cellular mixed stratified cementum33, what can in part be cervical-most adjacent to the root margin24-26. explained by the presence of uncalcified spaces, such as On Western blots, cementum protein extracts did not cross- lacunae and by the uncalcified core of Sharpey’s fibers. react with amelogenin antibodies25. These studies in Chemical analysis and physicochemical studies indicate conjunction with a literature review together confirmed the that the mineral component is the same as in other calcified

theory of cementum as a dental follicle derived connective tissues; that is, hydroxyapatite (Ca10(PO4)6(OH)2), with small tissue that forms subsequent to HERS disintegration. amounts of amorphous calcium phosphates present34. Due Briefly, cementogenesis begins with the deposition of a to its lower crystallinity of the mineral component matrix on the dentin surface by Hertwig’s epithelial root (compared to other hard tissues), cementum has a greater sheath, disruption of the HERS, migration and organization capacity for adsorption of fluoride and other elements over by ectomesenchymal cells from the , and their time but also more readily decalcifies in the presence of subsequent differentiation into cementoblasts25,27. The acidic conditions1. matrix they produce surrounds cells producing cementum, As in other hard tissues, the hydroxyapatite of cementum the cementoblasts, and the cementoblasts are generated is not pure, but contains other elements (ions) incorporated by differentiation of progenitor cells, which in turn are into mineral phase during mineralization, depending on their believed to arise from the dental follicle. concentration in the fluid environment. Over time, the The fate of HERS following the onset of cementogenesis is concentration may change by additional uptake or also controversial. Traditional thinking proposed that HERS substitution by other ions. Thus, cementum contains 0.5- disintegrated into small clusters and/or strands of epithelial 0.9% magnesium35-36, about half that in dentin, and it is cells that survived indefinitely in the periodontal ligament. lower at the surface than in deeper layers of cementum. More recent studies have suggested that epithelial cells of The significance of the low Mg+2 content in cementum the Hertwig’s root sheath undergo epithelial /mesenchymal remains obscure, but agrees with the notion that the transformation into fibroblasts and cementoblasts, that composition of cementum is more similar to bone tissue deposit acellular and cellular cementum, respectively1,28. The than to dentin. In contrast, the distribution of fluoride shows possibility that some epithelial cells of the root sheath the opposite trend. Cementum have a high fluoride content undergo epithelial/mesenchymal transformation and compared to other mineralized tissues (up to 0.9% ash subsequently secrete cementum matrix must be investigated weight), and this concentration shows a general increase further. There is evidence that cells of the inner layer of the with age and vary with fluoride supply to the individual, as root sheath become incorporated in cellular cementum or it is in bone, dentin and enamel as well37. Cementum also trapped between cementum and dentin during formation of contains 0.1-0.3% sulfur as a constituent of the organic the apical part of the root29. The only incontrovertible fact matrix38, and a number of trace elements may be present in is that many cells of the HERS retain an epithelial phenotype, concentrations detectable by electron microprobe analysis, and survive in periodontal ligament as the epithelial rests in particular Cu+2, Zn+2 and Na+2 36; however, their of Malassez1,6. distribution and significance do not seem to have been studied in detail. Composition Since cementum is not a uniform, mineralized connective Organic composition tissue, differences in the proportional composition of the The organic matrix of cementum is composed primarily of chemical constituents exist between the cementum varieties. . Type I collagen plays structural as well as Thus, the percentages of its chemical components may vary morphogenic roles and provides scaffolding for mineral from sample to sample, particularly in different species. crystals; it is the major component, accounting for 90% of Biochemical studies have shown that cementum has a similar all collagens. The type III collagen, which coats type I composition to bone. To about equal parts per volume, collagen fibrils, accounts for only 5%3. cementum is composed of water, organic matrix and mineral. Cementum contains two major non-collagenous proteins, About 50% of the dry mass is inorganic, and consists of bone sialoprotein (BSP) and osteopontin (OPN). Both are hydroxyapatite crystals. The remaining organic matrix phosphorilated and sulfated glycoproteins. These proteins, contains largely collagens, glycoproteins and proteoglycans1. which are prominently expressed in acellular extrinsic fiber cementum and acellular afibrillar cementum, bind tightly to Mineral composition the collagenous matrices and hydroxiapatite, and they Cementum is generally less mineralized than root dentin possess cell attachment properties through the Arg-Gly-Asp

654 Braz J Oral Sci. 4(12): 651-658 Dental cementum reviewed: development, structure, composition, regeneration and potential functions

(RGD) sequence, that binds to integrins39-40. Both proteins now considered to be an insulin-like growth factor1-like are expressed during early tooth root development by cells molecule, with similar properties to those of IGF-1, but along the root surface. Root surface cells express the BSP, larger than IGF-1 in molecular size51. The second molecule and it is also present in mature teeth. In contrast, OPN is is a collagenous protein referred to as cementum attachment present within the periodontal ligament region of the mature protein (CAP). Antibodies to CAP immunostain only teeth. These two proteins are believed to play a major role in cementum and not other periodontal components or other the differentiation of the cementoblasts progenitor cells to tissues52. In bovine tooth germs, the CAP is expressed by cementoblasts3. The BSP is believed to have adhesion cementoblasts, and in cementum its expression pattern is function to root surface cells and to participate in initiating different from that of type I collagen53. CAP promotes the mineralization. It is chemotatic to pre-cementoblasts and adhesion and spreading of mesenchymal cells; however, it promotes their adhesion and differentiation41. Many cells promotes the adhesion of mineralized-tissue-forming cells, express the OPN during periods of cementogenic activity. It preferentially54-55. regulates cell migration, differentiation, and survival through the interaction with integrins, and also participates in Cementum and Periodontal Regeneration inflammation by regulating monocyte- activation, Although the extent of injury and the amount of lost tissue phagocytosis, and nitric oxide production. In teeth and that must be filled are important determinants, whether a cementum, it may regulate biomineralization by at least two damaged tissue heals by regeneration or repair depends mechanisms: regulating bone cell differentiation and matrix on two crucial factors: the availability of needed cell types mineralization42. and the presence or absence of the cues, and signals Fibronectin43, which is believed to bind cells to the necessary to recruit and stimulate these cells. , and tenascin are present in HERS during One major goal of regenerative periodontal therapy is new differentiation, and later at the attachment site cementum formation and restoration of soft tissue of periodontal ligament to the root surface. Osteonectin, attachment to the cementum. This process requires osteocalcin and laminin are other matrix components found cementoblasts, and the origin of cementoblasts and the in cementum. Osteonectin is expressed by cementoblasts molecular factors regulating their recruitment and producing cellular extrinsic fiber cementum and are not fully understood. In vivo animal intrinsic fiber cementum44. Osteocalcin appears to be models to evaluate cementogenesis during tooth involved in the mineralization process45. The proteoglycans, development, the expression pattern of specific matrix small proteins that are widely distributed in mammalian molecules, and in vitro studies about the effects of species, are also present in cementum46. Biochemical analysis cementum components on periodontal cells have provided of extracts of human cementum have identified chondroitin important knowledge on how cementum components can sulfate, dermatan sulfate and hyaluronic acid47. regulate cementum regeneration3, 56-59. Although cementum The enzyme alkaline phosphatase is believed to participate formation in rodents differs from that in humans18, the in cementum mineralization. In rat molars, the enzyme is observation of Liu et al (1997)60 indicates that periodontal heterogeneously distributed in the periodontal ligament, ligament may be one source of cementoblasts progenitor with the highest activity being found adjacent to alveolar cells in adult humans. These investigators demonstrated bone and cementum. The enzyme activity adjacent to cellular that a small proportion of clones of cells cultured from intrinsic fiber cementum is higher than that to acellular human periodontal ligament form cementum-like mineralized extrinsic fiber cementum, and the thickness of the later nodules in culture, and also produce cementum-specific correlates positively with the enzyme activity48. markers61. Cementoblasts may also be derived from stem Several polypeptide growth factors with ability to promote cells present in the periodontal ligament, gingiva, and proliferation and differentiation of putative cementoblasts alveolar bone, when the pool of available progenitors is are found in cementum matrix. These include BMP-2, -3 likely to be reduced or absent; however, the molecules and –4, PDGF, α-and β-FGFs, TGFβ, PTH and IGF-13-4,49. responsible for recruiting and differentiating these cells While these molecules have been called growth factors, and the mechanisms involved remain to be better their role during tooth development does not appear to be investigated3. related to proliferative activity. In fact, some TGFbs and A variety of chemotatic factors, adhesion molecules, growth PTH-related protein may have a role in regulating cell factors, and matrix constituents participate together in the differentiation and subsequently mineralization49-50. recruitment of cementoblasts progenitors, their expansion It is important to know that many of these components are and differentiation. For example, cementum contains also present in bone; however, molecules unique to molecules that promote chemotatic migration, adhesion, cementum have also been described. One of these is an proliferation and differentiation of some periodontal cells IGF-1 isoform, referred to as cementum (CGF), types better than others, and these molecules are not

655 Braz J Oral Sci. 4(12): 651-658 Dental cementum reviewed: development, structure, composition, regeneration and potential functions

detectable in other periodontal structures51-52,55,61-63. proven cementum-like tissue following in vivo Adhesion molecules that cause negative selection by transplantation into immunodeficient mice44,72. excluding unwanted cells are also present in cementum54. One of the most important benefits of the establisment of Thus, the cementum microenvironment contains all the such combined in vitro/in vivo models is that they will allow components necessary for cell recruitment, proliferation and for elucidations of the relationship between differentiation, and molecules from the circulation are not and cementoblasts, besides providing more information necessary44. regarding the specific mechanisms involved in maintenance All these observations underscore the importance of of cementum structure and function in humans on the restoring or providing the cementum microenvironment to cellular and molecular level. Similarly, the in vitro/in vivo initiate and promote new cementum formation. The integrity system can also be used for further elucidation of the modes of cementum is altered by periodontal disease due to of action of current available regenerative products, such deposition of bacterial endotoxins64, and diseased as Emdogain, with apparent cementum-growth-promoting cementum is removed during periodontal therapy65. Dentin activities. not covered by cementum undergoes resorption. Root This knowledge probably will allow for the development of conditioning does not restore the original composition of strategies for regenerating cementum with cell-based cementum local environment, and instead expose molecules, therapies, by means of recruiting cells with cementoblastic especially type-I collagen, which has poor cell specificity65. potential, inducing their differentiation and excluding The application of some growth factors is not likely to unwanted osteoblastic precursors. Furthermore, the use of provide the complete repertoire of the needed molecules, these models allows for direct in vivo human since the concentration and type of growth factor on experimentation, which may be particularly important, cementum change continuously during the healing because cementum has been reported to differ between process66. Similarly, while barrier membranes can facilitate species in some aspects of its physiology9-11. In principle, population of the site by needed cells, they are not likely to cementogenic cells can be isolated from a relatively small provide the local environment for their differentiation67. specimen, expanded in culture, and subsequently Further, providing enamel proteins68, while likely to be transplanted to the same patient. Primary human conductive to early cementogenesis, may not provide cementoblasts can be efficiently grown from dissected appropriate environment for recruiting cementoblasts fragments of healthy root cementum treated with progenitors in adults and for their differentiation. Indeed, colagenase: the culture conditions are fairly standard and extracellular matrix components are expressed during it is possible to obtain large numbers (in the range of 108- periodontal healing67,69; however, whether all molecules are 109) of committed cementogenic cells from a single tooth44,72. expressed in adequate temporal sequence is not known. However, a significant drawback of this model is the All these factors together may explain why cementum requirement that healthy teeth be extracted for the cultures regeneration is not always predictable for the available to be established, since it is still not clear whether regenerative procedures. cementogenic cells can be obtained, by this approach, of Recent in vivo studies using rat periodontal defect models, aged and/or diseased patients. These drawbacks will have shown that both BSP and OPN are expressed by cells probably lead, in near future, to the investigation of sources linked to formation of mineralized tissues, while other than cementum for cell isolation (i.e.: periodontal osteopontin is also expressed by cells within the newly ligament, gingiva, bone marrow). formed periodontal ligament69. Future research directed at Once the cementogenic cells are selected and the molecules overexpressing or blocking expression of these molecules critical for cementum regeneration are identified, the next during periodontal wound healing should provide additional step may be to develop an efficient and relatively simple information required to establish the real function of these delivery system to be used for periodontal regeneration. molecules in mineralized tissues and also determine the This probably may be one of the major problems, since value of using such agents clinically for enhancing cementum is a mineralized “interface” tissue, connecting regeneration of periodontal tissues3. mineralized dentin and non-mineralized fibrous periodontal ligament – what implies that a successful delivery system Future directions on Cementum research should somehow reconcile these two distinct environments. The development of the cementum research in the cellular In this concern, molecules such as CAP show some and molecular fields is promising, since several laboratories promise60-62, due to its mineral-binding domain. Several have reported successful isolation and propagation of cells integrins as well as their natural ligands (e.g. collagens, (from both animal and human sources) exhibiting an BSP, OPN) are known to be expressed by cementoblastic apparent cementoblastic phenotype in vitro70-72. These cells cells67, implying that they may be important regulators of have been shown, reproducibly, to form histologically cementogenesis. This also indicates that targeting integrin 656 Braz J Oral Sci. 4(12): 651-658 Dental cementum reviewed: development, structure, composition, regeneration and potential functions

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