Human Cementoblasts Express Enamelassociated Molecules In

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J. NuÇez1, M. Sanz1, Human cementoblasts L. Hoz-Rodríguez2, M. Zeichner-David3, H. Arzate2 1Facultad de Odontología, Universidad express enamel-associated Complutense de Madrid, EspaÇa, 2Laboratorio de Biología Periodontal y Tejidos Mineralizados, Facultad de Odontología, Universidad Nacional molecules in vitro and Autónoma de MØxico, MØxico and 3Center for Craniofacial Molecular Biology, University of in vivo Southern California, Los Angeles, CA, USA

Nun˜ez J, Sanz M, Hoz-Rodrı´guez L, Zeichner-David M, Arzate H. Human cementoblasts express enamel-associated molecules in vitro and in vivo. J Periodont Res 2010. doi: 10.1111/j.1600-0765.2010.01291.x. Ó 2010 John Wiley & Sons A/S

Background and Objective: Cementum is a mineralized tissue that facilitates the attachment of periodontal ligament to the root and surrounding alveolar bone and plays a key role in the regeneration of periodontal tissues. The molecular mechanisms that regulate the proliferation and differentiation of cementoblasts, however, have not been elucidated to date. Enamel molecules are believed to regulate cementoblast differentiation and to initiate the formation of acellular extrinsic fiber cementum. The purpose of this study was therefore to isolate and culture human root-derived cells (HRDC) in order to determine whether they are able to express both cementum and specific enamel proteins and subsequently to confirm these findings in vivo. Material and Methods: Human root-derived cells were isolated and expanded in vitro. Cells were characterized using RT-PCR, immunostaining, western blotting and by examination of total mRNA to determine the expression of cementum and enamel markers. Human periodontal tissues were also examined for the expression of enamel-related proteins by immunostaining. Results: We showed that HRDC express mRNA corresponding to ameloblastin (AMBN), amelogenin (AMEL), enamelin (ENAM), tuftelin (TUFT) and cementum-associated molecules such as cementum protein 1 (CEMP1) and cementum attachment protein (CAP). Western blotting revealed that HRDC Dr Higinio Arzate, Laboratorio de Biología express both AMEL and AMBN gene products, as well as the cementum markers Periodontal y Tejidos Mineralizados, Facultad de CEMP1 and CAP. In vivo, we have showed that AMBN and AMEL are expressed Odontología, Universidad Nacional Autónoma de MØxico, Cd. Universitaria, C.P. 04510, by cementoblasts lining cementum, paravascular cells and periodontal ligament MØxico D.F. cells. Tel/Fax: +52 55 5622 5563 e-mail: [email protected] Conclusion: These results suggest that enamel-associated and cementum-associ- Key words: cementum; cementogenesis; ated proteins could act synergistically in regulating cementoblast differentiation cementum attachment protein; cementum and cementum deposition and offer new approaches on how the cementogenesis protein 1; amelogenin; ameloblastin process is regulated. Accepted for publication March 12, 2010

Cementum is a mineralized tissue that (1). The molecular mechanisms that source to cementoblasts and osteo- facilitates the attachment of periodon- regulate the proliferation and differen- blasts because they share phenotypic tal ligament to the root and sur- tiation of cells within the periodontal and structural features (2). There is rounding alveolar bone and plays a key ligament to become cementoblast cells also debate whether cementoblasts are role in both periodontal development are not yet completely understood. In a unique cell type or transformed and regeneration of periodontal tissues fact, some authors attribute a common osteoblasts (3). Cementoblasts share 2 Nun˜ez et al. several molecular characteristics with this study was to isolate and culture were cultured as previously described, osteoblasts including the production human root-derived cells (HRDC) in then collected, centrifuged and dis- of type I collagen and several noncol- order to determine whether they are solved in lysis buffer containing 1% lagenous proteins such as bone sialo- able to express both cementum and sodium dodecyl sulfate and a protease protein, osteocalcin and osteopontin specific enamel proteins and to confirm inhibitor cocktail (8). Equal amounts (4). Cementum, however, does not these findings in vivo. of total protein (10 lg per lane) were have the lamellar organization found separated by gel electrophoresis, blot- in bone: it is an avascular tissue; it ted onto Immobilon-P [poly(vinylidene Material and methods also lacks innervation and physiologi- difluoride)] membranes, as described cal remodelling and does not contain elsewhere (6), and incubated with rab- Cell culture bone marrow. Recently, it has been bit antiserum to AMEL, AMBN, or reported that the periodontal ligament Human root-derived cementoblast cells human recombinant cementum protein contains multipotent stem cells that were isolated from healthy human teeth 1 (diluted 1:500) or with a mouse have the potential to differentiate into extracted for orthodontic reasons and monoclonal anti-CAP (diluted 1:500) cementoblast-like cells in vitro and to according to the protocols approved by (Santa Cruz Biotechnology, Inc., Santa generate cementum and periodontal the Internal Review Board of the Cruz, CA, USA). After washing, ligament-like tissue in vivo (5). The Universidad Complutense de Madrid, membranes were incubated with per- molecular factors that regulate the Espan˜ a. Teeth were processed imme- oxidase-conjugated goat anti-rabbit differentiation of these populations of diately after extraction. The culture IgG secondary antibody or rabbit stem cells are, however, unknown. One and expansion of HRDC were per- anti-mouse IgG secondary antibody. of the major difficulties in studying formed according the procedures Detection was performed as previously cementoblasts is the lack of specific previously published by Nun˜ ez et al. described (6). markers. Recently, two genes encoding (9). Human alveolar bone-derived cementum proteins have been isolated: osteoblasts (HABDC) were also iso- Immunostaining cementum attachment protein (CAP) lated using standard methods previ- and cementum protein 1 (CEMP1) ously reported (10). Cultures from both Immunoexpression of AMEL, AMBN, (6). They have been demonstrated as isolates belonging to either second or CAP and CEMP1 was also determined cementoblast-specific products and third passages were used for the in HRDC and HABDC cultured consequently reliable specific markers experiments described below. in vitro. Double-immunofluorescence of these cells. staining was performed using mouse The clinical importance in under- monoclonal antibody against bovine RT-PCR standing the origin, differentiation and CAP (Santa Cruz Biotechnology, Inc.), behaviour of these cells lies in the fact The expression of AMEL, AMBN, rabbit polyclonal antibody against that regeneration of periodontal tissues ENAM, tuftelin 1 (TUFT1) CAP human CEMP1 and polyclonal anti- lost during periodontitis is based on and CEMP1 at the mRNA level in bodies against mouse AMEL and the formation of new cementum. It is HRDC and HABDC was established AMBN, as described elsewhere (12). believed that EMD can regulate by RT-PCR. Previously published pri- Cells were plated at a low density in cementoblast differentiation and initi- mer sequences and reaction conditions eight-well Lab-Tek chamber slides, ate the formation of acellular extrinsic were used to amplify AMEL, AMBN, cultured for 7 d, fixed and incubated fiber cementum (7,8). In fact, a treat- TUFT, ENAM, glyceraldehyde-3- with the antibodies against the proteins ment concept based on the application phosphate dehydrogenase (GAPDH), under study. Negative controls were of EMD aimed for new cementum CEMP1 and CAP (11). The reaction achieved by omitting the primary formation and periodontal regenera- products were analyzed by electro- antibody or by incubating with normal tion has been extensively used in phoresis of 15-lL samples in 2% aga- rabbit or mouse serum. The spatial clinical periodontics and its efficacy rose gels containing 0.5 lg/mL of expression of AMEL and AMBN in has been proven in multiple clinical ethidium bromide. The identity of the human periodontal tissues was ana- trials. EMD contains enamel proteins, PCR products was confirmed by lyzed by immunostaining. Human mainly amelogenin (AMEL), and the sequencing. dental tissues were obtained from a expression of enamel proteins by autopsy of a 29-year-old man, follow- cementoblast cells has also been a ing the policies of the Research Review Western blot subject of debate. The expression of Board of the Instituto Nacional de ameloblastin (AMBN) during cemen- After it was determined that HRDC Cancerologı´ a, Me´ xico City. The speci- tum formation has been demonstrated. express cementum and enamel-associ- mens containing periodontal structures However, the expression of AMEL and ated molecules at the mRNA level, we were fixed, decalcified, embedded in enamelin (ENAM) by cementoblasts determined whether the proteins were paraffin, sectioned and mounted on has not been completely accepted, also expressed. The expression of glass slides (13). Slides were incubated again because of the lack of specific proteins was analyzed by western with the corresponding antibodies. markers for these cells. The purpose of blotting. The HRDC and HABDC Sections incubated with normal rabbit Human cementoblasts express enamel-associated molecules 3 serum or lacking first antibody were blotting was used to determine the genin expression was also located in used as controls. expression of cementum and enamel- periodontal ligament subpopulations related proteins The results shown in and in cells with a paravascular Fig. 1B indicate that all proteins tested location (Fig. 3B). Alveolar bone and Results – AMEL (lane 1), AMBN (lane 2), osteoblasts were negative since they CEMP1 (lane 3) and CAP (lane 4) – did not express amelogenin (Fig. 3B) as Expression of cementum and revealed a main protein band at 27, 62, was cellular cementum (data not enamel-related molecules at the 55 and 50 kDa, respectively. Human shown). As the human specimens mRNA level alveolar bone-derived cells were nega- presented root resorption, AMEL The expression of cementum and tive for these proteins (data not expression was localized to the enamel-related molecules in HRDC shown). ÔcementoidÕ phase covering the resorp- and HABDC was first determined Immunolocalization of the CAP tion area and cells that could represent by RT-PCR. Our results show that protein in HRDC showed immunore- putative cementoblasts depositing AMEL, AMBN, ENAM, TUFT1, activity in the cell cytoplasm and cementum matrix onto the dentin in the CAP and CEMP1 amplicons were all nucleus (Fig. 2A,D,G). Double stain- cervical area (Fig. 3G). Ameloblastin expressed by HRDC. However, these ing for AMEL, AMBN and CEMP1 expression was found in cells in amplicons were not detected in reac- demonstrated that CAP co-localizes the vicinity of acellular cementum tions using total RNA obtained from with enamel-associated proteins and (Fig. 3D). Very few cells are in intimate HABDC (Fig. 1A). CEMP1 (Fig. 2E,H). Human alveolar contact with cementum and groups of bone-derived cells were negative for cells representing pre-cementoblasts these proteins (data not shown). Im- stained strongly (Fig. 3E). Cells with a Expression of cementum and munodetection of AMEL and AMBN paravascular location were also enamel-related molecules at the in human sections showed the expres- immunopositive for AMBN (Fig. 3E). protein level sion of AMEL in cementoblasts and a Cementoblasts located in cellular Total protein extracted from HRDC slightly positive immunoreactivity in cementum, cementocytes, alveolar and HABDC and subjected to sodium the cementum mineralized matrix bone and osteoblasts did not express dodecyl sulfate–polyacrylamide gel (Fig. 3A), mainly in the acellular AMBN. electrophoresis followed by western cementum region (Fig. 3A). Amelo- Discussion

(B) kDa 1234 In the present study we successfully (A) HRDC HABDC bp 120 isolated and established a cementoblast 90 GAPDH 600 cell line derived from normal human root cementum. These cells are truly CEMP1 433 64 cementoblasts because they express (at 50 the mRNA and protein levels) the only CAP 161 44 cementum markers available to date, AMEL 384 namely CAP and CEMP1 (8). These molecules have been shown to play an AMBN 555 30 important role during cementoblast differentiation and biomineralization ENAM 522 of cementum extracellular matrix (14). TUFT 494 Recently, it has been shown that CEMP1 possesses a strong affinity for hydroxyapatite and promotes octacal- 23 cium phosphate crystal nucleation (15). These molecules are also expressed by paravascular cells within the periodontal ligament that represent ances- Fig. 1. (A) The RT-PCR amplification of cementum attachment protein (CAP), cementum protein 1 (CEMP1), amelogenin (AMEL), ameloblastin (AMBN), enamelin (ENAM) and tors of cementoblasts, osteoblasts tuftelin (TUFT) transcripts in human root-derived cementoblasts (HRDC) and human and periodontal ligament fibroblasts. alveolar bone-derived osteoblasts (HABDC). The use of gene-specific primer sets for RT-PCR CAP and CEMP1 represent biologi- amplification generates a 433-bp amplicon for CEMP1, a 161-bp amplicon for CAP, a 384-bp cal markers for cementoblasts and amplicon for AMEL, a 555-bp amplicon for AMBN, a 522-bp amplicon for ENAM and a cementoblast-progenitor cells because 494-bp amplicon for TUFT. The HABDC did not express any of the transcripts. The glyc- CEMP1 co-localizes with periodontal eraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene was used as a control. ligament stem cells expressing STRO1 (B) Western blot analyses indicating the expression of protein species of 27 kDa (AMEL, lane (16). Furthermore, human osteoblast 1), 62 kDa (AMBN, lane 2), 55 kDa (CAP, lane 3) and 50 kDa (CEMP1, lane 4). cells do not express these proteins, 4 Nun˜ez et al.

CAP CEMP1 MERGE cementum formation and periodontal ligament repair in both experimental animals and humans (20,21). It was later demonstrated that AMBN is expressed by HertwigÕs epithelial root sheath cells (HERS) cells, both in vivo and in vitro. Our results clearly show that human root cementum-derived A B C cementoblasts express not only AMBN, but also other enamel-associ- CAP AMBN MERGE ated proteins such as AMEL, ENAM and TUFT. In addition, the expression of AMEL and AMBN by cementoblasts was confirmed in human tissues using immunohistochemistry. To our knowledge, this is the first report that demonstrates the expression of specific enamel-associated molecules D E F by human cementoblasts, in vitro and CAP AMEL MERGE in vivo. Both AMEL and AMBN promote cell attachment and proliferation of periodontal ligament cells in vitro. Leucine-rich AMEL peptide has been suggested to act as a signalling mole- cule capable of inducing the osteoblast phenotype in various types of cells, G H I including mouse embryonic stem cells (22). Consequently, it is possible that these proteins might participate during cementoblast differentiation in the adult periodontium by inducing undiff- erentiated mesenchymal cells into the cementoblast phenotype. Interestingly, cementoblasts, cementoid phase, mineralized matrix of human cementum, J K periodontal ligament cell subpopulations and putative stem cells located Fig. 2. Double staining and co-localization of cementum attachment protein with cementum paravascularly are positive for AMEL. protein 1 (CEMP1), amelogenin (AMEL) and ameloblastin (AMBN) in human root-derived Our results also support the role of cementoblasts (HRDC) and human alveolar bone-derived osteoblasts (HABDC). Cementum AMEL during cementum repair/ attachment protein (CAP) localizes to the cell cytoplasm (A, D and G). CEMP1 localizes to regeneration, because areas of root the cell cytoplasm and nucleus (B). Co-localization of CEMP1 and CAP indicates that CAP resorption and the matrix being stains fiber-like cell structures as well as the cytoplasm and the nucleus (C). Ameloblastin deposited by cementoblasts expressed stains the cell cytoplasm (E) and co-localizes with CAP at the same cell structures (F). AMEL. This finding is in agreement Amelogenin stains the cytoplasm and the nucleus and co-localizes with CAP at the same cell with other authors reporting that structures (H), except that it does not stain fiber-like structures (I). Representative negative HERS cells produce cementum-related controls using normal rabbit (J) or mouse (K) serum are negative. Magnification: · 40. Scale bar = 100 lm. and/or enamel-related proteins (1,3), which play a pivotal role in the differentiation of cementoblasts that which confirms that they have a In previous reports it was shown specifically produce acellular extrinsic different cell phenotype. Nevertheless, that antibodies to enamel proteins fiber cementum (6,7). Our results show the expression of CAP, CEMP1 and cross-react with proteins extracted that enamel-related proteins are only AMEL in the nucleus of HRDC could from mouse cementum, suggesting that expressed by cells associated with the be associated with the role of these cementum contains a unique class of acellular cementum. While the origin proteins as Ôgrowth factorsÕ related to enamel-associated or enamel-like pro- of enamel proteins in cementum could proliferation and differentiation proteins (18,19). Furthermore, EMDs be attributed to the stem cells in cesses (11,17). have been used to induce de novo the periodontal ligament, the exact Human cementoblasts express enamel-associated molecules 5

AMEL AMEL determine how the cementogenesis D D process is regulated and point out the AB PLC role of these proteins during peri-

PLC odontium homeostasis and regenera- CEM tion of periodontal structures.

Acknowledgements

CTB CEM This study was supported by funds CTB from DGAPA-UNAM (IN200908), BV CONACyt 48638 and 130950 to H.A.

A B C AMBN AMBN References D D PLC PLC 1. Bosshardt DD, Zalzal S, McKee MD, Nanci A. Developmental appearance and distribution of bone sialoprotein and BV osteopontin in human and rat cementum. CEM Anat Rec 1998;250:13–33. CTB 2. Hammarstro¨ m L, Alatli I, Fong CD. Origins of cementum. Oral Dis 1996;2: CEM CEM CTB PLC 63–69. 3. Narayanan AS, Bartold M. Biochemistry of periodontal connective tissues and BV their regeneration: a current perspective. Connect Tissue Res 1996;34:191–201. D E F 4. Booshardt DD. Are cementoblasts a sub- population of osteoblasts or a unique phenotype?. J Dent Res 2005;84:390–406.

CEM 5. Seo BM, Miura M, Gronthos S et al. BV Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004;364:149–155. 6. Alvarez-Perez MA, Narayanan S, Zeich- CTB CEM ner-David M, Carmona-Rodrı´ guez B, CTB Arzate H. Molecular cloning, expression and immunolocalization of a novel human cementum-derived protein (CP-23). Bone G D H D 2006;38:409–419. 7. Hammarstro¨ m L. Enamel matrix, cemen- Fig. 3. Immunostaining of human periodontal tissues shows amelogenin staining of ce- tum development and regeneration. J Clin mentoblasts, cementoid phase and periodontal ligament cell subpopulations (A). Alveolar Periodontol 1997;24:658–668. 8. Hammarstrom L. The role of enamel bone is negative. Putative stem cells with a paravascular location and periodontal ligament ¨ matrix proteins in the development of cell subpopulations stain strongly with polyclonal anti-amelogenin (B). Polyclonal anti-a- cementum and periodontal tissues. Ciba meloblastin cross-reacts with cell clusters in the vicinity of acellular cementum and with cells Found Symp 1997;205:246–255. close to the periodontal ligament blood vessels (D and E). The root resorption area shows 9. Nu´ n˜ ez J, Sanz-Blasco S, Vignoletti F et al. that cells and the matrix they are depositing immunoreact with polyclonal anti-amelogenin 17ß-estradiol promotes cementoblast pro- (G). Histological hematoxylin and eosin-stained sections show the anatomical aspects of liferation and cementum formation in experimental slides described above (C, F and H). Magnification in A, C, D and F: · 20 experimental periodontitis. J Clin Period- (scale bar = 100 lm). Magnification in B and D: · 40 (scale bar = 50 lm). Magnification ontol 2010 (in press). in G and H: · 100 (scale bar = 20 lm). AMBN, ameloblastin; AMEL, amelogenin; BV, 10. Arzate H, Alvarez-Perez MA, Aguilar- blood vessel; CEM, cementum; CTB, cementoblast; D, dentin; PLC, periodontal ligament Mendoza ME, Alvarez-Fregoso O. Human cementum tumor cells have different cells. features from human osteoblastic cells mechanisms that regulate cementoblast in charge of synthesizing cellular in vitro. J Periodontal Res 1998;33:249–258. differentiation are not known. Never- cementum (23,24). 11. Carmona-Rodrı´ guez B, Alvarez-Pe´ rez theless, it has been suggested that Taken together, our results suggest MA, Narayanan AS et al. Human cementum protein 1 induces expression of HERS cells undergo ecto-mesenchymal that enamel-associated proteins and bone and cementum proteins by human transformation and acquire morpho- cementum proteins could act synergis- gingival fibroblasts. Biochem Biophys Res logical and phenotypic changes leading tically, regulating cementoblast differ- Commun 2007;358:763–769. to acellular cementum formation, while entiation and cementum deposition. 12. Hosoya A, Nakamura H, Ninomiya T neural crest-derived cementoblasts are These data offer new approaches to et al. Immunohistochemical localization 6 Nun˜ez et al.

of alpha-smooth muscle actin during rat 16. Garcia de Paula e Silva FW, Ghosh A, serumless, chemically-defined medium. molar tooth development. J Histochem Arzate H, Kapila S, Bezerra da Silva LA, J Periodontal Res 1989;24:28–40. Cytochem 2006;54:1371–1378. Kapila Y. Calcium hydroxide promotes 20. Hammarstro¨ m L, Heul L, Gestrelius S. 13. Shi SR, Cote C, Kalra KL, Taylor CL, cementogenesis and induces cementoblas- Periodontal regeneration in a buccal Tandon AK. A technique for retrieving tic differentiation of PDL cells in a dehiscence model in monkeys after appli- antigens in formalin-fixed, routinely acid- CEMP-1 and ERK-dependent manner. cation of enamel matrix proteins. J Clin decalcified, celloidin embedded human Calcif Tissue Int 2010 (in press). Periodontol 1997;24:669–677. temporal bone sections for immunohisto- 17. Zeichner-David M, Chen LS, Hsu Z, 21. Heul L. Periodontal regeneration with chemistry. J Histochem Cytochem 1992;40: Reyna J, Caton J, Bringas P. Amelogenin enamel matrix derivative in one human 787–792. and ameloblastin show growth-factor like experimental defect. A case report. J Clin 14. Alvarez Pe´ rez MA, Pitaru S, Alvarez activity in periodontal ligament cells. Eur Periodontol 1997;24:693–696. Fregoso O, Reyes Gasga J, Arzate H. J Oral Sci 2006;114(suppl 1):244–253. 22. Warotayanont R, Zhu D, Snead ML, Anticementoblastoma-derived protein anti- 18. Slavkin HC, Bessem C, Fincham AG et al. Zhou Y. Leucine-rich amelogenin peptide body partially inhibits mineralization on a Human and mouse cementum proteins induces osteogenesis in mouse embryonic cementoblastic cell line. J Struct Biol immunologically related to enamel pro- stem cells. Biochem Biophys Res Commun 2003;143:1–13. teins. Biochim Biophys Acta 1989;25: 2008;367:1–6. 15. Villarreal-Ramı´rez E, Moreno A, 12–18. 23. Thomas HF. Root formation. Int J Dev Mas-Oliva J et al. Characterization of 19. Slavkin HC, Bringas P Jr, Bessem C et al. Biol 1995;39:231–237. recombinant human cementum protein 1 HertwigÕs epithelial root sheath differen- 24. Zeichner-David M, Oishi K, Su Z et al. (hrCEMP1): primary role in biomineral- tiation and initial cementum and bone Role of HertwigÕs epithelial root sheath ization. Biochem Biophys Res Commun formation during long-term organ culture cells in tooth root development. Dev Dyn 2009;19:384. of mouse mandibular first molars using 2003;228:651–663.