Objective: To perform a comparative study of the cellular proliferation in the peripheral

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1 Cellulr prolifertion mrkers in peripherl nd Brun Gonçlves GARCIA 1, Ptríci Crlos CALDEIRA 1 2, Suzn Cntnhede Orsini Mchdo de SOUSA 3 4 5, Ricrdo Alves MESQUITA 6 1- MSc, Deprtment of Orl Surgery nd Pthology, School of Dentistry, Federl University of Mins Geris, Belo Horizonte, MG, Brzil. 2- PhD, Deprtment of Stomtology, School of Dentistry, Pontifíci Universidde Ctólic do Prná, Curitib, PR, Brzil. 3- PhD, Deprtment of Orl Pthology, School of Dentistry, University of São Pulo, São Pulo, SP, Brzil. 4- PhD, Deprtment of Pthology, Biologicl Sciences Institute, Federl University of Mins Geris, Belo Horizonte, MG, Brzil. 5- Deprtment of Orl Surgery nd Pthology, School of Dentistry, Federl University of Mins Geris, Belo Horizonte, MG, Brzil. 6- Deprtment of Orl Surgery nd Pthology, School of Dentistry, Federl University of Mins Geris, Belo Horizonte, MG, Brzil. Corresponding ddress: Ptríci Crlos Cldeir - Universidde Federl de Mins Geris - Fculdde de Odontologi - Deprtmento de Clínic, Ptologi e Cirurgi Odontológics - Av. Antônio Crlos, 6627, Sl Pmpulh Belo Horizonte - MG - Brsil - Phone: Fx: e-mil: pt_cldeir@yhoo.com.br ABSTRACT Objective: To perform comprtive study of the cellulr prolifertion in the peripherl used for the sttisticl nlyses. Results: Mesenchyml component of the centrl lesions presented higher men number of AgNOR per nucleus nd PCNA index thn did the nd epithelil components presented similr men numbers of AgNOR per nucleus nd PCNA index in the OdF, s well s similr men number of AgNOR per nucleus in the POF. Conclusions: The mesenchyml component my well ply role in the differences between the biologicl behviour of the centrl lesions s compred to the peripherl lesions. Moreover, considering tht the epithelil nd mesenchyml components in odontogenic the true role of the epithelil components, which re believed to be inctive in nture, s well s in the development nd biologicl behviour of these lesions. Key words: INTRODUCTION 6 lrge mount of pprently inctive odontogenic epithelium. OdF presents slow-growing, progressive but pinless swelling, often with corticl expnsion or tooth displcement. A recurrence rte of 13% fter enucletion hs been reported in the literture 6,18,22 (POF) is the rre peripherl counterprt of OdF. POF is n pprently innocuous, elevted gingivl lesion tht hs yet to produce conclusive dt regrding its exct prognosis 1,11,12 benign connective-tissue mtrix nd islnds, or trbecule, of new bones 13,25. Curettge or rdicl surgicl resection is the most common tretment for OsF, depending on the lesion s size. Recurrence rtes vried from 6% to 28% for mndible lesions, while the recurrence rtes for mxillry lesions re unknown 18. Nevertheless, the peripherl ossifying rective nture ssocited with minerliztion nd derived from the periodontl ligment cells. J Appl Orl Sci. 106

2 crows nd rough restortions re considered to be locl irritnts 7,9. Locl surgicl excision is the most common tretment for PEOF nd the recurrence rte is pproximtely 16.0% 7. Nucleolr orgnizer regions (NOR) represent the loops of DNA which ctively trnscribe to ribosoml RNA, thus trnscribing to ribosomes nd ultimtely to protein. The NOR re ssocited with cidic rgyrophilic non-histonic proteins which cn be viewed through the AgNOR technique 20,24. Studies hve pplied this technique s useful method to evlute the differences mong cellulr prolifertion indexes in non-neoplstic rective lesions, s well s in benign or mlignnt neoplsms 3,9,15,17,23. The proliferting cellulr nucler ntigen (PCNA) is 36-kD cidic non-histone nucler protein which cts s n uxiliry protein for DNA delt polymerse, which is n bsolute requirement for DNA synthesis. Its distribution in the cell cycle increses through the G 1 phse, peks t the G 1 /S interphse nd decreses in the G 2 phse. Immunohistochemicl nlysis of PCNA hs lso been used s n uxiliry tool in the evlution of cellulr prolifertion indexes in lesions with vrible biologicl behviour 5,17. Although PCNA is considered to be cellulr prolifertion mrker, it hs been estblished tht growth nd technicl fctors, repir processes, biologicl hlf-lives of pproximtely 20 h nd cytokines relesed by the PCNA immunoexpression 5,16. OdF, POF, OsF nd PEOF contin similr histomorphologicl fetures, but present different 1,6,7,25. Since mjor theme of discussion, dditionl informtion concerning the prolifertion indexes is wrrnted in n ttempt to better clrify the differences in biologicl behviour mong OdF, POF, OsF nd PEOF. Also, nother importnt fct is the comprtive nlysis of cellulr prolifertion between the POF nd PEOF, which were considered the sme pthology in the pst 12. Therefore, the core ims of this study re: 1) to determine the cellulr prolifertion of OdF, POF, OsF nd PEOF nd 2) to compre the cellulr prolifertion indexes mong these lesions. MATERIAL AND METHODS Ethics sttement The present study s protocol ws pproved by the Committee of Bioethics in Reserch from the Universidde Federl de Mins Geris (UFMG, COEP 124/07). Specimens Specimens of the OdF (4 cses), POF (9 cses), OsF (7 cses) nd PEOF (8 cses) were retrieved UFMG (Belo Horizonte, Brzil) nd from the Orl Pthology Service of the University of São Pulo (São Pulo, Brzil). The criteri for dignosis of OdF, POF nd OsF were in ccordnce with the WHO OdF cn pper in two ptterns: the epithelium-poor type nd the epithelium-rich type 6. In this study, two cses of OdF were of the epithelium-rich type nd two cses were of the epithelium-poor type. Criteri to identify the PEOF were in ccordnce with Buchner nd Hnsen 7 (1987). AgNOR technique The AgNOR technique ws performed ccording to the stndrdized method of Trerè 24 (2000). embedded blocks were de-wxed nd hydrted. The sections were immersed in sodium citrte buffer (10 mm, ph 6.0) nd boiled t 120 C for 20 min. These were then cooled down to room temperture nd wshed with distilled wter. The sections were immersed in geltine nd silver nitrte solution in the drk t room temperture for 25 min. Immunohistochemistry Immunohistochemistry ws performed using the streptvidin-biotin stndrd protocol. Sections of blocks were de-wxed nd hydrted. The specimens were immersed in 10 mm citrte buffer (ph 6.0, 20 min t 98 C) for ntigen retrievl. The endogenous peroxidse ctivity ws blocked using 0.3% hydrogen peroxide. Sections were incubted with primry ntibody PCNA (PC10, MO879, Dko Corportion, Crpinteri CA, USA) for 18 hours t room temperture. Next, the sections were treted with LSAB â +system, HRP Peroxidse Kit (KO675, Dko Corportion, Crpinteri CA, USA) nd 3.3 -diminobenzidine tetrhydrochloride chromogen (D5637, DAB; Sigm Chemicl, St Louis MO, USA). Sections of orl squmous cell crcinom were used s the positive controls. Anlysis of AgNOR nd PCNA indexes Fibroblsts (mesenchyml component) were evluted in four groups of lesions. The epithelil cells of the islnds or strnds of odontogenic epithelium in the POF nd OdF were lso evluted. mterils presented in the POF nd PEOF were not included in this nlysis. AgNOR prmeters were estblished in 100 cells for ech cse using KS300 softwre coupled to Crl Zeiss Imge Anlyzer (Crl Zeiss, Oberkochen, J Appl Orl Sci. 107

3 Bden-Württemberg, Germny). The number, re nd contour index of AgNOR were obtined from digitl imges tken by JVC TK-1270/RGB homogeneous dots (Figure 1A). The men numbers of AgNOR per nucleus, re nd contour index for ll cses were determined. The contour index vried from 0.76 to Vlues ner 1 corresponded to round structure with regulr contour nd vlue distnt from 1 indicted n irregulr structure. Brown nuclei, regrdless of stining intensity, were considered PCNA-positive cells (Figure 1B). An index (IP) ws determined considering the number of PCNA positive cells per 500 cells in ech cse. This count ws performed t 400x Axiostr , Crl Zeiss, Oberkochen, Bden-Württemberg, Germny). The Kruskl-Wllis nd the Mnn-Whitney tests were used to compre the AgNOR nd IP dt. The sttisticl nlysis ws performed by the BioEstt softwre nd the lph level ws set t RESULTS Figure 1- AgNOR nd proliferting cellulr nucler ntigen (PCNA) stining. A- AgNOR in the peripherl intr-nucler homogeneous dots in the epithelil nd mesenchyml components. The rrow heds identify islnds of odontogenic epithelium (AgNOR technique, nuclei in the epithelil nd mesenchyml components protocol, 400x) The AgNOR nd IP men in the mesenchyml higher thn in the POF nd PEOF, respectively. The AgNOR re of the centrl lesions ws sttisticlly smller thn those found in the peripherl lesions per nucleus, re nd IP in the mesenchyml component between the POF nd PEOF presented result ws observed between the OdF nd OsF (P>0.05, Tble 2). The men number of AgNOR per nucleus in in the COF thn in the POF, while the AgNOR re of the POF ws sttisticlly higher thn those Tble 1- Comprtive nlysis of dt concerning the men number of AgNOR per nucleus, re nd proliferting cellulr POF men OdF men PEOF men OsF men Men number of AgNOR 1.26 (1.10/1.50) 1.49 (1.40/1.70) * 1.25 (1.10/1.40) 1.48 (1.30/1.70) * per nucleus b AgNOR re (μm2) c 1.46 (1.40/1.90) 1.21 (1.10/1.40) * 1.62 (1.39/1.86) 1.25 (1.00/1.53) * IP d 43.6 (11.6/58.2) 61.2 (56.0/67.6) * 47.6 (40.2/55.6) 57.1 (50.8/62.8) * b c d J Appl Orl Sci. 108

4 Tble 2- Comprtive nlysis of dt concerning the men number of AgNOR per nucleus, re nd proliferting cellulr nucler ntigen (PCNA) index (IP) in the mesenchyml component between peripherl odontogenic (POF) nd peripherl Men number of AgNOR per nucleus POF men PEOF men OdF men OsF men 1.26 (1.10/1.50) 1.25 (1.10/1.40) (1.40/1.70) 1.48 (1.30/1.70) AgNOR re (μm 2 ) 1.46 (1.40/1.90) 1.62 (1.39/1.86) (1.10/1.40) 1.25 (1.00/1.53) IP 43.6 (11.6/58.2) 47.6 (40.2/55.6) (56.0/67.6) 57.1 (50.8/62.8) Mnn-Whitney test. Tble 3- Comprtive nlysis of dt concerning the men number of AgNOR per nucleus, re nd proliferting cellulr nucler ntigen (PCNA) index (IP) in the epithelil (e) component between peripherl odontogenic (POF) nd odontogenic Men number of AgNOR per nucleus epof men eodf men 1.25 (1.07/1.51) 1.45 (1.40/1.51) * AgNOR re (μm 2 ) 1.47 (1.30/1.65) 1.23 (1.07/1.48) * IP 78.5 (34.2/99.1) (45.8/94.6) Tble 4- Comprtive nlysis of dt concerning the men number of AgNOR per nucleus, re nd proliferting cellulr nucler ntigen (PCNA) index (IP) between mesenchyml (m) nd epithelil (e) components in the peripherl odontogenic Men number of AgNOR per nucleus mpof men epof men modf men eodf men 1.26 (1.10/1.50) 1.25 (1.07/1.51) (1.40/1.70) 1.45 (1.40/1.51) AgNOR re (μm 2 ) 1.46 (1.40/1.90) 1.47 (1.30/1.65) (1.10/1.40) 1.23 (1.07/1.48) IP 43.6 (11.6/58.2) 78.5 (34.2/99.1) * 61.2 (56.0/67.6) (45.8/94.6) presented similr dt in the epithelil components of the POF nd OdF (P>0.05, Tble 3). Epithelil nd mesenchyml components of the POF nd OdF except for the IP men in the POF, in which the epithelil component presented higher IP (Tble 4). DISCUSSION Although the lesions evluted in this study exhibited similr histomorphologicl fetures, the pproprite dignosis nd differentition mongst them is possible. Therefore, the current study serves to provide informtion on cellulr prolifertion. The low number of cses in this study is due to the rrity of these lesions, especilly the OdF nd POF, which re rre benign odontogenic tumours 6,18. The current study demonstrted differences in the cellulr prolifertion of these four lesions, which is importnt in understnding their biologicl behviour. The morphometric study of AgNOR is relted to the degree of cellulr prolifertion in nonneoplstic rective nd neoplstic lesions 2. Benign neoplsms nd non-neoplstic rective lesions re chrcterized by low numbers of AgNOR per nucleus, lrge res nd regulr shpes or contour indexes 8,10,17. These fetures of the men number of AgNOR per nucleus could be observed in ll evluted lesions, defending the concept tht these Investigtions of cellulr prolifertion using PCNA J Appl Orl Sci. 109

5 in orl diseses my well bring bout informtion concerning prolifertive ctivity. Mesquit, et l. 17 (1998) nd Ono, et l. 19 (2007) demonstrted, by mens of the AgNOR nd IP nlyses, tht the cellulr prolifertion in the OsF ws higher thn in the PEOF. These results were re-shown in the current study, emphsizing the non-neoplstic rective nture of the PEOF. Other studies hve demonstrted tht non-neoplstic rective lesions, e.g mesothelium, present lower AgNOR counts thn do benign neoplsms with the sme cell components 10. It could be observed tht the cellulr prolifertion of the POF proved to be less thn the OdF but similr to the PEOF. These dt suggest similr cellulr prolifertion for both peripherl lesions, even though POF is in fct considered to be neoplstic lesion 6. AgNOR nlysis nd IP hve been performed on odontogenic cysts nd tumours 15,16. Mrtins, et l. 15 (2001) evluted AgNOR in melobstic nd the mesenchyml components present similr cellulr prolifertion. This fct is in ccordnce with the epithelil nd the mesenchyml components re considered neoplstic 21. In the current study, mesenchyml components presented similr AgNOR nd IP vlues in the OdF nd similr men number of AgNOR per nucleus in the POF. This indictes tht both the epithelil nd mesenchyml components in the POF nd OdF present similr cellulr prolifertion. the higher IP, in contrst to the smller AgNOR vlues, in the epithelil component of the POF. the POF my be responsible for this observtion 14. Therefore, it is importnt to evlute the true role of cytokines in this type of lesion, s well s the utility of PCNA nlysis s cellulr prolifertion In conclusion, the mesenchyml component my well ply role in the differences between the biologicl behviour of centrl lesions, s compred to peripherl lesions. Moreover, considering tht the epithelil nd mesenchyml components in odontogenic fibroms presented similr prolifertion index, further reserch is wrrnted to understnd the true role of epithelil components, which re believed to be inctive in nture, s well s in the development nd biologicl behviour of these lesions. ACKNOWLEDGMENTS The present reserch ws supported by grnts from the Conselho Ncionl de Desenvolvimento #472045/2011-3). Sous SCOM nd Mesquit RA re reserch fellows of CNPq. REFERENCES 1- Aleddini M, Slehizdeh S, Bghii F, Etemd-Moghdm S. Irnin popultion. J Orl Mxillofc Surg. 2010;68: Allison RT, Spencer S. Nucleolr orgnizer regions in odontogenic cysts nd meloblstoms. Br J Biomed Sci. 1993;50: Aror B, Kumr S, Jin R. Morphometric evlution of nucleolr orgniser regions in rective nd neoplstic lymph node lesions. J Indin Med Assoc. 2006;104: Ayres M, Ayres JR, Ayres DM, Sntos AS. Bioestt 3.0: Belém: Lither Mciel; Brboz CA, Pereir Pinto L, Freits RA, Cost AL, Souz LB. Proliferting cell nucler ntigen (PCNA) nd p53 protein expression in meloblstom nd denomtoid odontogenic tumor. Brz Dent J. 2005;16: Brnes L, Everson JW, Reichrt P, Sidrnsky D. World Helth Hed nd Neck Tumors. Lyon: IARC Press; Buchner A, Hnsen L. The histomorphologic spectrum of 1987;63: Cbrini RL, Schwint AE, Mendez A, Femopse F, Lnfrnchi H, Itoiz ME. Morphometric study of nucleolr orgnizer regions in humn orl norml mucos, ppillom nd squmous cell crcinom. J Orl Pthol Med. 1992;21: overgrowths of the gingiv. Brz Orl Res. 2012;26(4): Fonsec LM, Crmo MA. AgNORs in hyperplsi, ppillom nd orl squmous cell crcinom. Brz Dent J. 2000;11: Grci BG, Johnn ACBR, Silveir-Júnior JB, Crvlho VM, Mesquit RA. Retrospective nlysis of peripherl odontogenic Gondivkr SM, Gdbil AR, Chole R, Prikh RV, Blsrf S. review. Orl Oncol. 2011;47: Hrrison RF, Reynolds GM, Rowlnds DC. Immunohistochemicl evidence for the expression of proliferting cell nucler ntigen (PCNA) by non-proliferting heptocytes djcent to metsttic Mrtins C, Crvlho YR, Crmo MAV. Argyophilic nucleolr orgnizer regions (AgNORs) in odontogenic myxom (OM) nd 16- Meer S, Glpin JS, Altini M, Colemn H, Ali H. Proliferting cell nucler ntigen nd Ki67 immunorectivity in meloblstoms. Orl Surg Orl Med Orl Pthol Orl Rdiol Endod. 2003;95: Mesquit RA, Souz SCOM, Arújo NS. Prolifertive ctivity 1998;27:64-7. J Appl Orl Sci. 110

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