Dentl Reserch Journl Originl Article The role of cyclosporine A on the periodontl tissues Mllpp Jysheel 1, Dhoom Singh Meht 1 1 Deprtment of Periodontology, Bpuji Dentl College nd Hospitl, Dvngere, Krntk, Indi ABSTRACT Received: Novemer 2012 Accepted: My 2013 Address for correspondence: Dr. M. Jysheel, Deprtment of Periodontology, Bpuji Dentl College nd Hospitl, Dvngere - 577 004, Krntk, Indi. E-mil: Jysheel6@ gmil.com Bckground: Cyclosporin A (CsA) is known immunosuppressive gent nd cn e considered s lifesving drug in the orgn trnsplnttion cses. However, it is ssocited with mny side-effects on different tissues nd ody orgns including the periodontl tissues. The present niml study ws undertken to evlute the effects of CsA trgeting the tissue trid of periodontl tissues, i.e., gingiv, lveolr one nd cementum in rts. Mterils nd Methods: Twelve 6-week-old mle Wistr rts weighing 150-200 g were considered for the cse-control study in rts. The rts were divided into 2 groups: (1) CsA (test) group (2) Sline (control) group nd were dministered the sme sucutneously dily once for 45 dys. Impressions were tken nd study csts were prepred on weekly sis for the morphometric nlysis. At the end of 45 dys, rts were scrificed nd specimens were nlyzed for histomorphometric nlysis. CsA nd sline groups were nlyzed to test of ssocition using the Student t-test t 99% confidence intervl. Results: The morphometric exmintion showed significnt gingivl overgrowth in the CsA group, wheres no such growth in the sline group. Similrly, on histomorphometric nlysis, there ws significnt loss of lveolr one in CsA group s compred with control. Furthermore, there ws lrge mount of cementum formtion ccompnied y insertion of new connective tissue fiers especilly in the cervicl region of the tooth in CsA group rts. Conclusion: CsA trgets the periodontl tissues (gingiv, lveolr one nd cementum) in different pttern. Its role in cementogenesis cn e utilized for periodontl regenertion, if its locl ppliction is testified nd verified in the future niml studies. Key Words: Alveolr one loss, cyclosporin A, gingivl overgrowth, new cementum formtion INTRODUCTION Cyclosporin A (CsA) is one of the sic immunosuppressive drugs widely used to prevent orgn trnsplnt rejection nd for the tretment of other immune diseses, such s Behçet s disese, rheumtoid rthritis, systemic lupus erythemtosus, Crohn s disese, ulcertive colitis nd insulin dependent dietes mellitus. The side-effects of Access this rticle online Wesite: http//:drj.mui.c.ir CsA therpy include gingivl overgrowth (GO), nephrotoxicity, heptotoxicity nd one loss. [1] Periodontium is trget tissue for cyclosporine therpy wherein it trgets specilly the tissue of the periodontium (gingiv, lveolr one nd cementum) in very unique wy. Though CsA hs negtive impct on gingiv (overgrowth) nd lveolr one (one loss), [2] it stimultes cementum formtion on the root surfces. [3] The CsA-induced GO hs een reported in oth niml models [4,5] nd humn clinicl cses. [6] The prevlence of CsA-induced GO, which ffects the qulity-of-life of ptients, rnges from 8% to 70% with men prevlence of 35%. [6] Bone loss is cliniclly importnt prolem ffecting 6% to 15% of ptients in 1 st yer fter orgn trnsplnttion. An incresed lveolr one destruction t periodontl sites ws found in rts 802 Dentl Reserch Journl / Novemer 2013 / Vol 10 / Issue 6
fter CsA exposure. [7] This phenomenon of incresed lveolr one loss my e relted to the current concept of osteoimmunology wherein there is n incresed recognition of the interctions etween cells of immune nd skeletl systems. [8] There is ccumulting evidence tht CsA hs negtive impct on one metolism in humn, [9-11] lthough the current literture regrding the effect of CsA on lveolr one is controversil nd indequte. Dentl cementum is minerlized tissue covering the root dentin nd plys key role in the ttchment of the tooth to the surrounding structures. This ttchment occurs y mens of functionl insertion of periodontl collgen fiers into the new cementum (NC). In recent niml study, it ws oserved tht the cyclosporine fed rts showed NC deposition on the root surfces with functionlly inserting collgen fiers. [3,12] The NC is deposited on the firillr extrinsic fier cementum, minly on the cervicl third of roots s irregulr lyers, spurs of vrious sizes nd shpes or in oth configurtions. [3] There re severl studies in the literture investigting the role of CsA on the periodontl tissue trid on the individul sis. [4-8,12-14] However, on literture serch nd to the est of our knowledge, there is no such study, in which the impct of CsA on ll the three periodontl trget tissues were investigted simultneously. Hence, the present cse-control niml study ws undertken to investigte the effect of cyclosporine therpy to mesure the GO morphometriclly nd evlute the lveolr one loss nd NC formtion histologiclly in Wistr rts. MATERIALS AND METHODS Selection of rts nd cre Twelve mle Wistr rts, ge 6 weeks nd weighing pproximtely 150-200 g were used for this cse-control niml study. Rts were housed in polypropylene cges using husk s their edding nd kept in the niml house of the JJM Medicl College, Dvngere. The niml house temperture ws mintined t 24 C nd light cycle (12 h light nd 12 h drk). Rts were fed with stndrd commercil lortory diet nd wter d liitum. Hndling of the rts ws crried out s per the guidelines given y Ntionl Centre for Lortory Animl Sciences. The ethicl pprovl ws otined from the Ethicl Committee of JJM Medicl College, Dvngere. CsA dministrtion After 1 week of cclimtiztion in the niml house, the rts were rndomly divided into two groups: six rts in ech group. The control group rts were injected with norml sline 10 mg/kg ody weight/ dy sucutneously nd test groups with 10 mg/kg ody weight/dy of CsA sucutneously for period of 45 dys. CsA ws procured from Novrtis Indi nd ws injected using tuerculin syringe. The rts were weighed once week in order to djust the dose of CsA. After the injections, the rts were given free ccess to food nd wter. Assessment of GO Impression mking nd preprtion of stone models To record the gingivl chnges in the mndiulr nterior region, impressions were mde prior to strt of the study with customized crylic trys nd stone csts were poured to serve s seline. The procedure ws crried out t the end of 45 dys nd stone csts were poured. Gingivl dimensions on the cst were mesured. Morphometric mesurement of GO The gingivl dimensions in the region of mndiulr incisors were mesured nd recorded [Figure 1]. Buccolingul dimension [BLi], mesiodistl dimension [MDi] nd verticl height dimensions [VHi] in the Figure 1: Gingivl dimension in () sline treted rts () cyclosporin A treted rts Dentl Reserch Journl / Novemer 2013 / Vol 10 / Issue 6 803
region of the interdentl ppill ws recorded s previously descried. [14] Assessment of lveolr one loss y histomorphometric nlysis Rts were scrificed t the end of the study period. The mndiles were crefully removed nd the sections involving the mndiulr molr teeth nd one were dissected out nd immeditely trnsferred into solution contining 10% neutrl formlin ( tissue fixtive gent) nd smples were sujected to histomorphometric nlysis. Morphometric nlysis To mesure the one loss morphometriclly, imges of the specimen were cptured using three chip chrge coupled deviceccd cmer ttched to stereomicroscope with 5 ojective. The mesurement ws crried out using the imge pro-plus softwre. The distnce ws mesured from the cusp tip to the crest of the lveolr one. Assessment of NC formtion Declcifiction procedure Declcifiction of the specimen ws crried out using the declcifying gent (formlin-nitric cid solution) nd this procedure ws crried out in room temperture of 37 C. Ech 100 ml solution consisted of formic cid-10 ml, Nitric cid-10 ml nd distilled wter-80 ml. The solution ws replced every 3 dys, wheres the completion of the declcifiction ws tested every dy using needle test. The test ws crried out the using shrp needle; when the needle penetrtes the one without ny pressure it confirms the end of declcifiction process (pproximtely 30 dys). After the declcifiction ws crried out, the specimen ws wshed first in tp wter overnight, then in the following order; 70% lcohol for 30 min, 80% lcohol for 1 h, 90% lcohol for 2 h, solute lcohol for overnight nd finlly treted with chloroform for 2 h. Tissues were emedded in prffin wx nd cut into slices in the thickness of 5 micron using hrd-tissue microtome. Tissue sections were sujected to H nd E stining nd viewed under stereomicroscope. Sttisticl nlysis Sttisticl nlysis ws performed; CsA nd sline groups were nlyzed to test of ssocition using Student t-test t 99% confidence intervl. RESULTS In the present niml study, the morphometric nlysis showed no GO in the sline treted rts [Figure 1] where s significnt (P < 0.001) increse in gingivl dimensions in oth mxillry nd mndiulr regions in the CsA treted rts [Figure 1]. Furthermore in CsA treted rts, the incresed size of the gingiv (oth mrginl nd interdentl ppill) produced distem formtion etween mndiulr nterior teeth. The chnge in the gingivl dimension etween CsA treted nd sline treted rts ws sttisticlly significnt with the P vlue of < 0.0001 [Tle 1]. Morphometric evlution of the dissected mndiulr re reveled significnt mount of lveolr one loss in the CsA group rts when compred to the sline treted rts [Figure 2]. However, some res lso showed concomitnt lveolr one formtion nd resorption. On histologicl exmintion, the lveolr one resorption s mesured from the cusp tip to the lveolr crest ws found to e sttisticlly significnt (P < 0.01) [Figure 3] in the CsA treted rts s compred with sline treted rts. Such resorption sites were more pronounced nd frequently seen in the CsA treted specimen thn in the sline treted specimen [Figure 4]. The histologicl exmintion lso reveled drkly stined thick lyer of mssive NC on the root surfces in the CsA treted rts s compred to sline treted rts. Acellulr type of cementum covered the coronl root dentin of the mndiulr first molrs of CsA group rts, wheres cellulr cementum ws more evident on the Tle 1: Chnge in the gingivl dimension (in millimeters) etween sline treted nd CsA treted rts with stndrd devition nd P vlue from t test Sl. no Gingivl dimension in sline treted rts (mesured in millimeter) Gingivl dimension in CsA treted (mesured in millimeter) Bli Mdi Vhi Bli Mdi Vhi 1 2 2 1.3 0.7 3.1 2.4 3.2 3 2.1 1.7 1 3.2 2.3 2.3 4 2 1.6 0.9 2.9 2.2 2.4 5 1.9 1.6 1 3.1 2.4 2.4 6 2.1 1.7 1 2.7 2 2.5 Averge 2.03 1.55 0.9 2.93 2.36 2.56 SD 0.082 0.16 0.13 0.24 0.30 0.33 t-test P vlue 0.000624 0.005369 0.000248 CsA: Cyclosporin A 804 Dentl Reserch Journl / Novemer 2013 / Vol 10 / Issue 6
Figure 2: Morphometric ssessment of lveolr one level () no chnge in sline treted rts () reduced in cyclosporin A treted rts Figure 3: Grph showing lveolr one loss in cyclosporin A treted nd sline treted rts Figure 5: Thickness of new cementum () no chnge in sline treted rts () incresed in cyclosporin A treted rts Figure 4: Histologicl ssessment of lveolr one level () no chnge in sline treted rts () reduced in cyclosporin A treted rts mesured in microns Figure 6: Grph showing new cementum formtion in cyclosporin A treted nd sline treted rts picl third of roots. In some segments, the cementum consisted of two or more lyers; the externl lyer showed strited structure nd presented suggestive imges of inclusion of collgen fiers originting from the underlying connective tissue. The djcent gingivl connective tissue ws composed of lrge mounts of dense collgen undles inserting into the NC [Figure 5]. The cementolsts were situted close to the cementum surfce in the periodontl ligment nd constituted ovoid or round, plump cells with sophilic cytoplsm. These cells were present singly or in groups of up to 4 cells. The cementocytes were present in the cementl lcuns. In the sline treted rts, norml thickness of cementum ws seen without ny oservle increse in the thickness of the cementum. In the CsA treted rts, the men increse in the thickness of cementum ws found to e 13.183 μ s compred to sline treted rts (4.733 μ). The difference etween these two groups ws sttisticlly significnt with the P vlue of < 0.0001 [Figure 6]. Dentl Reserch Journl / Novemer 2013 / Vol 10 / Issue 6 805
DISCUSSION Jysheel nd Meht: Cyclosporin A trgeting periodontl tissues Though the role of cyclosporine A on the individul periodontl tissues hs een evluted in severl niml nd humn studies, the ssessment of its effects on ll the three periodontl tissues (gingiv, lveolr one nd cementum) in single experimentl study mkes it unique from other studies. In the present morphometric nd histologicl nlysis on rts, CsA ws found to ct differentilly on ll three periodontl trget tissues: GO, lveolr one loss nd NC formtion, fter n experimentl period of 45 dys. These findings support the oservtions mde y other uthors in their ddressing this issue. [4-8,12-14] The CsA-induced GO hs een oserved in vrious niml models [4,5] nd humn clinicl studies. [6] In the present morphometric ssessment of the drug induced GO model, cliniclly evident increse in the dimensions of gingiv with significnt chnge in the morphology ws oserved in the CsA group rts s compred to the sline treted rts, in 45 dy experimentl period. Though the exct mechnism is not cler, it hs een proposed tht the drug cts directly or indirectly on the growth nd function of oth gingivl firolsts nd collgen fiers through its ltertion in vrious growth fctors nd cytokines. [15] The potent CsA effect on TGF-β trnscription nd secretion hs een oserved in vitro in humn T lymphocytes, mouse proximl tuulr cell lines nd tuule-interstitil firolsts nd in vivo in mouse nd rt kidneys. [16] It is possile tht CsA cts through similr pthwy of development nd regression of CsA-induced GO during tretment. There re evidences to show tht CsA cuses incresed one turnover, with higher resorption thn formtion, resulting in one loss. [7,9,10,17] CsA-induced osteopeni hs een reported to e ssocited with n incresed incidence of one frctures. [17] Studies hve shown tht CsA therpy in rts hs lso cused incresed osteoclsi nd decresed one formtion t periodontl sites. [7] However in contrst to these findings, there re reports of significntly less lveolr one loss nd more one formtion in the experimentl periodontitis model in rts treted with CsA, iphsic effects of CsA on osteolst differentition nd one formtion ws oserved. [15-17] Studies hve postulted tht CsA exerts its osteopenic effect through the T-cell rther thn directly on one. [9] Other hypothesis my e tht, CsA medites its osteopenic effect y interfering in the cytokine ctivity on oth osteoclsts nd osteolsts t the one microenvironment; thus, influencing one remodeling. [17] One recent study suggested tht folic cid my hve protective effect on the CsA-induced lveolr one loss. [10] In the present morphometric nd histologicl study, evlution of the specimens reveled significnt mount of lveolr one loss in the CsA group rts s compred to control (sline) group; though, in some res, there ws lso n evidence of concomitnt formtion nd resorption of lveolr one. These results re in greement with the findings of erlier studies. [7,9,10,17] Cementum plys key role in the periodontl ttchment pprtus through the gingivl nd periodontl connective tissue fier insertion. However in periodontl disese, this ttchment pprtus including the cementum is dversely ffected owing to the inflmmtory process resulting in the suspect integrity nd stility of the tooth. Hence, one of the min ojectives of periodontl therpy includes the regenertion nd reformtion of these lost periodontl tissues including the cementum. The present histologicl study reveled lrge mount of NC formtion minly in the cervicl res with insertion of newly formed collgen fiers into it ut no such chnges were seen in the sline group [Figure 5]. The NC is chrcterized y (1) its lrge volume (2) irregulr outline (3) rther infrequent oservtions of incrementl outlines nd (4) presence of gloulr odies. Connective tissue djcent to the newly formed cementum shows densely pcked collgen fiers nd were seen to e functionlly inserting into the newly formed cementum. This finding ws in ccordnce with the oservtions mde in the previous studies. [12-14] However, present study stnds different in terms of providing oth quntittive nd qulittive dt of the CsA-induced NC formtion. TGF-β over-production due to CsA my e one of the mechnisms tht could explin the greter thickness of cementum formtion. [17] Another report demonstrted tht CsA my influence oth prolifertion nd differentition of humn periodontl ligment cells, which my ply n importnt role in the homeostsis of periodontl tissue. [1] Frequently, new ttchment requires NC formtion to replce disesed root surfces contminted with the cteril endotoxins, which re eliminted during periodontl therpy. The formtion nd regultion of NC in humns is not so cler. The connective tissue mtrix of cementum sequesters growth fctors such s the TGF-, firolst growth fctors nd cementum 806 Dentl Reserch Journl / Novemer 2013 / Vol 10 / Issue 6
derived growth fctor s well s ttery of other polypeptides, including osteopontin, one siloprotein- II nd cementum-specific ttchment protein, which medite cell dhesion nd spreding. These molecules ffect the migrtion, ttchment nd prolifertion of periodontl cells nd their mtrix synthesis nd more importntly, they mnifest cell specificity nd tissue specificity mong the sme cell type. The extrcellulr mtrix of cementum hs the potentil to regulte the differentition of precursor cells into cementolsts. Thus, cementum components re cple of providing informtionl signls for the recruitment, prolifertion nd differentition of periodontl cells nd regulte the regenertion of cementum s well s djcent periodontl components. [11,18] One of the experimentl studies demonstrted tht CsA dministrtion leds to the formtion of new cementum like islets (NCLI) inside the gingivl connective tissue. These islets were locted djcent to lood vessels ner the root surfces. NCLI were found to e engulfed y the multinucleted cells due to their loction ner the lood vessels. [19] Previous investigtions reveled tht NC formed during CsA dministrtion is mintined even fter suspension of the tretment. [20] There is potentil for future studies on different doses of CsA on gingiv, lveolr one nd NC. From this experimentl study, it cn e concluded tht periodontl regenertion, which is n ultimte gol of ny periodontl therpy cn e chieved through CsA induced cementogenesis. Since systemic dministrtion CsA t the clinicl level to chieve cementogenesis nd periodontl regenertion will e ethiclly unwrrnted nd uncceptle, further niml studies cn e conducted to ssess the effect of locl ppliction of CsA on the plned root surfces would result in cementogenesis nd periodontl regenertion. Hence within the limits of this experimentl study, it cn e concluded tht CsA in immunosuppressive dose on rts hs got its negtive effect on gingiv nd one, chrcterized y GO nd lveolr one loss. However, it hs lso got one positive effect on cementum where it cuses NC formtion with the insertion of new collgen fiers. When ll these effects of CsA re considered, it cn e concluded tht CsA hs its trget on periodontl tissue cusing GO, lveolr one loss nd NC formtion nd this could e new trid of CsA effects on periodontium. CONCLUSION CsA trgets the periodontl tissues (gingiv, lveolr one nd cementum) in different pttern. Its role in cementogenesis cn e utilized for periodontl regenertion if its locl ppliction is testified nd verified in the future niml studies. ACKNOWLEDGEMENTS We would like to cknowledge the help nd coopertion given y Dr. Ahmed Muji, Professor nd Hed, Deprtment of Orl Pthology nd Microiology for crrying out the histopthologicl work for this study. REFERENCES 1. Spolidorio LC, Spolidorio DM, Holzhusen M. Effects of long-term cyclosporin therpy on the periodontium of rts. J Periodontl Res 2004;39:257-62. 2. Nieh S, Fu E, Chng HL, Wng SL, Wikesjö UM. Histopthologic ltertions of periodontium in cyclosporin-treted rts. Is the periodontium trget tissue for the drug? J Clin Periodontol 1996;23:730-6. 3. Aynoglou CM, Lesty C. New cementum formtion induced y cyclosporin A: A histologicl, ultrstructurl nd histomorphometric study in the rt. J Periodontl Res 1997;32:543-56. 4. Prhu A, Meht DS. A morphologic comprison of gingivl chnges influenced y cyclosporin nd tcrolimus in rts: An experimentl study. J Periodontol 2006;77:265-70. 5. Cetinky BO, Acikgoz G, Keles GC, Ays B, Korkmz A. The effect of cyclosporin A on lveolr one in rts sujected to experimentl periodontl disese. Toxicol Pthol 2006;34:716-22. 6. Afonso M, Bello Vde O, Shili JA, Sposto MR. Cyclosporin A-induced gingivl overgrowth in renl trnsplnt ptients. J Periodontol 2003;74:51-6. 7. Fu E, Hsieh YD, Nieh S, Wikesjö UM, Liu D. Effects of cyclosporin A on lveolr one: An experimentl study in the rt. J Periodontol 1999;70:189-94. 8. Murthy MB. Osteoimmunology Unleshing the concepts. J Indin Soc Periodontol 2011;15:190-8. 9. Buchinsky FJ, M Y, Mnn GN, Rucinski B, Bryer HP, Romero DF, et l. T lymphocytes ply criticl role in the development of cyclosporin A-induced osteopeni. Endocrinology 1996;137:2278-85. 10. Mohmmdi A, Omrni L, Omrni LR, Kini F, Eshrghin A, Azizi Z, et l. Protective effect of folic cid on cyclosporineinduced one loss in rts. Trnspl Int 2012;25:127-33. 11. Andrukhov O, Mtejk M, Rusch-Fn X. Effect of cyclosporin A on prolifertion nd differentition of humn periodontl ligment cells. Act Odontol Scnd 2010;68:329-34. 12. Aynoglou CM. Evidence tht cyclosporin A dministrtion induces the formtion of new cementum-like islets inside the gingivl connective tissue. J Periodontl Res 1998;33:166-71. Dentl Reserch Journl / Novemer 2013 / Vol 10 / Issue 6 807
13. Spolidorio LC, Spolidorio DM, Holzhusen M, Nssr CA, Nssr PO. Cyclosporin A-induced new cementum formtion: A morphometric evlution in the peripicl region of rts. Brz Dent J 2007;18:24-8. 14. Fu E, Nieh S, Chng HL, Wng SL. Cyclosporin A-induced gingivl overgrowth in rts: Mcroscopic nd microscopic oservtions. Int J Periodontics Restortive Dent 1996;16:278-91. 15. Che HJ, H MS, Yun DH, Pe HO, Chung HT, Che SW, et l. Mechnism of cyclosporine-induced overgrowth in gingiv. J Dent Res 2006;85:515-9. 16. Spolidorio LC, Spolidorio DM, Holzhusen M, Nssr PO, Nssr CA. Effects of long-term cyclosporin therpy on gingiv of rts Anlysis y stereologicl nd iochemicl estimtion. Brz Orl Res 2005;19:112-8. 17. Yeo H, Beck LH, McDonld JM, Zyzfoon M. Cyclosporin A elicits dose-dependent iphsic effects on osteolst differentition nd one formtion. Bone 2007;40:1502-16. 18. Serkov N, Christins U. Trnsplnttion: Toxicokinetics nd mechnisms of toxicity of cyclosporine nd mcrolides. Curr Opin Investig Drugs 2003;4:1287-96. 19. Aynoglou CM. Phgocytosis of new cementum-like islets formed inside the gingivl connective tissue in cyclosporin-a treted rts. J Periodontl Res 1999;34:65-9. 20. Aynoglou CM, Lesty C. Mintennce of new cementum formed during cyclosporin A dministrtion fter suspension of the tretment. J Periodontl Res 1997;32:614-8. How to cite this rticle: Jysheel M, Meht DS. The role of cyclosporine A on the periodontl tissues. Dent Res J 2013;10:802-8. Source of Support: We would like to thnk JJM medicl College, Dvngere, Indi for Animl House fcility nd Bpuji Dentl College & Hospitl, Dvngere, Indi for the Lortory fcility. Conflict of Interest: None declred. Announcement Android App A free ppliction to rowse nd serch the journl s content is now ville for Android sed moiles nd devices. The ppliction provides Tle of Contents of the ltest issues, which re stored on the device for future offline rowsing. Internet connection is required to ccess the ck issues nd serch fcility. The ppliction is comptile with ll the versions of Android. The ppliction cn e downloded from https://mrket.ndroid.com/detils?id=comm.pp.medknow. For suggestions nd comments do write ck to us. 808 Dentl Reserch Journl / Novemer 2013 / Vol 10 / Issue 6