Orl Surgery, Orl Medicine, Orl Rdiology, 2015, Vol. 3, No. 1, 20-24 Avilble online t http://pubs.sciepub.com/orl/3/1/5 Science nd Eduction Publishing DOI:10.12691/orl-3-1-5 The Effect of Mouth Rinsing with Stbilized Chlorine Dioxide on Periodontitis Shuichi Tsubur 1,2,*, Kimio Uchiym 2,3, Mnbu Ymd 2, Osmu Mimori 2, Shigeru Hiym 2, Ktsum Ktoh 2, Mski Shibt 2 1 Deprtment of Biochemistry, The Nippon Dentl University School of Life Dentistry t Niigt, Hmur-cho, Chuo-ku, Niigt, Jpn 2 Deprtment of Dentistry nd Orl surgery, NHO Tochigi Medicl Center, Nktomtsuri, Utsunomiy, Tochigi, Jpn 3 Clinicl Reserch Deprtment, NHO Tochigi Medicl Center, Nktomtsuri, Utsunomiy, Tochigi, Jpn *Corresponding uthor: tshu@uctv.ne.jp Received July 05, 2015; Revised July 26, 2015; Accepted August 02, 2015 Abstrct MATATACORO TM (contining 10 mg/l stbilized chlorine dioxide: MA-T, no lcohol, nd is n odorless ntibcteril gent) is useful for steriliztion nd disinfection. The im of this study ws to investigte whether MA-T is n pproprite mouth rinse for ptients selected from whom undergoing initil periodontl tretment. The effect of rinsing with MA-T ws compred with tht with Concool TM (contining 0.05% chlorhexidine gluconte: CX), Neosterine Green TM (contining 0.02% benzethonium chloride: NG), nd purified wter (PW). MA-T rinsing resulted in mrked chnge in the BANA-score nd in the VAS scle. The men BANA scores (score ± SD) on dy 0 nd dy 30 were 1.55±0.50 nd 0.4±0.50 (p<0.001) for MA-T, 1.65±0.48 nd 0.75±0.44 for CX, 1.43±0.50 nd 0.88±0.61 for NG, nd 1.83±0.38 nd 1.48±0.55 for PW, respectively. The introrl feeling cused by mouth rinsing ws self-ssessed using the VAS scores (men ± SD) on dy 0 nd dy 30; they were 48.23±14.70 nd 74.43±7.33 (26.2 mm increse) for MA-T, 50.45±11.94 nd 62.23±8.88 (11.8 mm increse) for CX, 54.13±8.11 nd 63.73±8.18 (9.6 mm increse) for NG, nd 54.70±6.06 nd 59.18±6.11 (4.5 mm increse) for PW, respectively. These results suggest tht MA-T induced mrked reduction of periodontopthic orl bcteri nd incresed feeling of freshness fter the tretment period. These results suggest tht MA-T is n pproprite mouth rinse for ptient with periodontitis. Keywords: stbilized chlorine dioxide, mouth rinse, periodontitis, orl bcteri, freshness Cite This Article: Shuichi Tsubur, Kimio Uchiym, Mnbu Ymd, Osmu Mimori, Shigeru Hiym, Ktsum Ktoh, nd Mski Shibt, The Effect of Mouth Rinsing with Stbilized Chlorine Dioxide on Periodontitis. Orl Surgery, Orl Medicine, Orl Rdiology, vol. 3, no. 1 (2015): 20-24. doi: 10.12691/orl-3-1-5. 1. Introduction Periodontitis, chronic inflmmtory disese of the tissues supporting the teeth, is cused by multiple fctors, including orl bcteri, nd is bsed on person s genetic profile nd environmentl fctors. [1] Therefore, there is no complete tretment technique for periodontitis. The first periodontl tretment is elimintion of periodontopthic bcteri from the subgingivl re. Orl hygiene guidnce s prt of generl tretment is usully selected s the first-choice tretment. The generl tretments for periodontitis consist of bsiclly periodontl initil tretments, such s scling, root plning, nd periodontl surgery, or combintion of surgicl nd chemicl methods including the use of ntibcteril drugs, prticulrly moxicillin, tetrcycline, nd zithromycin. [2,3] However, the use of ntibcteril drugs s the conventionl tretment method is ssocited with number of risks, including the production of drug-resistnt bcteri nd llergic rections in elderly individuls [4]. On the other hnd, it is well known tht the use of mouth rinses hs n effective nd distinct role in the mngement of periodontitis both in the erly stge nd fter generl tretments. [5,6,7] The most populr products for mouth rinsing re those contining chlorhexidine gluconte or benzethonium chloride with lcohol. However, they hve n unplesnt tste, their formultions hve hrsh tste, they stin teeth, nd they excessively stimulte the surfce of the orl mucos becuse of the high lcohol content. Therefore, lterntive mouth rinses not contining lcohol re needed for the tretment of periodontitis. The commercilized solution MATATACORO TM (MA- T: ACENET Inc., Tokyo, Jpn) contining 10 mg/l stbilized chlorine dioxide, which neither stins nor hs hrsh tste, hs ntibcteril efficcy without lcohol. Stbilized chlorine dioxide is compound with ntimicrobil properties ginst not only orl bcteri but lso vriety of bcteri. [8] Though orl rinsing with stbilized chlorine dioxide is expected to reduce the number of bcteri in the mouth, [9] there hve been no reports on the clinicl ctivity of this product. This is the first clinicl study to evlute whether the orl dministrtion of MA-T reduces the levels of periodontopthic bcteri. The effect of MA-T on periodontl inflmmtion ws exmined in chronic periodontitis ptients.
Orl Surgery, Orl Medicine, Orl Rdiology 21 2. Mteril nd Methods 2.1. Ptients nd Study Design From Jnury 2013 to November 2014, 160 subjects (80 mles nd 80 femles; non-smokers; ge, 46.4 ± 15.0 yers (men ± SD); ge rnge 25-73 yers) with chronic periodontitis nd no other severe generl diseses were selected s prticipnts. The inclusion criteri for pplicnts were: (1) hving t lest 20 nturl teeth, nd (2) t lest eight teeth with probing periodontl pocket depth (PD) of > 4 mm on molr teeth. Teeth with excess mobility nd/or bscess formtion were excluded from the study. All prticipnts were selected from ptients referred to the uthors clinics. The study protocol ws pproved by the Committee of Ethics Affirs of NHO Tochigi Medicl Center nd ws conducted ccording to the principles outlined in the Declrtion of Helsinki for experiments involving humn subjects. The subjects provided their written, informed consent before prticipting in the 30- dy study nd were confirmed to meet the following criteri: (1) received initil periodontl tretment, such s dentl clening; (2) took no commercilized mouth-rinse within the previous month; nd (3) took no ntibiotic drugs within the previous month. Dentl clening served s dentl prophylxis to remove ll supr-gingivl plque, stin, nd clculus before the study. After the strt of this study, ll prticipnts did not received professionl prophylxis or dditionl brushing using ultrsonic toothbrushes or power toothbrushes. A rndomized, double-blind, plcebo-controlled study design with four prllel groups ws used. The subjects were seprted rndomly into four groups to receive MATATACORO TM [MA-T] (n = 40), Concool TM (contining 0.05% chlorhexidine gluconte) [CX] (n = 40), Neosterine Green TM (contining 0.02% benzethonium chloride) [NG] (n = 40), nd purified wter [PW] group (n = 40), respectively (Tble 1). Tble 1. Distribution of study prticipnts Group N Sex Age, yers (men ± SD) MA-T 21 Mle 46.4 ± 15.0 19 Femle 47.0 ± 14.4 CX 18 Mle 48.3 ± 13.2 22 Femle 46.4 ± 14.3 NG 22 Mle 45.4 ± 15.2 18 Femle 46.2 ± 13.2 PW 19 Mle 44.9 ± 15.5 21 Femle 45.1 ± 15.3 After the initil periodontl tretment, mouth rinsing ws done with MA-T, CX, NG, or PW (30 ml) for 30 seconds every morning nd evening for 30 dys. Tooth brushing ws done three times per dy without ny toothpste, nd none of the teeth received ny tretment during this study. All prticipnts used stndrdized tooth brushes. During the study, prticipnts kept their usul dietry hbits for tooth brushing, but they were instructed to refrin from using ny other commercil mouth rinses. Four nterior or premolr nd molr teeth were selected for ssessment of clinicl prmeters nd subjected to microbil tests; these teeth hd pocket deeper thn 4 mm nd similr probing depths nd gingivl inflmmtion levels. 2.2. VAS Scle The intr-orl feeling cused by mouth rinsing ws selfssessed with the use of Visul Anlog Scle (VAS), in which the ptients quntified their freshness response by mking mrk on 100-mm-long line tht ws nchored by word descriptors t ech end: unplesnt t the left end nd very refreshed t the right end. The ptients mrked the point on the line tht described their perception of the feeling. Ptients were sked: How fresh do you feel tody? Plce verticl mrk on the line below to indicte how you feel tody. The VAS scle ws determined by mesuring the distnce in millimeters from the left-hnd end of the line to the point tht the ptient mrked.[10,11] Intr-orl feeling ws mesured for ll of the mouth nd ws cquired by one clinicin on dy 0, dy 14, nd dy 30. It ws evluted by the difference of VAS scores between dy 0 nd dy 14 or dy 30. 2.3. Clinicl Prmeters Probing Pocket Depth (PPD), Bleeding on Probing (BOP), nd the Gingivl Index (GI) were ssessed for ll prticipnts on six sites per tooth t bseline (dy 0), dy 14, nd dy 30. PPD nd BOP were ssessed using mnul periodontl probe (CP10SE; HuFriedy, Chicgo, IL, USA). For ssessing PPD, periodontl probe equipped with 0.5-mm-dimeter tip ws inserted into the gingivl crevice nd swept from the distl to the mesil spect of the tooth t depth of pproximtely 1 mm nd n ngle of pproximtely 60 while mintining contct with the sulculr epithelium. BOP ws then recorded nd scored s present/bsent by running the probe 1-2 mm into the gingivl crevice. Gingivitis of the buccl nd lingul mrginl gingiv nd the interdentl ppille of ll scorble teeth ws scored using the Loe-Silness GI on 4- point scle from 0 (bsence of inflmmtion) to 3 (severe inflmmtion) [12]. 2.4. Bcteril Assy The BANA test (Knowell Therpeutic Technologies Inc., Toronto, Cnd) is chir-side dignostic system tht is highly sensitive nd specific for determining the presence of red-complex periodontl pthogens (Porphyromons gingivlis, Treponem denticol, nd Tnnerell forsythis). These bcteri hve been implicted in periodontl disese nd its progression.[13] The products of this rection cn be demonstrted by the color rection on regent strip s blue-blck product. Assessments were mde s the reltive intensity of three ctegories of the blue-blck color chnge (strong positive, positive, or negtive). Plque ws collected for BANA nlysis from the sme tooth sites tht were used for ssessing the clinicl prmeters t dy 0, dy 14, nd dy 30. After incubtion of plques smples for 5 min t 35 C with Evn s blck dye solution, nphthylmine, which is relesed s result of the presence of ny BANAhydrolyzing bcteril species, diffuses to form permnent blue-blck color. The reltive intensity of the formed blue color (strong positive, positive, or negtive) ws ssessed.
22 Orl Surgery, Orl Medicine, Orl Rdiology 2.5. Sttisticl Anlysis All dt nlysis ws performed using the Mnn- Whitney U-test with IBM SPSS sttistics ver.19. Differences of p < 0.05 were considered significnt. 3. Results A totl of 160 prticipnts with chronic periodontitis were selected ccording to the study design, nd ll prticipnts completed the study. Between dy 0 nd dy 30, men difference of VAS scores were ech 26.2mm(MA-T), 11.8mm(CX), 9.6mm(NG) nd 4.5mm(PW). MA-T ws significntly more difference compred with PW. CX nd NG were significntly more difference compred with PW. The improvement of VAS scores between dy 0 nd dy 14 or dy 30 re shown in Tble 2. The men PPD chnges from dy 0 to dy 30 re shown in Tble 3. The BOP scores from dy 0 to dy 30 re shown in Tble 4.The men GI scores from dy 0 to dy 30 re shown in Tble 5. PPD, BOP, nd GI ll decresed to dy 30, but mrked differences were not seen between MA-T nd CX, NG, nd PW, respectively. The men BANA scores from dy 0 to dy 30 re shown in Tble 6. MA-T ws significntly more effective in lowering BANA scores t dy 14 (p<0.01) nd dy 30 (p<0.001) thn CX, t dy 30 (p<0.001) thn NG, nd t dy 14 (p<0.005) nd dy 30 (p<0.001) thn PW. Tble 2. Men difference of VAS scle (scores ± SD) with Mttcoro TM, CX, NG, nd PW Given these results, MA-T ppers to reduce periodontl orl bcteri fter periodontl tretment nd increses the feeling of freshness fter the tretment period. 4. Discussion In chronic periodontitis, it hs been thought tht mintining dequte levels of orl hygiene using mechnicl methods lone is impossible, nd this hs led to the djunctive use of ntimicrobil mouth rinses in orl hygiene regimens to help prevent nd control chronic periodontitis [15]. Recently, 0.02% chlorhexidine mouth rinse hs become widely used in mny countries. However, becuse of certin side effects ssocited with this gent, in prticulr llergic rections, tooth stining, clculus formtion, nd tste berrtions, it ws deemed desirble to use lower doses of this gent, if possible. Thus, there is need for lterntive mouth rinses for the tretment of periodontitis. This ws the first clinicl study of the effects of stbilized chlorine dioxide (MA-T) on periodontitis in humn subjects. It hs neither smell nor hrsh tste, nd despite its mildness, which is similr to tht of wter, significnt improvements in VAS scle scores were seen compred to other grgles. In this study, we estimted tht MA-T hs strongly improved on VAS score, nd CX nd NG lso showed similr results. Ech mteril hs powers decresing orl bcteri by long term use. However MA-T hs neither smell nor hrsh tste, nd hs especilly the mildness those of similr to wter, MA-T showed remrkble chnge on VAS score thn the others. Thus, in totlly, MA-T is more suitble mouth rinse thn other mterils. The mechnism, in which MA-T would cuse fresher tste or dvntged mteril, hs not been uncler. It might to be tht MA-T hs neither smell nor hrsh tste nd re neutrl ph, nd the chnge of BANA score indicted the trget bcteri inside the periodontl pocket were decresed by the MA-T rinsing. Robert et l. reported [16] tht phosphte-buffered chlorine dioxide solution ws effective in short-term trils for control of wterline contmintion in ultrsonic dentl scling units. Furthermore, John et l. reported[17] tht 0.04% stbilized chlorine dioxide, 3% sodium hypochlorite, nd 2% chlorhexidine were effective s endodontic irrignts in polymicrobil biofilm. However, both studies used bovine tooth model system, not humn subjects in the clinicl setting. From the beginning, stbilized chlorine dioxide ws developed for dentl units nd ultrsonic scler wterline contmintion with potentil pthogens such s lph-hemolytic streptococci nd some Grm-negtive rods. [18,19,20,21] Stbilized chlorine dioxide solution is more fvorble for some ptients with periodontitis, better thn other mterils. MA-T hs no objectionble pungent odor or bitter tste in its use s lvge with ultrsonic scler wter line. Thus, it might be useful nd effective for use s mouth rinse. MA-T is expected to be more vluble for mouth rinsing in ptients who hve been treted for chronic periodontitis for long periods or who hve xerostomi becuse it provides wek stimulus nd flvor. The present results suggest tht MA-T could be effective for improving conditions in the periodontl
Orl Surgery, Orl Medicine, Orl Rdiology 23 pocket nd orl cvity of ptients with chronic periodontitis. To minimize potentil bis from this study, double-blind, prllel, rndomized, simple clinicl study ws performed, which included 160 subjects of both sexes rnging in ge from 23 to 84 yers. In order to confirm the results of the present study, further essentil microbil experiments hve to be performed on isolted periodontl pthogens; it is especilly importnt to exmine redcomplex pthogens in culture nd monitor their growth in the presence of MA-T, CX, NG, nd PW. Red-complex pthogens re mong the most recognizble precursors inititing cscde of inflmmtory meditors, nd n importnt chnge is the shift in the composition of microflor due to mny fctors, which results in microbil communities dominted by nerobic bcteri. Tble 3. Men Probing Pocket Depth (mm ± SD) with Mttcoro TM, CX, NG, nd PW Group N Dy 0 Dy 14 Dy 30 MA-T 40 5.00 ± 0.75 4.65 ± 0.77 4.48 ± 0.75 CX 40 4.90 ± 0.71 4.40 ± 0.71 4.03 ± 0.70 NG 40 4.53 ± 0.68 3.88 ± 0.61 3.50 ± 0.51 PW 40 4.90 ± 0.59 4.50 ± 0.60 4.38 ± 0.59 Tble 4. Men Bleeding on Probing (BOP ) scores ± SD with Mttcoro TM, CX, NG, nd PW Group N Dy 0 Dy 14 Dy 30 MA-T 40 1.63 ± 0.49 1.38 ± 0.49 1.13 ± 0.52 b CX 40 1.60 ± 0.50 1.15 ± 0.53 0.65 ± 0.58 b NG 40 1.45 ± 0.50 1.23 ± 0.48 0.88 ± 0.56 b PW 40 1.80 ± 0.41 1.50 ± 0.55 1.35 ± 0.53 BOP scoring: 0 is non-bleeding to slight bleeding, tooth site of 0-30%; 1 is moderte bleeding, tooth site of 31-60%; 2 is severe bleeding, tooth site of more thn 60%. b : Significnt difference from dy 0, p<0.05 The present results suggest tht MA-T hs the cpcity to control chronic or cute periodontitis. Bcteriotherpy by mouth rinsing provides more promising new reserch field of dentl science thn conventionl orl helth tretment. MA-T showed mrked chnges in the BANA test, but PPD, BOP, nd GI did not show mrked chnges between MA-T nd CX, NG, PW (Tble 3-Tble 5). Tble 5. Men Gingivl index (GI ) scores ± SD with Mttcoro TM, CX, NG, nd PW Group N Dy 0 Dy 14 Dy 30 MA-T 40 1.65 ± 0.74 1.25 ± 0.67 1.05 ± 0.68 c CX 40 1.83 ± 0.59 1.28 ± 0.60 0.83 ± 0.59 c NG 40 1.65 ± 0.74 1.20 ± 0.72 0.88 ± 0.61 c PW 40 1.95 ± 0.55 1.55 ± 0.55 1.25 ± 0.59 c GI scores: 0 is norml, 1 is slight gingivl inflmmtion (no bleeding using instrument), 2 is moderte gingivl inflmmtion (bleeding using instrument), 3 is gingivl bscess, bleeding c : Significnt difference from dy 0, p<0.05 In the present study, the effect of MA-T on periodontopthic orl bcteri ws evluted, nd it ws found tht MA-T dministrtion successfully decresed the numbers of periodontopthic bcteri in dentl plque fter 30 dys. MA-T is mildly effective for periodontl tissues, such s gingivl epithelium nd gingivl cpillries. Thus, the men PPD, BOP, nd GI did not show mrked chnges. Otherwise, the men BANA-score showed mrked chnges with MA-T, better thn CX, NG, nd PW. The trget bcteri were significntly decresed fter 30 dys in the MT group (Tble 6). It is likely tht MA-T could ply greter role in situtions requiring improvement of gingivl creviculr fluid ctivity, lthough the mechnisms re still uncler. As the MA-T mouth rinse is reltively simple tretment method, MA- T is useful s homecre mteril for preventing periodontl diseses. It is lso well known tht reduction of periodontl pthogens is importnt for clinicl improvement nd to decrese the risk of disese relpse. [14] Therefore, mouth rinsing with MA-T fter initil tretment with periodontl mechnicl debridement might represent n effective pproch for the tretment of periodontitis. Tble 6. Men BANA-score ± SD with Mttcoro TM, CX, NG nd PW BANA scores: 0 is negtive, 1 is positive, 2 is strong positive MA-T ws significntly more effective with lowering BANA scores t dy14 nd dy 30 compred with CX, t dy 30 compred with NG, t Dy14 nd Dy 30 compred with PW. *: p<0.05 Therefore, more reserch is needed to identify pproprite effector strins for mouth rinsing specificlly designed to prevent nd tret periodontl disese. The present experimentl protocol did not include ny orl hygiene instruction before tretment or t bseline; however, subjects in ll groups might hve systemticlly ltered their orl hygiene regimens due to the observtion of routine dentl work. Mny fctors, including ttention bis, contribute to perceived plcebo effects in clinicl trils. However, this is n importnt first step, nd one tht provides vluble informtion s to reltive levels of ctivity ginst orl bcteri in very stndrdized lbortory setting.
24 Orl Surgery, Orl Medicine, Orl Rdiology 5. Conclusions MA-T is n effective orl mouth rinse for ptients with periodontitis. MA-T mouth rinsing could be useful for the tretment of inflmmtion nd the clinicl symptoms of periodontitis. The mechnism underlying the ntibcteril effect of the MA-T mouth rinse is proposed to involve the inhibition of proteses tht originte from orl bcteri. Furthermore, MA-T mouth rinse my contribute to humn helth cre with respect to not only orl diseses, but lso the control of host immunologicl responses. Acknowledgments The uthors would like to thnk Dr Hiromi Shimomur for helpful nd criticl discussions. Sttement of Competing Interests The uthors hve no competing interests. List of Abbrevitions MA-T : MATATACORO TM CX : Concool TM NG : Neosterine Green TM PW : Purified wter VAS : Visul Anlog Scle PPD : Probing Pocket Depth BOP : Bleeding on Probing GI : Gingivl Index References [1] Nishimur, F., Iwmoto, Y., Mineshib, J. et l, Periodontl disese nd dibetes mellitus: the role of tumor necrosis fctorlph in 2-wy reltionship, J Periodontol, 74(1). 97-102. Jn. 2003. [2] Becker, W., Becker, B.E., Cffesse, et l, A longitudinl study compring scling, osseous surgery, nd modified Widmn procedures: results fter 5 yers, J Periodontol, 72(12). 1675-84. Dec. 2001. [3] vn Winkelhoff, A.J., Rms, T.E. nd Slots, J., Systemic ntibiotic therpy in periodontitis, Periodontol 2000, 10. 45-78. Feb. 1996. [4] Slots, J. nd Rms, T.E., Antibiotics in periodontl therpy: dvntges nd disdvntges, J Clin Periodontol, 17(7). 479-93. Aug. 1990. [5] Cincio, S.G., Luciello, F., Shibly, O., et l, The effect of n ntiseptic mouthrinse on implnt mintennce: Plque nd periimplnt gingivl tissues, J Periodontol, 66(11). 962-65. Nov. 1995. [6] Christine, H., Chrles, R.D.H., Nresh, C., et l, Comprtive efficcy of n ntiseptic mouthrinse nd n ntiplque/ntigingivitis dentifrice, J Am Dent Assoc, 132(5). 670-75. My. 2001. [7] Chrles, C.H., Mostler, K.M., Brtels, L.L., et l, Comprtive ntiplque nd ntigingivitis effectiveness of chlorhexidine nd n essentil oil mouthrinse: 6-month clinicl tril, J Clin Periodontol, 31(10). 878-84. Oct. 2004. [8] Wirthlin, M.R., Ahn, B.J., Enriquez, B., et l, Effects of stbilized chlorine dioxide nd chlorhexidine mouthrinses in vitro on cells involved in periodontl heling, J West Soc Periodontol Periodontl Abstr, 54(3). 67-71. 2006. [9] Wirthlin, M.R., Choi, J.H. nd Kye, S.B., Use of chlorine dioxide mouthrinses s the ultrsonic scling lvge reduces the vible bcteri in the generl erosols, J West Soc Periodontol Periodontl Abstr, 54(2). 35-44. 2006. [10] Hollnd, G.R., Nrhi, M.N., Addy, M., et l, Guidelines for the design nd conduct of clinicl trils on dentine hypersensitivity, J Clin Periodontol, 24(11). 808-13. Nov. 1997. [11] Gould, D., Kelly, D., Goldstone, L., et l, Exmining the vlidity of pressure ulcer risk ssessment scles: Developing nd using illustrted ptient stimultions to collect the dt, J Clin Nursing, 10(5). 697-706. Sep. 2001. [12] Loe, H. nd Silness, J., Periodontl disese in pregnncy. 1. Prevlence nd severity, Act Odontol Scnd, 21.533-51. Dec. 1963. [13] Loesche, W.J., Bretz, W.A., Loptin, D., et l, Multi-center clinicl evlution of chirside method for detecting certin periodontopthic bcteri in periodontl disese, J Periodontol, 61(3).189-96. Mr. 1990. [14] Hffjee, A.D., Cugini, M.A., Dibrt, S., et l, Clinicl nd microbiologicl fetures of subjects with dult periodontitis who responded poorly to scling nd root plnning, J Clin Periodontol, 24(10). 767-76. Oct. 1997. [15] Anne, D.H, G. Torresyp, nd Sigmund S.S, Clinicl chnges following four different periodontl therpies for the tretment of chronic periodontitis: 1-yer results, J.Clinic Periodontol, 34. 243-253. July. 2007. [16] Robert, W., Gryson, W. nd Mrshll Jr., Evlution of ultrsonic scling unit wterline contmintion fter use of chlorine dioxide mouthrinse lvge, J Periodontol. 72(3). 401-10. Mr. 2001. [17] Lundstrom JR, Willimson AE, Vilhver AL, et l. Bctericidl ctivity of stbilized chlorine dioxide s n endodontic irrignt in polymicrobil biofilm tooth model system, J Endod, 36(11). 1874-78. Nov. 2010. [18] Fitzgibbon, E.J., Brtzoks, C.A., Mrtin, M.V., et l, The source, frequency nd extent of bcteril contmintion of dentl unit wter system, Br Dent J. 157(3). 98-101. Aug. 1984. [19] Krpy, R.I., Plmondon, T.J., Mills, S.E., et l, Combining periodic nd continuous sodium hypochlorite tretment to control biofilms in dentl unit wter systems, J Am Dent Assoc, 130(7). 957-65. Jul. 1999. [20] Willims, H.N., Pszko-Kolv, C., Shhmt, M., et l, Moleculr techniques revel high prevlence of Legionell in dentl units, J Am Dent Assoc, 127(8). 1188-93. Aug. 1996. [21] Tippett, B.F., Edwrds, J.L. nd Jenkinson, H.F., Bcteril contmintion of dentl unit wter lines- possible source of cross-infection, NZ Dent J, 84(378). 112-13. Oct. 1988.