TIME TO SHIFT: FROM TO ROOT SURFACE MARILOU CIANTAR Prim Dent J. 2014;3(3):38-42

and scientific basis of the aetiology ABSTRACT of periodontal diseases (caused by the bacterial biofilm) and the clinical Non-surgical periodontal treatment has traditionally been based on the notion methods sometimes employed in treating that bacterial plaque (dental biofilm) penetrates and infects dental . the disease (removal of and Removal of this infected cementum via scaling and root planing (SRP) was ‘infected’ cementum). considered essential for re-establishing periodontal health. In the 1980s the concept of SRP was questioned because several in vitro studies showed that It was not until the 1980s that the the biofilm was superficially located on the root surface and its disruption and traditional therapeutic method of scaling removal could be relatively easily achieved by ultrasonic instrumentation of and root planing (SRP) employed in daily the root surface (known as root surface debridement (RSD). Subsequent in vivo clinical practice was questioned by a studies corroborated the in vitro findings. There is now sufficient clinical evidence series of studies. The rationale behind to substantiate the concept that the deliberate removal of cementum by SRP is these studies was to ascertain whether no longer warranted or justified, and that the more gentle and conservative the deliberate removal of tooth (root) approach of RSD should be implemented in daily periodontal practice. tissue during SRP, in order to achieve periodontal health, could be justified. The results of these studies, both in vitro and in vivo, showed that the biofilm, rather eriodontology has been than calculus or ‘infected’ cementum, extensively researched over the was responsible for disease and that P past 50 years. This includes the it was superficially located on the root fields of periodontal pathology and surfaces of periodontally involved teeth periodontal therapy. The seminal and could be easily removed (via root work performed by Löe et al in the surface debridement (RSD) in order to mid-1960s1 confirmed the cause-and- achieve periodontal health; thus the effect relationship between bacterial deliberate removal of root substance biofilms (then referred to as dental via SRP appeared to be unjustified. plaque) and the host inflammatory response. Periodontal therapy has Treatment of periodontal focused on the importance of removing diseases: a historical ‘accretions’ and ‘infected cementum’ perspective from the root surface, with particular The first microscopic evidence of emphasis on removing calculus and bacterial accretions around teeth infected cementum as part of cause- was brought to light by Antony van related (whether non-surgical and/or Leeuwenhoek in 16832 some 200 surgical) periodontal therapy. This mode years prior to the key study by Löe et al of treatment persists to some extent (1965)1 that established the cause-and- today, even though the biofilm has been effect relationship between established as the principal cause of and the initiation of periodontal diseases. periodontal diseases. This has led to a In this study, a group of dental students rift between the theoretical knowledge who had good periodontal health were

KEY WORDS AUTHOR Marilou Ciantar BChD(Hons), MSc, PhD, Root Surface Debridement, Scaling MFDS, MFD, FFD and Root Planing, Dental Biofilm, Specialist Periodontist and Oral Surgeon, Blackhills Specialist Dental Referral Clinic, Dental Calculus, Non-surgical Aberruthven, Perthshire Periodontal Therapy

38 PRIMARY DENTAL JOURNAL asked to refrain from daily toothbrushing SRP or RSD? of calcified deposits (calculus) on teeth has for three weeks. Periodontal indices were Traditional non-surgical periodontal been the gold standard for periodontal monitored before and after these three therapy encompasses therapy for centuries. Indeed, there was weeks, after which toothbrushing was instructions and SRP performed as initial a time when this was the mainstay of reinitiated. The clinical data collated at therapy and then routinely repeated, non-surgical periodontal treatment, with the end of the study showed that dental usually on a three-monthly basis. The the emphasis placed on the removal of plaque accumulation caused gingival frequency at which this is performed all calculus, rather than the dental biofilm. inflammation and its removal led to is generally determined by the patient’s resolution of clinical symptoms and susceptibility to disease; in other Both supragingival and subgingival restitution of periodontal health. Although words, the more susceptible the patient, calculus form following accumulation this was the first scientific study that the more often SRP is repeated. All of undisturbed biofilm. The exact role showed the interaction between dental clinicians agree that the patient’s role in of subgingival calculus in the initiation biofilm and the host response, the need periodontal therapy is crucial; effective and progression of for removal of dental deposits around daily removal of biofilm performed by remains debatable.10 Clinicians are teeth had long been observed and the patient not only leads to resolution aware that there is considerable acknowledged. Indeed, a primitive form of ,1 but also profoundly variation between patients in the of the modern-day dates back impacts on the successful management amount of calculus formation, with some some 3500BC; the toothbrush, as we of periodontitis.7 Furthermore, effective being very prone to extensive calculus know it today, originated in China about daily removal of supragingival plaque formation, whereas others manifest 1600. In his 1728 thesis Le Chirurgien by the patient prevents recolonisation of very little. Often the volume of calculus Dentiste, Pierre Fauchard acknowledged periodontal pockets. But to what extent formation does not appear to be the need for ‘oral cleanliness’ and is the repeated planing of the root commensurate with the extent of disease proposed that teeth should be cleaned surface justified, given that this is a present; thus patients with aggressive periodically by the dentist.3 This mode destructive process that removes tooth periodontitis often display negligible of treatment was propagated well into structure? It is useful to define first what calculus formation. There is also the 19th and 20th centuries as the non- is meant by ‘scaling’ and ‘root planing’. considerable variation between ethnic surgical4 and surgical5 treatment of patient groups in the amount of calculus pyorrhoea alveolaris. Application of Scaling formation.11-13 Other factors that affect these scraping treatment protocols was Scaling has been defined as the amount of calculus formation include facilitated by the development of sharp instrumentation to remove all age, gender, diet, location in oral cavity, periodontal instruments by GV Black supragingival uncalcified and calcified oral hygiene, bacterial composition, (1915),6 which formed the basis of accretions and all gross subgingival host responses and access to root planing as a therapeutic technique. accretions.8,9 This mechanical removal professional treatment.14

REFERENCES effect of a plaque control program oral hygiene before 40 years of age. 1996;7(2 Spec No):58-64. on tooth mortality, caries and J Periodontal Res. 1979;14:526-40. 15 Clerehugh V, Worthington HV, 1 Löe H, Theilade E, Jensen SB. periodontal disease in adults. J Clin 12 Gaare D, Rolla G, Aryadi FJ, van der Lennon MA, Chandler R. Site Experimental gingivitis in man. Periodontol. 2004;31:749-57. Ouderaa F. Comparison of the rate of progression of loss of attachment J Periodontol. 1965;36:177-87. 8 O’Leary TJ. The impact of research formation of supragingival calculus in over 5 years in 14- to 19- year 2 Dobell C. Antony van Leeuwenhoek on scaling and root planing. an Asian and a European population. old adolescents. J Clin Periodontol. and his “Little Animalcules”. New J Periodontol. 1986;57:69-75. In: ten Cate JM, editor. Recent 1995;22:15-21. York: Dover Publications; 1960. 9 Claffey N, Polyzois I. Non-surgical Advances in the Study of Calculus. 16 Mombelli A, Nyman S, Brägger U, 3 Viau G. The manuscript of Fauchard. therapy. In: Lindhe J, Lang NP, Oxford: IRL Press; 1989 p. 115-22. Wennström J, Lang NP. Clinical and Dent Cosmos. 1923;65:823-6. Karring T, editors. Clinical 13 Anerud A, Löe H, Boysen H. The microbiological changes associated 4 Rehwinkel FH. Proceeding of and Implant natural history and clinical course with an altered subgingival dental societies. Dent Cosmos. Dentistry. 5th ed. Oxford: Blackwell of calculus formation in man. J Clin environment induced by 1877;19:567-79. Munksgaard; 2008. p. 766-79. Periodontol. 1991;18:160-70. periodontal pocket reduction. 5 Stones HH. The surgical treatment 10 Mandel ID, Gaffar A. Calculus 14 White DJ, McClanahan SF, J Clin Periodontol. 1995;22:780-7. of pyorrhoea alveolaris. Proc R Soc revisited: a review. J Clin Lanzalaco AC, Cox ER, Bacca L, 17 Albander JM, Kingman A, Brown Med. 1923;25:886-92. Periodontol. 1986;13:249-57. Perlich MA, et al. The comparative LJ, Löe H. Gingival inflammation 6 Black GV. Special Dental 11 Anerud A, Loe H Boysen H, efficacy of two commercial tartar and subgingival calculus as Pathology. Chicago, IL: Medico- Smith H. The natural history of control dentifrices in preventing determinants of disease progression Dental Publishers; 1915. periodontal disease in man. calculus development and facilitating in early-onset periodontitis. J Clin 7 Axelsson P, Lindhe J. The long-term Changes in gingival health and easier dental cleanings. J Clin Dent. Periodontol. 1998;25:231-7.

V O L 3 N O 3 AUGUST 2014 39 TIME TO SHIFT: FROM SCALING AND ROOT PLANING TO ROOT SURFACE DEBRIDEMENT

Figure 1: Periodontal healing in the presence of subgingival calculus

The pathological significance of calculus in the initiation and progression of periodontal disease has been implied based on a number of cross-sectional and longitudinal studies that have shown an association between the presence of calculus and periodontal disease;15-18 however, a cause-and-effect relationship between calculus and periodontal disease has not been established. This is because of the difficulty in performing such a study given that calculus is invariably covered with a dental biofilm, epithelial attachment is observed in the our inability to remove all calculus, our which in itself initiates an inflammatory presence of calculus (Figure 1) once patients still seem to improve following response in the host.1 the biofilm has been removed from the periodontal treatment – highlighting the surface of the calculus.21,22 Clinical importance of biofilm removal during Although calculus has a porous evidence of periodontal healing is evident therapy. The clinical benefit of scaling to structure and has been shown to harbour in daily practice even though complete remove calculus stems from the fact that several periodontal pathogens such as removal of calculus evades even the calculus interferes with the patient’s daily Aggregatibacter actinomycetemcomitans, most experienced clinicians using a plaque control regimen; in other words, and Treponema diverse range of instruments.23-25 it presents an obstacle to self-performed denticola,19 the essentially inert nature biofilm control and also serves as an of calculus has been shown in a number As clinicians, we invariably leave behind ideal substrate for bacterial colonisation. of studies. For example, it has been extensive calculus deposits after scaling26 Subgingival calculus is no different in shown that autoclaved calculus does and the amount of residual calculus this respect from supragingival calculus. not initiate an inflammatory response or seems to be proportional to the pocket It should therefore be remembered that cause abscess formation.20 Furthermore, depth and to tooth type, with molars subgingival calculus forms as a result histological evidence is available to show manifesting the greatest degree of of the disease process rather than that periodontal healing via a normal residual calculus.27 However, despite being the cause of it.28

18 Griffiths GS, Duffy S, Eaton KA, 21 Listgarten M, Ellegaard B. Electron surgical evaluation. J Periodontol. removal and the prevention of Gilthorpe MA, Johnson NW. microscope evidence of a cellular 1986;57:672-80. its formation. Periodontol 2000. Prevalence and extent of lifetime attachment between junctional 25 Schwarz F, Bieling K, Venghaus S, 2011;55:167-88. cumulative attachment loss (LACL) epithelium and dental calculus. Sculean A, Jepson S, Becker J. Influence 29 Adriaens PA, DeBoever JA, Loesche at different thresholds and J Periodont Res. 1973;8:143-50. of fluorescence-controlled Er:YAG laser WA. Bacterial invasion in root associations with clinical variables: 22 Lang NP, Mombelli A, Attsrom R. radiation, the Vector system and hand cementum and radicular dentine changes on a population of young Oral biofilms and calculus. In: instruments on periodontally diseased of periodontally disease teeth male military recruits over 3 years. Lindhe J, Lang NP, Karring T, editors. root surfaces in vivo. J Clin Periodontol. in humans: a reservoir of J Clin Periodontol. 2002;28:961-9. Clinical Periodontology and Implant 2006;33:200-8. periodontopathic . J 19 Calabrese N, Galgut P, Morden N. Dentistry. 5th ed. Oxford: Blackwell 26 Caffesse RG, Sweeney PL, Smith BA. Periodontol. 1988;59:222-30. Identification of Aggregatibacter Munksgaard; 2008. p. 183-206. Scaling and root planing with and 30 Aleo JJ, De Renzis FA, Farber PA. actinomycetemcomitans, Treponema 23 Rabbani GM, Ash MM Jr, Caffesse without periodontal flap surgery. J In vitro attachment of human denticola and Porphyromonas R. The effectiveness of subgingival Clin Periodontol. 1986;13:205-10. gingival fibroblasts to root surfaces. gingivalis within human dental scaling and root planing in calculus 27 Fleischer HC, Mellonig JT, Brayer J Periodontol. 1975;46:639-45. calculus: a pilot investigation. J Int removal. J Periodontol. 1981;52:119-23. WK, Gray JL, Barnett JD. Scaling 31 Shapiro L, Lodate FM, Courant PR, Acad Periodontol. 2007;9:118-28. 24 Gellin RG, Miller MC, Javed T, and root planing efficacy in Stallard RE. Endotoxin determinations 20 Allen DL, Kerr DA. Tissue response Engler WO, Mishkin DJ. The multirooted teeth. J Periodontol. in gingival inflammation. J in the guinea pig to sterile and effectiveness of the Titan-S sonic 1989;60:402-9. Periodontol. 1974;43:591-6. non-sterile calculus. J Periodontol. scaler versus curettes in the removal 28 Jepsen S, Deschner J, Braun A, 32 Aleo JJ, De Renzis FA, Farber PA, 1965;36:121-6. of subgingival calculus. A human Schwarz F, Eberhard J. Calculus Varboncoeur AP. The presence of

40 PRIMARY DENTAL JOURNAL Root planing or subgingivally, was pivotal to the as had been surmised. A second in Root planing has been defined as treatment of periodontitis. vitro study40 demonstrated the ease instrumentation to remove the microbial with which endotoxin could be removed flora on the root surface or lying free Studies have shown that cementum from the root surface of periodontally in the pocket, all flecks of calculus and from periodontally involved teeth involved teeth which had been extracted. all contaminated cementum and dentine. had a significantly higher content of This study used the LAL test to assay The aim is to remove the softened lipopolysaccharide when compared the amount of endotoxin recovered from cementum so that the root surface is to non-periodontally involved teeth or the root surfaces of teeth which had made hard and smooth.8,9 The rationale control teeth (ie teeth extracted for non- been extracted for periodontal reasons. for root planing was based on the notion periodontal reasons) when assayed Endotoxin quantification took place that once the root surface became using the Limulus amoebocyte lysate following rinsing and brushing the roots exposed to the subgingival environment (LAL) test.37 However, the extent to which of these teeth (akin to root debridement) in periodontitis, it underwent both endotoxin was adsorbed by cementum versus stripping the entire root surface structural and pathological changes. or whether it was only surface-move (akin to root planing). The results of Structural and topographical changes back became a contentious issue. this study showed that over 99% of have been described, including the Advocates of SRP claimed (and still endotoxin was removed following formation of resorption lacunae that do, to some extent) that the cementum rinsing and brushing only. These results could potentially lead to entry of became infected with endotoxin during were corroborated by findings from bacteria and their products into the disease process, and that this layer other in vitro 40,41 and in vivo studies.42 cementum and radicular dentine.29 of infected tooth structure had to be These collective findings demonstrated Pathological changes in cementum were removed to achieve a biocompatible unequivocally that bacterial endotoxin implied, based on the assumption that root surface to allow healing to take was superficially located on cementum bacterial toxins or lipopolysaccharide place, citing periodontal healing and that it could be relatively easy (endotoxin) released by Gram-negative following SRP as evidence for this. to remove by simple measures not bacteria were adsorbed into the root involving extensive removal of surface. This led to the concept of In the 1980s, several investigators38,39 cementum. cementum becoming ‘infected’ and started to question the extent of therefore incompatible with attaching penetration of endotoxin into cementum Root surface debridement to healthy gingival/periodontal tissue.30 and therefore the actual clinical need The ease with which bacterial to remove cementum as part of the endotoxin can be removed from Endotoxin has several noxious properties: therapeutic process, as performed periodontally involved root surfaces38-42 it is a potent inflammatory agent31 and during SRP. An in vitro study38 used has profound clinical implications. an inhibitor of cell proliferation, cell extracted teeth immersed in a solution Root planing can only be effectively viability and gingival fibroblast of endotoxin for periods ranging performed with sharp hand instruments, reattachment to root surfaces.30,32 It between two to12 weeks, after in order to plane off the so-called also inhibits bone growth33 and induces which the teeth were subjected to infected cementum. However, if bone resorption34,35 and collagenase radiographic and immunofluorescence cementum does not become infected production by endotoxin-activated techniques for localisation of the then removal of the root surface macrophages.36 It was therefore endotoxin. This showed that the contaminants can be achieved with inferred that removal of the bacterial endotoxin was located on the surface a much lighter form of instrumentation endotoxin, located either supragingivally of the root and was not adsorbed, that does not remove tooth structure.

biologic activity of cementum-bound Boyan BD. Mechanisms of alveolar The distribution of bacterial cementum in healing following endotoxin. J Periodontol. 1974;45:672-5. bone destruction in periodontitis. lipopolysaccharide (endotoxin) in treatment of periodontal disease. 33 Iino Y, Hopps RM. The bone-resorbing Periodontol 2000. 1997;14:158-72. relation to periodontally involved A clinical study. J Clin Periodontol. activities in tissue culture of 36 Wilson M. Biological activities of roots surfaces. J Clin Periodontol. 1988;15:464-8. lipopolysaccharides from the bacteria lipopolysaccharides from oral 1986;13:748-51. 43 Smart GJ, Wilson M, Davies EH, Actinobacillus actinomycetemco- bacteria and their relevance to the 40 Hughes FJ, Smales FC. Kieser JB. The assessment of mitans, Bacteroides gingivalis and pathogenesis of . Immunohistochemical investigation ultrasonic root surface debridement ochracea isolated Sci Prog. 1995;78:19-34. of the presence of and distribution by determination of residual from human mouths. Arch Oral Biol. 37 Nishimine D, O’Leary TJ. Hand of cementum associated lipopoly- endotoxin level. J Clin Periodontol. 1984;19:59-63. instrumentation versus ultrasonics saccharides in periodontal disease. 1990;17:174-8. 34 Bom-van Noorloos AA, van der in the removal of endotoxin from J Periodontal Res. 1986;21:660-2. 44 Ramfjord SP, Knowles JW, Nissie RR, Meer JWM, van de Gevel JS, root surfaces. J Periodontol. 41 Hughes FJ, Auger DW, Smales FC. Burgett FG, Shick RA. Results following Schepens E, van Steenbergen YJM, 1979;50:345-9. Investigation of the distribution of three modalities of periodontal therapy. Burger E. Bacteroides gingivalis 38 Nakib NM, Bissada NF, Simmelink cementum-associated J Periodontol. 1975;46:522-6. stimulates bone resorption via JW, Goldstine EN. Endotoxin lipopolysaccharidess in periodontal 45 Lindhe J, Westfelt E, Nyman S, interleukin-1 production by penetration into root cementum of disease by scanning electron Socransky SS, Haffajee AD. Long- mononuclear cells: the relative role periodontally healthy and diseased microscope immunohistochemistry. term effect of surgical/non-surgical for B. gingivalis endotoxin. J Clin human teeth. J Periodontol. J Periodontal Res. 1988;23:100-6. treatment of periodontal disease. Periodontol. 1990;17:409-13. 1982;53:368-78. 42 Nyman S, Westfelt E, Sarhead G, J Clin Periodontol. 1984;11:448-58. 35 Schwartz Z, Goultschin J, Dean DD, 39 Moore J, Wilson, M, Kieser JB. Karring T. Role of “diseased” 46 Badersten A, Nilveus R, Egelberg J.

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A less invasive and gentler form of using plain ultrasonic tips; use of Conclusion root surface instrumentation, which if diamond-coated ultrasonic tips should Non-surgical periodontal therapy is used correctly does not result in tooth be avoided as this will lead to significant effective because it removes the bacterial structure removal, can be provided root surface loss.48 The ultrasonic biofilm that is the primary aetiological by ultrasonic instrumentation. Smart handpiece should be used on a low factor initiating periodontal diseases; et al (1990)43 showed that by using to medium power setting and the tip removal of other secondary factors, such ultrasonic instruments and adopting applied parallel to the tooth surface as dental calculus, benefit the patient a ‘conservative instrumentation regime using multiple overlapping strokes.49 because calculus encourages plaque of overlapping strokes and light Excessive instrument pressure, prolonged retention and interferes with effective pressure’ for a limited period of time, contact time or increased tip-to-tooth biofilm removal. Once disease extends periodontally involved root surfaces angle will all cause root damage. to involve the root surface, removal of can be rendered free of bacterial the subgingival biofilm is crucial for the endotoxin. Furthermore, this study The advantages of ultrasonic root long-term success of periodontal therapy. showed that light ultrasonic debridement are multiple: Removal of the subgingival biofilm instrumentation for less than 1s/mm2 1 It is up to 10 times more conservative should be performed both by the patient, of root surface was all that was of root surface tissue.50,51 Other via effective supragingival cleaning and required to remove all bacterial studies differ in this regard,52 subgingival root brushing, and by the contaminants. This amounts to about possibly due to use of different types operator by RSD. Previous traditional 17 seconds of instrumentation for an of instruments/instrument settings. methods such as SRP, which focused average pocket. This technique became 2 Micro-ultrasonic tips allow better on removing the ‘infected’ cementum, known as root surface debridement, access to the base of deep (>6mm) inevitably involved excessive removal the aim of which is remove bacterial periodontal pockets53,54 and within of root tissue and it is questionable contaminants (principally the bacterial furcations.55 whether such techniques, although biofilm) without the intentional removal 3 Being a non-invasive process, local effective, should have any place in of tooth structure. Studies have provided anaesthesia is usually not required. modern periodontal therapy. Based evidence for the importance of the 4 Greater cost effectiveness; manual on the available scientific evidence, removal of subgingival plaque in the instrumentation takes 20–50% longer it is time to shift from SRP to RSD. treatment of periodontitis,44-46 and to achieve the same clinical result.56-58 clinical studies in both animals and 5 Greater comfort for the patient59 humans 42,47 have provided scientific and, possibly, the operator. evidence for RSD as a therapeutic 6 The possibility of full-mouth modality for periodontal treatment. treatments, particularly if local Ideally, RSD should be carried out anaesthesia is not employed.

Effect of nonsurgical periodontal J Periodontol. 1998;69:547-53. 1 With unmodified and modified Periodontol. 1996;1:443-90. therapy. J Clin Periodontol. 49 Ritz L, Hefti AF, Rateitschak KH. ultrasonic instruments. Int J 57 Cobb CM. Clinical significance of 1987;14:425-43. An in vitro investigation on the loss Periodontics Restorative Dent. non-surgical periodontal therapy: 47 Nyman S, Sarhead G, Ericsson J, of root substance in scaling with 1992;12:310-23. an evidence based perspective of Gottlow J, Karring T. Role of various instruments. J Clin 54 Barendregt DS, van Der Velden, scaling and root planing. J Clin “diseased” root cementum in Periodontol. 1991;18:643-47. Timmerman MF, van der Wijden F. Periodontol. 2002;29:Suppl 2:6-16. healing following treatment of 50 Busslinger A, Lampe K, Beuchat M, Penetration depths with an 58 Tunkel J, Hienecke A, Flemming TF. periodontal disease. An Lehmann B. A comparative in vitro ultrasonic mini insert compared A systematic review of efficacy of experimental study in the dog. J study of the magnetostrictive and with a conventional curette in machine driven and manual Periodontal Res. 1986;21:496-503. piezoelectric ultrasonic scaling patients with periodontitis and in subgingival debridement in the 48 Kocher T, König J, Hansen P, instruments. J Clin Periodontol. periodontal maintenance. J Clin treatment of chronic periodontitis. J Rühling A. Subgingival polishing 2001;28:642-9. Periodontol. 2008;35:31-6. Clin Periodontol. 2002;29(S3):72-81. compared to scaling with steel 51 Obeid PR, Bercy P. Loss of tooth 55 Leon IE, Vogel RI. A comparison of 59 Ioannou I, Dimitriadis N, curettes: a clinical pilot study. J substance during root planing with the effectiveness of hand scaling Papadimitriou K, Sakellari D, Vouros Clin Periodontol. 2001;28:194-9. various periodontal instruments: and ultrasonic debridement in I, Konstantinidis A. Hand instrument- 48 Flemming TF, Petersilka GJ, Mehl an in vitro study. furcations as evaluated by ation versus ultrasonic debridement A, Hickel R, Klaiber B. Working Clin Oral Invest. 2005;9:118-23. differential dark field microscopy. in the treatment of chronic periodontitis: parameters of a magnetostrictive 53 Dragoo MR. A clinical evaluation J Periodontol. 1987;58:86-94. a randomized clinical and ultrasonic scaler influencing root of hand and ultrasonic instruments 56 Cobb CM. Non-surgical pocket microbiological trial. J Clin substance removal in vitro. on subgingival debridement. therapy: mechanical. Ann Periodontol. 2009;36:132-41.

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