jcd xvii.1.covers

Journal of
THE INTERNATIONAL JOURNAL OF APPLIED DENTAL RESEARCH Volume XXII
Robert C. Emling, EdD Caren M. Barnes, RDH, MS Annerose Borutta, Prof.Dr.med.habil.
Robert L. Boyd, DDS, MEd Kenneth H. Burrell, DDS, MS Mark E. Cohen, PhD David Drake, MS. PhD Subgingival Delivery of Oral Debriding Agents: Heinz Duschner, Prof.Dr.
William Michael Edgar, PhD, DDSc, FDSRCS A Proof of Concept Denise Estafan, DDS, MS Stuart L. Fischman, DMD Tanya Dunlap, PhD Duane C. Keller, DMD
Rosa Helena Miranda Grande, DDS, PhD Perio Protect LLC
John J. Hefferren, PhD St. Louis, MO, USA
Elliot V. Hersh, DMD, PhD Mark E. Jensen, DDS, PhD Milton V. Marshall, PhD
Carl J. Kleber, MSD, PhD University of Texas Health Science Center at Houston
Israel Kleinberg, DDS, PhD, DSc Houston, TX, USA
Karl F. Leinfelder, DDS, MS J. William Costerton, PhD
Jonathan Mann, DMD, MSc Kenneth Markowitz, DDS Allegheny General Hospital and Allegheny-Singer Research Institute
Milton V. Marshall, PhD, DABT Pittsburgh, PA, USA
Pier Francesco Porciani, MD, MScD Christoph Schaudinn, PhD
Howard M. Proskin, PhD Mark S. Putt, MSD, PhD Robert Koch Institute
Bruce R. Schemehorn, MS Warren Scherer, DDS Betty Sindelar, PT, PhD John R. Cotton, PhD
Jon B. Suzuki, DDS, PhD, MBA Jason M. Tanzer, DMD, PhD Norman Tinanoff, DDS, MS Athens, OH, USA
Henry O. Trowbridge, DDS, PhD Richard I. Vogel, DMD James S. Wefel, PhD Anthony E. Winston, BSc Wayne T. Wozniak, PhD Stefan Zimmer, Prof. Dr. med dent.
Stephen M. Siegel The Journal of Clinical Dentistry (ISSN 0895-8831) is published by Professional Audience Communications, Inc., P.O. Box 243, Yardley, PA 19067.
POSTMASTER; Send address changes to P. O. Box 243, Yardley, PA 19067.
Copyright 2011 by the YES Group, Inc. All rights reserved. No part of this publication may be reproduced without written permission from the publisher.
Subgingival Delivery of Oral Debriding Agents: A Proof of Concept Tanya Dunlap, PhD Duane C. Keller, DMD
Perio Protect LLC
St. Louis, MO, USA
Milton V. Marshall, PhD
University of Texas Health Science Center at Houston
Houston, TX, USA
J. William Costerton, PhD
Allegheny General Hospital and Allegheny-Singer Research Institute
Pittsburgh, PA, USA
Christoph Schaudinn, PhD
Robert Koch Institute
Betty Sindelar, PT, PhD John R. Cotton, PhD
Athens, OH, USA
• Objective: This study is a proof of concept to determine the efficacy of a custom-fabricated tray in placing antimicrobial and debriding
agents in the periodontal pockets of persons with active gingival infections. Localized subgingival delivery of antimicrobial andantibiotic agents is routinely employed as adjunctive therapy for the treatment and management of periopathogens associated withperiodontal disease. Because these delivery techniques often face time constraints and impose temporary restrictions on patient brush-ing and flossing, a custom-formed prescription dental tray can be used to deliver and maintain medications in periodontal pockets between office visits and without brushing or flossing restrictions. The ability of this tray to maintain sufficient concentrations of medi -cation in the periodontal pockets to have a therapeutic effect is evaluated here with theoretical modeling and practical application. • Methods: Hydrogen peroxide is an oral debriding agent and oral wound cleanser with antimicrobial properties. The debriding effect
of 1.7% hydrogen peroxide gel was tested in vitro on Streptococcus mutans biofilm using glass carriers for collection. Diffusionmodeling tested the potential of the customized tray to place hydrogen peroxide gel into the sulcus in the presence of crevicularfluid flow. Changes in periodontal microflora with scanning electron microscopy analysis of in vivo paper point site sampling wereanalyzed before and after a thin ribbon of 1.7% hydrogen peroxide gel (approximately 0.7 gm) and a subtherapeutic dose (three drops)of Vibramycin® (50 mg/5 ml) were placed via Perio Trays® into periodontal pockets, ranging from 4–8 mm at daily prescribed inter -vals for two to five weeks. • Results: In vitro results indicate that 1.7% hydrogen peroxide gel breaks down the exopolysaccharide slime and cell walls of S. mutans,
and begins to debride the cells from glass carriers within 10 minutes. Diffusion modeling indicates that hydrogen peroxide can pene -trate into the deeper pockets (9 mm), but also its concentration in these deep pockets will increase over wearing time in the absenceof degradation by peroxidases and catalase. Site sampling data confirm diffusion modeling results, with evidence that medicationdelivered with the prescription tray reduced subgingival bacterial loads and enhanced healing of corresponding oral tissues. • Conclusion: The prescription Perio Tray effectively placed medication in the gingival sulcus. Mathematical modeling indicated
Perio Tray placement of hydrogen peroxide gel in periodontal pockets with depths up to 9 mm over 15 minutes treatment time wastheoretically possible. Pathology reports reveal reductions in subgingival bacterial loads and improvements in pretreatment pocketdepths of up to 8 mm after 1.7% hydrogen peroxide and Vibramycin Syrup were prescribed for use with the Perio Tray. The in vitroanalysis indicating that hydrogen peroxide is the active and effective oral debriding agent needs to be confirmed with additionalstudies.
(J Clin Dent 2011;22:149–158)
in tooth loss.4,5 A local periodontal inflammatory response may Periodontal disease is the host response to oral biofilm microb ial also adversely affect the host systemic immune response and "triggers"1-3 that can result in localized tissue inflammation, gin- general health.6-8 For clinical practitioners, these localized and gival ulcerations with bleeding, tissue destruction, and bone loss systemically adverse effects are underscored by the prevalence leading to deep periodontal pocket formation that can culminate of periodontal disease and the insufficiencies inherent in current The Journal of Clinical Dentistry
Vol. XXII, No. 5
treatment methods, of which scaling and root planning (SRP) is When surgery is not required or after it has been performed, the accepted gold standard in non-surgical treatment. The bene - adjunctive chemical therapies can enhance treatment out- fits of SRP9,10 are well recognized, but significant limitations comes.10,11 Recently, new attention has been paid to peroxide usage occur with SRP, including mechanical inability to remove all as a viable subgingival1,31-34 and supragingival35 antibiofilm bacterial cells, resulting in biofilm regeneration that requires agent. Aqueous ≤ 3% hydrogen peroxide is a known oral de- repetitive mechanical procedures and the risks of bacteremia briding agent and wound cleanser.36 It has been formulated in associated with mechanical debridement and scaling. Faced with mouthrinses, dentifrices, and gels for topical application, most these limiting situations, practitioners employ adjunctive thera- commonly for tooth whitening.37,38 Researchers are also inter- pies11 or surgery. ested in the disinfectant properties of hydrogen peroxide. The hy- Periodontitis is a persistent inflammatory response to bacter- droxyl radical formed from hydrogen peroxide decomposition, ial growth in slime-enclosed communities that, like all classic especially in the presence of iron (Fe3+), has been shown to kill biofilms, resists clearance by host defenses and systemic anti - 99.99% of oral periopathogens and 99.999% Streptococcus biotic therapy.1,4,12,13 The ability of biofilms to persist in spite of mutans (S. mutans) bacteria within three minutes.39 activated host responses lies at the root of their persistence.
One problem with chemotherapeutic treatment is the delivery Physical biofilm debridement has developed as the gold standard and maintenance of peroxides in the sulcus. The sulcus is a in the treatment of biofilm infections as it is in dentistry with unique space for chemotherapeutic treatment modalities because SRP, but it is impossible to eliminate all bacteria in the biofilm it is accessible topically, but the salient problem of overcoming with SRP and recolonization can occur.14-16 gingival crevicular fluid flow tends to limit chemical contact in Mechanical debridement can have the specific limitation of the gingival space. The most effective topical administration of stim ulating biofilm regeneration. In one study, mechanical peroxides for biofilm management appears to be tray delivery of removal of 50% of the initial biofilm resulted in a four-fold a gel formulation.33,35,40 If peroxides can debride subgingival increase in biofilm growth. Subsequent 75% removal of the re- planktonic cells of the biofilm and significantly reduce the pe- growth resulted in a three-fold increase over that present from the ripheral elements of biofilms, the peroxides may shift biofilm first regrowth under magni fic ation analysis.17 A biofilm that is communities into a defensive growth mode, limiting their ability mechanically disturbed can thus increase its reproductive capa- to reproduce or trigger inflammation. bilities in response to the physical forces used to perturb it. This study, to evaluate the potential of a custom-fabricated Given the limitations with mechanical procedures, adjunctive dental tray to retain medication in the sulcus a sufficient amount antibiotic therapies are employed to improve treatment out- of time for the medication to have a therapeutic effect, has three comes.18,19 Antibiotics effectively kill individual planktonic cells distinct parts. The first shows in vitro debridement results of and some of the peripheral, actively dividing bacteria in the 1.7% hydrogen peroxide gel. The second is a theoretical exercise biofilm, but have little effect on the dormant core enclosed in the evaluating the potential for prescription Perio Tray® (Perio Protect, protective slime matrix.20,21 In order for the antibiotics to work LLC, St. Louis, MO, USA) delivery of 1.7% hydrogen peroxide more effectively, the matrix has to be removed and the dormant gel into the sulcus against the force of crevicular fluid flow. The core stimulated; however, when the matrix is mechanically de- third evaluates the practical application and efficacy of using cus- brided, the remaining biofilm cells are stimulated, prompting rapid tomized trays for localized subgingival delivery of medication regeneration until the biofilm returns to a protected stable popu- based on pathology reports documenting in vivo subgingival lation stasis for which additional SRP is often necessary in a cycli- biofilm changes after medication is placed into periodontal pock- cal fashion.22 Therefore, most patients with chronic or aggressive ets via prescription Perio Trays.
periodontitis have SRP performed every three to six months.
Risks of bacteremia associated with mechanical debridement Material and Methods
and scaling23,24 increase with repetitive use of SRP. For most In Vitro Debridement with 1.7% Hydrogen Peroxide Gel
healthy adults, the host immune system is capable of managing In vitro assessments were conducted to confirm the debriding the inflammatory response induced by bacteremia during perio - action of 1.7% hydrogen peroxide gel (Dakota Pharmacy, Bis- dontal procedures, but for the millions of immunocompromised marck, ND, USA) on oral biofilm using the LIVE/DEAD® sys- individuals and adults with diabetes, cardiovascular disease, tem developed by Molecular Probes (Invitrogen, Carlsbad, CA, joint replacements, and other inflammatory illnesses, an increase USA).41 The assessment of viability involves staining prepara- in the chronic systemic inflammatory burden may pose additional tions with propidium iodide, which penetrates the compromised health risks.25 Given these potential risks, it would be beneficial bacterial wall of dead bacteria and binds to their DNA so the cells to have a treatment modality that could reduce localized perio - appear in a rich red color. Bacteria with intact cell walls exclude dontal inflammation before mechanical debridement, and thus propidium iodide, and are stained green by the Syto 9 counter decrease the possibility of bacteremia.
stain. Bacteria that are injured with partially compromised cell For some cases, surgery is necessary. When pathogenic bac- walls stain an orange color. The viability of bacteria is deter- teria are capable of penetrating phagocytic and non-phagocytic mined by assessing the proportion of stained red, orange/yellow, cells, they evolve to survive within the host cells,26-28 which can and green bacteria, recorded at the moment at which the popu- result in the development of host granulomatous tissue.29,30 In lation was stained. these cases, the only appropriate therapy is surgical removal of S. mutans (strain UA 159) were inoculated in Brain Heart Infusion the internally infected tissues. (OXOID LTD., Basingstoke, UK) with 2% sucrose (Bethesda Vol. XXII, No. 5
The Journal of Clinical Dentistry
Research Laboratories, Gaithersburg, MD, USA), into MatTek These morphotypes were counted in each biofilm colony at three glass bottom microwell plates (MatTek Corporation Ashland, standard areas of 100 µm2 and averaged. The results were multi - MA, USA), which were incubated for 24 hours at 37oC, 5% CO plied by the colony area and height. The sum for every morpho - of an orbital shaker. After 24 hours under a laminar hood, me- type over all colonies yielded the total amount of bacteria per dia from each plate were removed and new media were replaced.
paper point.
Plates were incubated for another 24 hours under similar condi- After initial SEM analysis of paper points, the periodontal tions to form a mature biofilm on the third day.
pockets were treated with 1.7% hydrogen peroxide gel and a sub- The plates were aseptically removed from the incubator, and clinical dose (three drops per tray) of Vibramycin® Syrup (50 exposed to the 1.7% hydrogen peroxide gel and to gel with all mg/5 ml, Pfizer, New York, NY, USA), topically placed via pre- excipients except hydrogen peroxide for five or 10 minutes prior scription Perio Trays. Paper point biofilm sampling was repeated to rinsing with sterile phosphate buffered saline (BioWhittaker/ two to five weeks after daily Perio Tray delivery of medication.
Lonza Walkersville, MD, USA) and exposure to the LIVE/DEAD® The accuracy of the SEM image analysis approach can be only BacLite™ (Invitrogen, Carlsbad, CA, USA) staining procedure.
described as a rough approximation whose exactness is not more A control series was similarly prepared, and all manipulations than one order of magnitude. Therefore, only significant changes were conducted to prevent the removal of biofilm by mechanical forces for comparison. Following the staining procedure, thestained plates were examined using a Leica TCS-SP2 confocal scanning laser microscope (CSLM).
In Vitro Debridement with 1.7% Hydrogen Peroxide Gel
When S. mutans biofilms generated in vitro were examined by the LIVE/DEAD technique, without gel treatment, the majority The modeling tests the theory that the prescription tray system of the millions of bacterial cells in these coherent and luxuriant results in a concentration of hydrogen peroxide (c) delivered and biofilms were alive (green) with uncompromised bacterial walls maintained in the gingival sulcus or periodontal pocket during the (Figure 1). In the micrograph, intact bacterial cells, approxi- period of treatment. A simple mass transport model was used to mately 0.61 µm in diameter, are seen to be embedded in an exo - estimate the ability of hydrogen peroxide to penetrate the perio - polysaccharide (EPS) matrix.
dontal pocket over time as a function of distance. In Vivo Subgingival Effects of Medication
Delivered with the Perio Tray
A retrospective review of pathology reports from a private general dental clinic identified records from four patient (threemen, one woman, age range 33–71 years) who had selectedtreatment with the custom-fabricated Perio Tray for delivery ofmedication before SRP, either because of previous mechanicalperiodontal treatment failure or because they refused SRP treat-ment. Because of their treatment status, the patients were askedto consent to biofilm samplings as a diagnostic adjunct to theirplan of care. Sterile Absorbent Points (#504 Henry Schein Inc.,Melville, NY, USA) were held in place for 10 seconds in 19 totalperiodontal pockets before chemotherapeutic treatment beganwith the Perio Tray, and again two to five weeks after dailytreatment began. None of the patients had had SRP three monthsprior to or during the course of this treatment.
Paper points were prepared for scanning electron microscopy (SEM) by dehydration in a graded ethanol series, critical pointdried, mounted on a stub, sputter coated with a 20 nm layer ofplatinum, and examined with an XL 30 S, FEG SEM (FEI Com- Figure 1. Confocal micrograph of untreated control S. mutans biofilm, showing
pany, Hillsboro, OR, USA) operating at 5 kV in the secondary large numbers of live (green) cells, with a few membrane-compromised (orange) electron mode.
All paper points were systematically scanned and documented S. mutans are a good choice for testing because, in contrast to with overview, regional, and detail images. The approximate many subgingival bacteria, this biofilm produces large amounts dimension of each biofilm colony could be calculated based on of EPS matrix that act as an additional protection barrier for the the overview and regional images. Furthermore, the approximate bacteria, increasing the challenge for matrix decomposition and number of bacteria/colony/layers was counted, and six basic debridement.42 In further contrast to subgingival bacteria, S. mutans morphotypes were defined (spiral-rods, short-rods [1:2 width: is a mostly aerobic growing organism, able to handle larger length], middle-long rods [> 1:2 – < 1:7], long rods [> 1:7], amounts of the peroxide, which makes it less susceptible to the flagellated rods, filamentous-rods, and coccus-like bacteria).
debriding action of the 1.7% hydrogen peroxide gel.
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When S. mutans biofilms were treated for five minutes with 1.7% hydrogen peroxide gel and examined using the LIVE/DEAD technique, bacterial wall integrity of only a small pro-portion of the biofilm cells were compromised, and only a smallproportion of them stained orange to yellow (Figure 2). However,when these biofilms were exposed to the 1.7% peroxide gel for10 minutes, virtually all of the bacterial walls were disrupted(red; Figure 3). Most of the cells disintegrated and released theirDNA as a tangled "skein." It can be concluded that, at some pointbetween five and 10 minutes, the chosen concentration of perox -ide eradicates virtually all of the cells in the S. mutans biofilm. Figure 3. Confocal micrograph of a S. mutans biofilm treated for 10 minutes
with the 1.7% hydrogen peroxide gel. All of the bacteria are degraded (red) and
only a few faint cell profiles remain because the cell walls have been compro-
mised and disintegrated, releasing their DNA as a tangled fibrous mass.
Figure 2. Confocal micrograph of S. mutans biofilm exposed to the 1.7% hy-
drogen peroxide gel for 5 minutes. Most of the coccoid cells are intact (green).
The number of membrane-compromised cells (yellow) is similar to that seen in
untreated biofilms.
As a control for this experiment, a gel with all excipients except the active ingredient, hydrogen peroxide, was used onS. mutans biofilm. Exposure to this gel for 10 minutes left thebiofilm almost completely unaffected (Figure 4) in that verylarge areas showed only living (green) cells. In some small areassome bacterial wells were compromised (orange), but these werein the same proportion seen in untreated biofilms.
These results demonstrate that a 10-minute exposure to a 1.7% hydrogen peroxide aqueous gel can debride bacterial cell wallswithin a typical dental biofilm. The modeling below evaluates the Figure 4. Confocal micrograph of S. mutans biofilm treated for 10 minutes with
potential of hydrogen peroxide gel delivery in the sulcus against a gel containing 0% hydrogen peroxide. Note the clear predominance of living(green) cells in this control preparation. gingival crevicular fluid, which occurs in the periodontal pocket.
mm) was used as the width of the cross-sectional area at the gingival-tooth interface. The depth of this space was con sidered In modeling the geometry of the area of diffusion, only the mo- as a range from 4–9 mm, reflecting the variation in pocket- probing lars of a typical adult mouth were considered. The length of the depth associated with periodontal disease severity. Because both cross-sectional area of this space was considered as the average lingual and buccal surfaces are considered with pocket-probing length of a molar from mesial to distal. After measuring six molars depth analyses, two rectangular spaces were included in this three times each, the average length was determined as 8.88 mm.
modeling to represent both of these surfaces.
Given that a dental probe is able to fit into the periodontal pocket Prior studies have demonstrated that GCF flow rate increases when disease is present, the width of a typical dental probe (0.83 with periodontal disease, exhibiting a range of 1.8 to 137.0 µl/h

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with a mean of 45.7 ± 35.7 µl/h.43 The flow rates were indica- simulated for 15 minutes of treatment. The plot in Figure 5 tive of pocket depths greater than 4 mm, bleeding on probing at shows the simulation.
greater than 40% of the sites, and clinical attachment levelsgreater than 4.5 mm. These characteristics are consistent with the"typical patient" receiving treatment with the prescription trays.44For subsequent calculations, the flow rate was converted to afluid velocity (v) by dividing the flow rate (Q) by the cross- sectional area (A) or v = Q / A This resulted in a velocity of 0.861 x 10-3 mm/s.
Reducing the problem to one dimension, the distance along the tooth root is considered the positive x direction, with the originat 0 mm pocket probing depth. The governing equation for thisdiffusion problem is then Figure 5. The simulated results show the concentration of hydrogen peroxide
into the pocket increases with the length of time the peroxide remains applied.
where c is the concentration of hydrogen peroxide as a functionof distance x (pocket depth) and time t.45 The velocity (v) flows This analysis indicates hydrogen peroxide held in the Perio out of the tooth, and the constant D is the coefficient of diffusion.
Trays can diffuse into the periodontal pockets over time. Even From prior studies, the coefficient of diffusion for a 10% solution with a relatively large GCF flow, the diffusion enters 9 mm deep of hydrogen peroxide was given as 1.48 cm2/day.46 Although the pockets within the 15-minute time period studied here. Thus, treating concentration of hydrogen peroxide is lower (1.7%), the throughout the time that a patient would wear the prescription Pe- 10% diffusion coefficient is a reasonable approximation for the rio Tray, the concentration of hydrogen peroxide in the deeper treatment concentration in this exercise. The first term on the right pockets improves, indicating that even with GCF there is an in- side of the equation reflects the process of convection where creasing concentration in the deeper areas.
fluid flow lowers the concentration of hydrogen peroxide, whilethe second term describes hydrogen peroxide diffusion against the In Vivo Subgingival Effects of Medication
concentration gradient. If the fluid velocity drops to 0, the above Delivered with the Perio Tray
equation reduces to Fick's law of diffusion. Tables I–IV detail microbial descriptions provided by SEM In order to solve the equation, initial and boundary conditions analysis in pathology reports for 19 total periodontal pockets had to be assumed: no concentration of hydrogen peroxide is pre- from four patients. Examples of SEMs from the pathology re- sent in the pocket at time = 0; the concentration of hydrogen per- ports are presented in Figures 6–9, and they are representative of oxide in the tray at x = 0 is c ; and no hydrogen peroxide leaves the results seen from each sampling site before and after treat- the base of the periodontal pocket, assumed to be x = 9 for this ment with 1.7% hydrogen peroxide gel and three drops of Vibra - problem (this is considered a no flux condition at x = 9). In ad- mycin Syrup delivered via the Perio Tray.
dition, the concentration of hydrogen peroxide in the tray c was Patient 1 had Type II periodontal disease on arrival at the modeled as a decreasing amount to account for the change in clinic. Pathology reports record sites analyzed evidenced bleed- degradation and outflow. Because of the changing hydrogen ing on probing before treatment, and absence of bleeding on peroxide concentration in the tray, no steady state will be probing after four times daily use of the Perio Tray subgingival placement of medication for five weeks. Pocket probing depth The time-dependent solution was coded into Matlab (Version decreased 1–2 mm during this time, and one of three sites had 7.4; Natick, MA, USA) using finite difference analogues, and no bacteria recovered after five weeks of treatment. Patient 2 had Retrospective Compilation of Microbiological Data from Pathology Reports for Male Patient with Type 2 Periodontal Disease Patient 1—Male, Periodontal Disease Type II—Treatment Duration 5 Weeks, 4 Day, 15 Minutes Microbial Description Based on Scanning Electron Micrograph Extensive presence of biofilm ( 5 107 bacteria) with notable high percentage of Treponema-like morphotypes.
Multilayered biofilm ( 5 105 bacteria) composed primarily of short rods, long rods and coccus-like morphotypes.
Relatively little amount of biofilm ( 5 104 bacteria) dominated by Coccus-like morphotypes.
No bacteria could be found on the entire paper point, only large numbers of eukaryotic cells.
Abundant biofilm ( 5 107 bacteria) in which coccus-like bacteria and rods of different length were found.
Polymicrobial biofilm ( 5 105 bacteria) with predominance of filamentous-like morphotypes, as formed by many Actinomyces species.
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Retrospective Compilation of Microbiological Data from Pathology Reports for Female Patient with Type 1 Periodontal Disease Patient 2— Female, Periodontal Disease Type I—Treatment Duration 5 Weeks, 2 Microbial Description Based on Scanning Electron Micrograph Dense, multi-layered biofilm ( 1 106 bacteria) in which only Treponema-like bacteria and rods of different length were found.
Only eukaryotic cells found; no evidence of bacteria.
Great number of Treponema-like bacteria in a very densely composed biofilm ( 1 105 bacteria).
Many eukaryotic cells of different types; no evidence of bacteria.
Patchy groups of bacteria ( 1 104 bacteria) together with eukaryotic cells.
Many large eukaryotic cells; no evidence of bacteria. Poly-microbial biofilm ( 5 106 bacteria) consisting to a large extent of Treponema-like morphotypes.
Few spots with filamentous and coccus-like bacteria biofilm ( 5 104 bacteria) together with many eukaryotic cells.
Table III
Retrospective Compilation of Microbiological Data from Pathology Reports for Male Patient with Type 4 Periodontal Disease Patient 3—Male, Periodontal Disease Type IV—Treatment Duration 2 Weeks, 6 Day, 15 Minutes Microbial Description Based on Scanning Electron Micrograph Multi-layered biofilm (5 106 bacteria) with a high number of Treponema-like morphotypes.
Only very sparse biofilm patches (1 103 bacteria) and many eukaryotic cells.
Biofilm (1 107 bacteria) with bacteria partly embedded in extracellular matrix.
Multi-layered biofilm ( 1 106 bacteria) predominantly composed of mid-long rods.
Poly-microbial biofilm (1 107 bacteria) with characteristic long rods.
Biofilm ( 1 105 bacteria) with different rod morphologies, coccus-like bacteria and filamentous bacteria.
Biofilm (5 105 bacteria) in which Treponema-like morphotypes contributed 50% of all bacteria.
Dense layer of eukaryotic cells; no evidence of bacteria.
Large, multi-layered biofilm (5 108 bacteria) with fusiform bacteria and Treponema-like morphotypes.
Large number of eukaryotic cells; no evidence of bacteria.
Biofilm (5 107 bacteria) in which fusiform bacteria and short rods were frequently found as well as coccus-like Eukaryotic cells; no evidence of bacteria.
Retrospective Compilation of Microbiological Data from Pathology Reports for Male Patient with Type 4 Periodontal Disease Patient 4—Male, Periodontal Disease Type IV—Treatment Duration 2 Weeks, 6 Day, 15 Minutes Microbial Description Based on Scanning Electron Micrograph Biofilm ( 1 107 bacteria) with large numbers of long and short rods, as well as Treponema-like bacteria.
Many eukaryotic cells; no evidence of bacteria.
Multi-layered biofilm ( 5 106 bacteria) in which Treponema-like morphotypes predominated.
Biofilm ( 5 104 bacteria) composed of a majority of Treponema-like bacteria.
Patches of biofilm ( 1 106 bacteria) with great diversity of rod-shaped bacteria (short, middle and long).
Biofilm amount decreased ( 1 104 bacteria), while the same morphotypes were observed.
Densely composed biofilm ( 1 106 bacteria) with all types of rod-like morphotypes.
Eukaryotic cells; no evidence of bacteria.
Biofilm ( 1 106 bacteria) embedded in its extracellular matrix.
Few patches of biofilm ( 5 103 bacteria) consisting of rod shaped bacteria with varying length.
Densely embedded biofilm ( 1 107 bacteria) revealing large numbers of Treponema-like morphotypes.
Eukaryotic cells and small biofilm colonies ( 5 103 bacteria) with rod shaped morphology.
Type I periodontal disease and was treated for 10 minutes twice after five weeks. Patient 3 had Type IV periodontal disease on ar- daily for five weeks; pocket probing depth decreased 2–3 mm at rival at the clinic, and was treated for 15 minutes six times daily the sites analyzed, no bleeding on probing was seen at any site, for two weeks; no bleeding on probing was seen, and pocket and no bacteria were recovered from three of four sites sampled probing depth decreased 1–5 mm at the sites analyzed after two

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Figure 6a. Patient 1 SEM from paper point sample 24 mb before treatment
Figure 6b. Patient 1 SEM from paper point sample 24 mb after five weeks' daily
indicates predominance of Treponema-like morphotypes. treatment shows multi-layered biofilm with short rods, long rods, and coccus-likemorphotypes. Figure 7a. Patient 2 SEM from paper point sample 14 mb before treatment
indicates dense multi-layered biofilm with Treponema-like bacteria and rods of
Figure 7b. Patient 2 SEM from paper point sample 14 mb after five weeks' daily
treatment. No bacteria were found. Figure 8a. Patient 3 SEM from paper point sample 31 mb before treatment in-
Figure 8b. Patient 3 SEM from paper point sample 31 mb after two weeks' daily
dicates multi-layered biofilm with high number of Treponema-like morphotypes. treatment indicates sparse biofilm and eukaryotic cells.

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Figure 9a. Patient 4 SEM from paper point sample 21 ml before treatment in-
Figure 9b. Patient 4 SEM from paper point sample 21 ml after two weeks'
dicates biofilm composed of rod-like morphotypes. daily treatment indicates presence of eukaryotic cells and no bacteria. weeks of Perio Tray delivery of medication. Additionally, no bac- reports were reviewed here self-reportedly did not strictly com- teria were recovered from paper points at three of five sites af- ply with treatment protocols; they missed one or more of the ter two weeks of treatment. Patient 4 had Type IV periodontal daily recommended treatments, or did not sustain treatment for disease at the time of treatment, and no bleeding on probing at the recommended 10 or 15 minutes. Nevertheless, the pathology three of six sites was seen after two weeks of six times daily use reports establish in vivo evidence of subgingival biofilm de- of medication in the Perio Tray for 15 minutes. Pocket probing bridement after Perio Tray placement of medication, even with- depth decreased 0–5 mm during this time; two treated sites had out strictly controlled conditions. no bacteria present after treatment. In all cases after treatment Direct visualization with SEM in these pathology reports differs when bacteria were observed on paper points, the recovery was from DNA analysis primarily in that standard DNA- Polymerase reduced compared to the initial sampling.
Chain Reaction tests (commercially available with OralDNA® Labs,Brentwood, TN, USA and Hain Diagnostics, LLC, Midland, TX, USA) evaluate a relatively small number of selected bacterial The mathematical diffusion modeling indicates hydrogen per- species, whereas SEM offers an indiscriminative view of the entire oxide can effectively be placed into deep periodontal pockets biofilm. Neither procedure can discriminate between living and (> 6 mm) with Perio Trays, and that concentration of hydrogen dead bacteria.
peroxide increases over time in the absence of degrading en- In addition to the evidence of biofilm suppression, an overview zymes like catalase or peroxidase. Even with a relatively large of the pathology reports indicates that the prescription tray deliv- GCF flow, the medication theoretically diffuses into 9 mm deep ery of 1.7% hydrogen peroxide and three drops of Vibra mycin pockets within the 15-minute time period evaluated. helped reduce pocket depths and bleeding on probing, a confir- Hydrogen peroxide (1.7%) was chosen as the treatment agent mation that the prescribed solutions were held in place long enough for this diffusion modeling exercise because in prior studies, for the medication to have a therapeutic effect. Yet because the re- aqueous solutions of hydrogen peroxide (> 1%) have been shown ductions were not uniform, the tray delivery of prescribed solutions to decrease plaque and gingivitis indices,47,48 to have antimicro- may only be suggested as an adjunct to a compre hensive treatment bial effects on bacteria associated with periodontal disease,49,50 plan implemented under the supervision of a dentist. In clinical and to enhance wound healing after gingival surgery.37 In addi- practice, use of the prescription tray delivery of medication is of- tion, the biofilm potential technique1,51 provides case study evi- ten followed by full mouth debridement, site- specific scaling as dence of subgingival biofilm suppression in 6 mm pockets after needed, site-specific surgery when needed, and prophylaxis. Perio Tray delivery (20 minutes, four times a day for five days) While the in vitro results indicate that hydrogen peroxide is of 1.5% hydrogen peroxide (Peroxyl®, Colgate-Palmolive Com- effective on oral biofilms, their debriding action on in vivo sub- pany, New York, NY, USA) alone, and then in combination with gingival biofilms cannot be confirmed with the pathology reports three drops of Sumycin® Syrup (125 mg/5ml, Par Pharmaceuti- because hydrogen peroxide and Vibramycin were both used.
cals, Woodcliff Lake, NJ, USA) for two five-day periods.34,52 Tetracyclines are commonly prescribed in the course of treatment While the debriding effects of hydrogen peroxide on oral for periodontal disease.55-59 For these patients, the sorbitol-based biofilm were confirmed with the in vitro study presented here and Vibramycin was prescribed for topical delivery for its anti- the previously published case study, the strictly controlled clin- inflammatory properties. Further testing needs to be completed ical environment of the previously published case study does not to confirm the specific therapeutic effects of individual solutions take into consideration patient compliance with a patient tray used with the prescription tray system.
delivery system. Real-world patients often do not adhere to pre- Additional testing is also needed to confirm the most appropri- scribed treatment regimens,53,54 and the patients whose pathology ate course of treatment with individual medications. If medication Vol. XXII, No. 5
The Journal of Clinical Dentistry
delivered subgingivally with the Perio Tray can decrease biofilm ment protocol: a blinded, randomized clinical trial. J Clin Periodontol 2010; populations, reduce pocket depths, and eliminate bleeding before mechanical debridement, then the risks of bacteremia with me- 16. Resposo S, Tobler J, Alfant B, Gollwitzer J, Walker C, Shaddox L. Poster presentation: Differences between Biofilm Growth Before and After Perio - chanical debridement may also be reduced, as may the extent and dontal Therapy. AADR 37th Annual Meeting. April 3, 2008. Abstract avail- scope of necessary mechanical debriding procedures. 17. Palmer RJ, Caldwell DE. A flowcell for the study of plaque removal and regrowth. J Micro Methods 1995;24:171-82.
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24. Morozumi T, Kubota T, Abe D, Shimizu T, Komatsu Y, Yoshie H. Effects of irrigation with an antiseptic and oral administration of azithromycin on Acknowledgment: This work was supported by Perio Protect, LLC.
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