Disinfection of Root Canal Systems

Disinfection of Root Canal SystemsThe Treatment of Apical Periodontitis

Edited by

Nestor Cohenca

Department of Endodontics and Pediatric Dentistry,University of Washington, Seattle, WA, USA

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Library of Congress Cataloging-in-Publication Data

Disinfection of root canal systems : the treatment of apical periodontitis / edited by Nestor Cohenca.

p. ; cm.

Treatment of apical periodontitis

Includes bibliographical references and index.

ISBN 978-1-118-36768-1 (cloth)

I. Cohenca, Nestor, 1968- editor of compilation. II. Title: Treatment of apical periodontitis.

[DNLM: 1. Periapical Periodontitis–therapy. 2. Root Canal Therapy. 3. Disinfection–methods. 4. Root Canal Irrigants.

5. Therapeutic Irrigation. WU 230]

RK351

617.6′342059–dc232014004701

A catalogue record for this book is available from the British Library.

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electronic books.

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Typeset in 9.5/11.5 pt PalatinoLTStd by Laserwords Private Limited, Chennai, India

1 2014

Contents

Contributors viiIntroduction ixPreface xiAcknowledgments xiii

Part 1: Background 1

1 Root Canal Infection and EndodonticApical Disease 3Nestor Cohenca and Ana MariaGonzález Amaro

2 The Anatomy of the Root CanalSystem as a Challenge to EffectiveDisinfection 15Eric Herbranson

3 Biofilms in Root Canal Infections 29Christine M. Sedgley and Rogério deCastilho Jacinto

4 Efficacy of Root Canal Disinfection 57Ashraf F. Fouad

5 Impact of Root Canal Disinfection onTreatment Outcome 71James D. Johnson and Natasha M. Flake

Part 2: Nonsurgical IntracanalDisinfection 89

6 Shaping the Root Canal System toPromote Effective Disinfection 91Ove A. Peters and Frank Paqué

7 Topical Disinfectants for Root CanalIrrigation 109Bettina Basrani and Markus Haapasalo

8 Fluid Dynamics of Irrigation withinthe Root Canal System 141Franklin R. Tay

9 Positive Pressure Irrigation 169Cesar de Gregorio, Carlos Heilborn, andNestor Cohenca

10 Apical Negative Pressure Irrigation(ANP) 189Nestor Cohenca, Cesar de Gregorio, andAvina Paranjpe

11 Disinfection of the Root CanalSystem by Sonic, Ultrasonic, andLaser Activated Irrigation 217Luc van der Sluis, Bram Verhaagen,Ricardo Macedo, and Michel Versluis

v

vi Contents

12 Ozonization and ElectrochemicalRoot Canal Disinfection 239Roberta Pileggi

13 Intracanal Medication in Root CanalDisinfection 247Lea Assed Bezerra da Silva, RaquelAssed Bezerra da Silva, PauloNelson-Filho, and Nestor Cohenca

14 Emerging Technologies in RootCanal Disinfection 277Anil Kishen, Annie Shrestha, and NestorCohenca

Part 3: Apical Response andSurgery 297

15 Healing of Apical Lesions: How DoThey Heal, Why Does the HealingTake So Long, and Why Do SomeLesions Fail to Heal? 299Zvi Metzger and Anda Kfir

16 Surgical Endodontics: TheComplimentary Approach 319Richard Rubinstein and AlirezaAminlari

Index 341

Contributors

Alireza AminlariPrivate Practice, Farmington Hills, MI, USA

Department of Cariology, University of Michigan,

Ann Arbor, MI, USA

Bettina BasraniDiscipline of Endodontics, Faculty of Dentistry,

University of Toronto, Toronto, ON, Canada

Rogério de Castilho JacintoDepartment of Semiology and Clinics, Federal

University of Pelotas, Pelotas, RS, Brazil

Nestor CohencaDepartment of Endodontics and Pediatric

Dentistry, University of Washington, Seattle,

WA, USA

NatashaM. FlakeDepartment of Endodontics, University of

Washington, Seattle, WA, USA

Ashraf F. FouadDepartment of Endodontics, Prosthodontics and

Operative Dentistry, University of Maryland,

Baltimore, MD, USA

Shimon FriedmanFaculty of Dentistry, University of Toronto,

Toronto, ON, Canada

AnaMaria González AmaroDepartment of Microbiology, University

Autonomous of San Luis Potosí, San Luis Potosí,

Mexico

Cesar de GregorioDepartment of Endodontics, University of

Washington, Seattle, WA, USA

Markus HaapasaloDepartment of Oral Biological andMedical

Sciences, Vancouver, BC, Canada

Carlos HeilbornPrivate Practice, Asunción, Paraguay

Eric HerbransonPrivate practice, San Leandro, California, USA

James D. JohnsonDepartment of Endodontics, University of

Washington, Seattle, WA, USA

Anda KfirDepartment of Endodontology, Tel Aviv

University, Tel Aviv, Israel

Anil KishenUniversity of Toronto, Toronto, ON, Canada

RicardoMacedoThe Academic Centre for Dentistry in Amsterdam

(ACTA), University of Amsterdam, Amsterdam,

The Netherlands

vii

viii Contributors

Zvi MetzgerDepartment of Endodontology, Tel Aviv

University, Tel Aviv, Israel

Paulo Nelson-FilhoDepartment of Pediatric Dentistry, School of

Dentistry of Ribeirão Preto. University of SaoPaulo, Riberao Preto, Brazil

Frank PaquéDepartment of Preventive Dentistry,

Periodontology, and Cariology, University ofZurich, Zürich, Switzerland

Avina ParanjpeDepartment of Endodontics, University of

Washington, Seattle, WA, USA

Ove A. PetersDepartment of Endodontics, University of the

Pacific, San Francisco, CA, USA

Roberta PileggiDepartment of Endodontics, University of Florida,

Gainesville, FL, USA

Richard RubinsteinPrivate Practice, Farmington Hills, MI, USADepartment of Cariology, Restorative Sciences, and

Endodontics, University of Michigan, AnnArbor, MI, USA

ChristineM. SedgleyDepartment of Endodontology, Oregon Health and

Science University, Portland, OR, USA

Annie ShresthaUniversity of Toronto, Toronto, ON, Canada

Lea Assed Bezerra da SilvaDepartment of Pediatric Dentistry, School of

Dentistry of Ribeirão Preto. University of SaoPaulo, Riberao Preto, Brazil

Raquel Assed Bezerra da SilvaDepartment of Pediatric Dentistry, School of

Dentistry of Ribeirão Preto. University of SaoPaulo, Riberao Preto, Brazil

Luc van der SluisDepartment of Conservative Dentistry and

Endodontics, University of Toulouse, Toulouse,France

Franklin R. TayDepartment of Endodontics, Georgia Regents

University, Augusta, GA, USA

Bram VerhaagenResearch Institute for Biomedical Technology and

Technical Medicine, University of Twente,Enschede, The Netherlands

Michel VersluisResearch Institute for Nanotechnology, Research

Institute for Biomedical Technology andTechnical Medicine, University of Twente,Enschede, The Netherlands

Introduction

During my first endodontic residency, my programdirector, Professor Ilana Heling, always remindedme that “bacteria” is the reason for all endodonticfailures. Back then (24 years ago), I knew all theliterature supporting the link between bacteria andapical pathosis but was naive enough to think thatbiomechanical instrumentation of the root canalwill take care of the microbiological status insidethe canal, and the immune system (the host) willdo the rest at the apical area.

A few years later, the endodontic worldunderwent a complete reshape of concept andarmamentarium. Among them being new tech-niques, Ni-Ti alloys, rotary systems, using nickel,and the operating microscopes. For all thoseendodontists trained in the early 1990s or earlier, itmeant to learn endodontics almost all over again,and so we did. However, the amazing technolog-ical advancement in endodontics had a cost tothe consumer, in this case, the endodontists andultimately the patient.

If we place the financial aspect aside, theseadvancements were supposed to produce bet-ter endodontic therapy, which is measured by ahigher outcome and more teeth being saved fromextractions. However, despite the “art and science”of current endodontic therapy, current outcomestudies have failed to demonstrate an increase inendodontic success. How come? Should we notexpect at least a slight improvement? After all, weshape better, we see better, definitely we spendmuch more, and yet studies demonstrate the same

outcome of apical healing. Something is wrong! In

business, we would have used the expression that

perhaps we invested our time and money on the

wrong market.

After reading most of the outcome studies pub-

lished between 2005 and 2008, I realized that we

will never get any better in healing apical periodon-

titis until we realize and accept who our real enemy

is, what our biological aim is, and what should be

our strategy to target the biological reasons of the

disease.

Thus, the purpose of this book is to provide the

reader with a unique perspective on how to heal

apical periodontitis. Successful endodontic therapy

depends on the removal of microorganisms and

their endotoxins from the root canal system. Toxic

metabolites and by-products released from orga-

nized biofilms within the canal diffuse into apical

tissues and elicit inflammatory responses and bone

resorption. Therefore, as endodontists, our main

goal should be to focus on the predictable elimina-

tion of microorganisms from the root canal system.

However, the inherent anatomy andmorphology of

the root canal system imposes additional inherent

challenges making this loyal task of disinfection

even tougher. Isthmuses, intercanal and intracanal

communications, accessory and lateral canals, cur-

vatures and oval-shaped canals are all part of the

anatomical challenges we need to overcome. Yet,

the goal remains the same: control of the infection.

Bacterial reduction to the extent of a negative

ix

x Introduction

culture should be considered the desired “clinicaloutcome” of endodontic treatment.

In 2004, Bergenholtz and Spangberg claimedthat studies must address the fundamental prin-ciples of endodontics, rather than the adoptionof cutting-edge technology that contributes tothe advancement of root canal therapy. To makethis statement even stronger, in 2007, Ng et al.published an excellent systematic review on theoutcome of primary root canal treatment andreported that the success rates had not improvedover the past 40 years. The aforementioned is incomplete agreementwith the finding byKakehashi,Stanley, and Fitzgerald. We simply detoured andlost perspective for a few decades, but I believethat it is never too late to make it right.

That is how and why I became interested in thefield of root canal disinfection and toward higherhealing of apical periodontitis. Another of mymentors, Professor Shimon Friedman – consideredone of the best in the field of endodontic out-come – once challenged me on this difficult orimpossible task. Although we are not done yet,the results of years of intense research conducted

by the authors of this book have produced a sig-nificant amount of new data that will provide thereader with a good and current understanding ofthe etiology of apical periodontitis and the tech-niques available to obtain a more efficient andpredictable disinfection toward better healing andgreater outcome. The book discusses the etiologyof endodontic disease, especially the endodonticbiofilm, and all therapies available to predictablydisinfect the root canal system thus increasingendodontic treatment outcome.

I would like to thank the Commissioner ofWiley, Mr. Rick Blanchette, for his vision, trust,and, most of all, his persistence. Last, my gratitudeto all authors and co-authors that without muchhesitation collaborated with their knowledge,experience, and valuable time. I am extremelyproud and blessed for the opportunity I had towork with leaders from around the world whocontributed to this book. Not only because ofexpertise, but because we share the same concernsand passion for endodontics. Together, we joinedforces to contribute to our specialty, our peers, andour patients.

Nestor CohencaSchool of Dentistry,

University of Washington,Seattle, WA, USA

Preface

The passage of time has brought about consider-

able evolution of the understanding of endodontic

disease and its primary cause and the modalities

applied to tackle that cause aswell as the limitations

of those modalities. Driven by an ever-increasing

volume of research, this process has seen several

shifts in focus that, in turn, have generated tech-

nological advances that benefitted almost every

aspect of how endodontic therapy is delivered. The

rapid pace of those advances challenged the tradi-

tional endodontic textbooks to keep up both with

the changing focus and the technologies developed

to benefit endodontic therapy.

The current focus is the disinfection of the root

canal system, widely recognized as a formidable

challenge. Confronting this challenge requires

specific knowledge, unique technologies devel-

oped that apply this knowledge, and updating the

clinicians on how to apply those developments

in practice. The textbook Disinfection of Root CanalSystems: The Treatment of Apical Periodontitis by

Dr. Nestor Cohenca is designed specifically to pro-

vide the knowledge base, the detailed information

on current and emerging technological advances,

and guidelines on how to clinically apply those

technologies. In this regard, it is a most timely addi-

tion to the endodontic texts available to clinicians,

students, and researchers.

Compared to a decade ago, the understanding of

canal disinfection has evolved in ways that impact

changes in devices, procedures, protocols, and

even concepts. For one, the limitation of endodon-tic instruments in disinfecting the root canals hasbeen well appreciated, shifting the focus to theanatomical intricacies of the root canal systemsas well as the microbial resilience as the ultimatechallenges in the clinical practice of endodontictherapy. As a result of this shift, in a relativelyshort period of time, we have seen development of(the list is not all-inclusive): innovative instrumentdesigns to better address root canal anatomy; newdevices for delivery of antimicrobial agents intoroot canal systems; new antimicrobial agents withdual and triple activity; harnessing of physicaleffects to distribute and activate antimicrobialagents; optimization of the light energy appliedto better address the microbial challenges withinthe root canal systems; and the use of nanotech-nologies to challenge microbial biofilms in waysnever tried before in endodontics. Faced withsuch rapid evolution, compiling a textbook thataims to capture the most recent innovations andconcepts is a challenge. On the one hand, the his-toric context is required to foster a comprehensiveunderstanding of biology and therapeutic concepts.On the other hand, the innovative content needsto remain contemporary for the next few yearsto come.

Tomeet those challenges, Dr. Cohenca teamed upwith a large panel of experts, allowing the readerto benefit from contributions by 30 educators,researchers, and clinicians from four continents.The result of this collective effort is a unique

xi

xii Preface

textbook that highlights like no other the mostcurrent concepts of endodontic therapy of theinfected teeth. With access to this collective inter-national expertise, the reader gains an in-depthand wide-ranging insight into the current state ofdisinfection of root canal systems. Under this onecover, the reader will find current research-basedinformation on endodontic infection and resultingdisease, microbial challenges facing the host andthe clinician, methods and limitations of assessingthe efficacy of root canal disinfection, ambivalentrelationship between disinfection and healingoutcomes of endodontic therapy, anatomic com-plexities of root canal systems, value of the shapingof root canals, applicable irrigating agents, chal-lenges inherent to flow dynamics in root canals,contrasting approaches to delivery and activationof irrigation agents within root canals, recent andemerging adjuncts to canal disinfection, intra-canal medication, kinetics of the healing processesof infection-related endodontic disease, and thecomplimentary surgical treatment procedure.The specific areas several of its chapters focuson and detailed reviews of research findings thatthe chapters provide are not commonly found inendodontic textbooks.

This new textbook is organized in a logicalsequence. Rather than assigning precedence totreatment techniques, the book first reviews thefoundations of biology, anatomy, researchmethods,and healing assessment, to highlight the challengesfacing the clinician and researcher in addressingthe disinfection of root canal systems. This is then

followed by a comprehensive coverage of theprincipal disinfection procedures, modalities thathave yet to enter the mainstream, and emergingconcepts. The lingering challenges of slow healingkinetics and surgical management when disinfec-tion is ineffective are then reviewed to provide anessential perspective.

In compiling this textbook, its editor engagedmany talented and knowledgeable individualswithin the endodontic community to contributechapters in their respective areas of undisputedexpertise. With collective depth and breadth ofexperience and knowledge in both endodonticscience and art, they possess wide-ranging insightinto endodontic research, education, and clini-cal practice. As a longtime educator, researcher,and clinician, I find it gratifying to see the talentharnessed to collaborate on this textbook.

This textbook has all the potential to become anindispensable resource for dentists and endodonticspecialists, endodontic specialty-program students,and for those engaged in endodontic research. Theywill find its content critical to fostering a soundunderstanding of the challenges underlining thedisinfection of root canal systems, and also as auseful guide to the most current devices, materials,and techniques developed tomeet those challenges.It will assist them in achieving sophistication intheir selected endeavors.

Shimon FriedmanFaculty of Dentistry, University of Toronto,

Toronto, Ontario, Canada

Acknowledgments

I must confess that I was blessed with one of thebest endodontic education any professional candream of. I was lucky enough to be at the rightplace and at the right time. Having mentors likeAdam Stabholz, Shimon Friedman, Ilana Heling,Anna Fuks, James Simon, James Johnson, MarthaSomerman, and Joel Berg impacted my career. Eachand every single one of them taught me somethingdifferent and shaped not only my professionalknowledge but also my personality. I want to takethis opportunity to thank every one of them andexpress my gratitude and love. I hope to be able tofollow their path and legacy.

Finally, I would like to acknowledge my grat-itude and love to my family. First, thanks to my

parents who during the first 20 years of my lifehave motivated me to study hard and excel. Sec-ond, I would like to thank my wife Ruti and mychildren Yair, Natalie, and Daniel who during thepast 25 years had to understand and support thehours and days I missed from their lives. I hopethat my love and dedication made it up and willserve them as a positive example that in life youneed to love what you do and then give your allwith passion! From my part, I know that withouttheir love and support I would have never becomewho I am and this book would have never beenwritten.

xiii

Part 1Background

1 Root Canal Infection and EndodonticApical Disease

Nestor CohencaDepartment of Endodontics and Pediatric Dentistry, University of Washington, Seattle,WA, USA

Ana Maria Gonzá lez AmaroDepartment of Microbiology, University Autonomous of San Luis Potosí, San LuisPotosí, Mexico

Infection control: why now?

The outcome of endodontic treatment dependson the microbiological status of the root canal. Ininflamed vital pulps, the infection is commonlylimited to the site of exposure causing a localizedinflammatory response (1, 2). However, whenaseptic technique is used, the effect of endodontictherapy is predictably high as demonstrated byseveral studies (3–7).

In infected necrotic pulps, microorganisms arepresent within the root canal system and denti-nal tubules, causing a apical inflammatory lesioncalled apical periodontitis. In these cases, endodontictreatment should be essentially directed towardthe prevention and control of pulpal and apicalinfections, as stated by Kakehashi, Stanley, andFitzgerald nearly 50 years ago (8). Unfortunately,the success of the therapy for these cases is 10–15%

Disinfection of Root Canal Systems: The Treatment of Apical Periodontitis, First Edition. Edited by Nestor Cohenca.© 2014 John Wiley & Sons, Inc. Published 2014 by John Wiley & Sons, Inc.

lower when compared to non-contaminated teeth(Table 1.1) (3–7, 9–11). What is more concerning isthe fact that this lower outcome has not changedor improved despite all the technological advance-ments the world of endodontics has seen (12). Howcome that despite the “art and science” of currentendodontic therapy, outcome studies have failedto demonstrate an increase in endodontic success?Why do we fail to predictably control the infectionafter so many years of research, experiment, andtreatment? The answer might be related to the factthat very few advances have ever targeted the realproblem, which continues to be microorganisms,especially in the apical third. The success of theendodontic treatment is possible only with anunderstanding of the molecular biology of thepathogens, their structures, synergies, and weak-nesses. No file will ever disinfect a root canal, nor isit designed for that purpose. Recognizing that our

3

4 Background

Table 1.1 Outcome of endodontic therapy based on the presence or absence of apical periodontitis.

Author and year Cases/cohort RecallWithout apicalperiodontitis (%)

With apicalperiodontitis (%)

Strindberg (1956) 258 6 months to 10 years 93 80

Kerekes and Tronstad (1979) 491 Norway, Dental school 3–5 years 92 89

Sjogren et al. (1990) 96 Sweden, Dental school 8–10 years (91%) 96 86

Friedman et al. (2003) 92 4–6 years (20%) 92 74

Farzaneh et al. (2004) 94 Toronto, Grad students 4–6 years (48%) 94 81

Orstravik et al. (2004) Norway, Dental school 0.5–4 years (83%) 94 79

Ng et al. (2011) 702 London Grad students 4 years (83%) 83

Ricucci et al. (2011) 816 Italy (Ricucci) 5 years (87%) 92.3 82.7

endodontic therapy will end in failure if we do notfind a method to completely destroy the microbeswithin the root canal system, infection controlmust be our main goal and concern. Therefore, weshould focus our research and development onefficient and predictable methods to control theinfection and improve the endodontic treatmentand healing of apical periodontitis.

Terminology and apical definitions

The term apical periodontitis has gained increasingsupport and is used widely in current literature.The American Association of Endodontists recentlypublished the revised Glossary of EndodonticTerms (13). Some of the terms defined in theglossary are as follows:

Normal apical tissues Teeth with normal apical tis-sues that are not sensitive to percussion or pal-pation testing. The lamina dura surrounding theroot is intact and the periodontal ligament spaceis uniform.

Symptomatic apical periodontitis Inflammation, usu-ally of the apical periodontium, producingclinical symptoms including painful response tobiting and/or percussion or palpation. It may ormay not be associated with an apical radiolucentarea.

Asymptomatic apical periodontitis Inflammation anddestruction of apical periodontium that is ofpulpal origin, appears as an apical radiolucentarea and does not produce clinical symptoms.

Acute apical abscess An inflammatory reaction topulpal infection and necrosis characterized byrapid onset, spontaneous pain, tenderness of thetooth to pressure, pus formation, and swelling ofassociated tissues.

Chronic apical abscess An inflammatory reactionto pulpal infection and necrosis characterizedby gradual onset, little or no discomfort, andthe intermittent discharge of pus through anassociated sinus tract.

In biological terms, apical periodontitis means“inflammation of the periodontium.” This is abroad term to describe an inflammatory reactionin the tissues, including lateral and furcal locationsof inflammation; it does not distinguish etymolog-ically pulp-induced periodontitis from marginallyderived periodontitis. More specific pathologiessuch as granulomas and cysts were excludedbecause they do not represent a “clinical or radio-graphic” diagnostic reality, but rather a diagnosisbased on histological findings. The prevalence ofapical periodontitis has increased throughout theyears (14, 15), even in the low caries-rate adultDanish population (16).

The evolution of endodonticmicrobiology

Miller, in 1890 (17), was the first to demonstratethe presence of bacteria in necrotic human pulptissue. However, the cause and effect relationship

Root Canal Infection and Endodontic Apical Disease 5

is attributed to Kakehashi et al. (8) who experi-mented with gnotobiotic (germ-free) and normalrats. Bacterial contamination in the orally exposedpulp tissue caused necrosis and apical pathosesin normal rats. The study is considered a classicreference as it initiated a new and bright era inendodontic microbiology.

In 1966, Moller (18) established the importanceof adequate isolation for microbiological samplingand various culture media for the recovery andidentification of anaerobic microorganisms, pro-viding more relevant information regarding thetype of bacteria present in root canal systems.Bergenholtz then demonstrated the presence ofbacteria in the traumatized teeth. Despite the factthat pulp chambers were not exposed, bacterialgrowth was observed in 64% of all samples. Theflora was dominated by anaerobic microorganismsincluding Bacteroides, Corynebacterium, Peptostrep-tococcus, and Fusobacterium (19). Two years later,Sundqvist (20) demonstrated the prevalence ofanaerobic bacteria in root canals, supporting theresults obtained by Bergenholtz.

In the 1980s, the studies were more focused onunderstanding the colonization and interactionswithin the endodontic microflora. Moller investi-gated the relationship between uncontaminatednecrotic pulp and apical tissues. The study main-tained uncontaminated necrotic pulp in the rootcanal during a period of at least 6 months, andevaluated changes in the microbial flora enclosedin the root canal and its capacity to induce api-cal periodontitis (21). Using 9 monkeys (Macacafascicularis), the pulp of 78 teeth was asepti-cally necrotized. Twenty-six of the pulp chamberswere sealed and the pulp chamber remained free.Fifty-two teeth were infected with the indigenousflora. Clinical, radiographic and microbiologicaldata was recorded before and after the completionof the study. The root canals were initially unin-fected sterile in the final samples. No inflammatoryreactions were found on the 26 control teeth. On theexperimental teeth inflammatory reactions wereobserved clinically (12/52 teeth) and radiographi-cally (47/52 teeth). An average of 8 to 15 bacterialstrains were identified as facultative anaerobicbacteria including enterococci, coliforms, andanaerobic bacteria such as Bacteroides, Eubacterium,Propionibacterium, and Peptococcus, Peptostreptococ-cus. Some anaerobic bacteria not present on the

initial microbiological test were isolated on thefinal samples. Histological examination of the api-cal tissue confirmed the presence an inflammatoryreaction to the bacterial contamination (21).

In 1982, Fabricius et al. investigated the pulpsof 24 root canals, 8 in each of the 3 monkeys thatwere experimented on. Teeth were mechanicallydevitalized and exposed to the oral flora for about1 week and thereafter sealed. Microbiologic sam-pling and analysis was performed in 16 teeth (of 2of the monkeys) after 7 days of closure (initial sam-ples). Afterward, inoculation pulps were sealed fora period of 6 months. Final sampling was takenfrom the main root canal, the dentin, and the apicalregion at the same sampling session. All microbio-logic analyses were carried out quantitatively. Finalroot canal samples from the apical region showeda predominance of obligate anaerobic nonsporu-lating bacteria; in fact 85–98% of the bacterial cellswere anaerobic. The most frequently found specieswere Bacteroides and Gram-positive anaerobicrods. A lower proportion of facultative anaerobicbacteria were found; this was most pronouncedfor coliform rods in comparison with the strains ofBacteroides melaninogenicus.

Today, electron microscopy has become a greattechnology in many areas of science. Nair (22)studied the structure of the endodontic flora, itsrelationship to the dentinal wall, microbial inter-actions, and dynamics of apical inflammatoryresponse. The study was performed on humanteeth with granulomas and cysts. The resultsshowed the presence of microorganisms in all thesamples. The flora consisted of cocci, bacilli, andspirochetes filamentous organisms. In most cases,the bacteria were restricted to the canal, but in 4granulomas and 1 cyst, the bacteria were foundin the lesion. There was a distinct bacterial plaqueadhering to the dentinal wall at the apical foramen.Nair describes this finding as a group or commu-nity of one ormore types of microorganisms as wellas bacterial condensate, suggesting the formationof plaque in the dentinal wall by the flora of theroot canal. This finding is considered to be thesubject that currently occupies many researchers:the presence of biofilm on root canal walls.

In 1990, Nair (23) analyzed nine therapy-resistantand asymptomatic human apical lesions (4–10years) removed during surgery using light andelectronmicroscopy. Six out of nine lesions revealed

6 Background

microorganisms in the apical root canal. Four con-tained bacteria and two contained yeasts. Of thethree cases with no microorganisms, one revealed aforeign body giant cell granuloma. In the majorityof therapy-resistant apical lesions, microorgan-isms (bacteria, yeast) and foreign body giant cellgranulomas play a significant role in treatmentfailures.

Although endodontic microbiology has evolvedsignificantly, it still lacks an understanding of theecology of the root canal and requires an analysisof the bond that develops between microorgan-isms and their surroundings. This relationship isan essential element that provides a glimpse intothe understanding of their behavior and ability toinvade an area that is rich in nutrients and whoseabiotic and biotic factors determine the distributionand quantity of living organisms that may sharethe root space. In the early 1990s, Sundqvist (24, 25)published a couple of reviews summarizing theavailable data. Bacterial flora of the root canal isdominated by obligate anaerobes, comprising upto 90% of the total population. Aerobic bacteria arerarely found initially in the infected root canals butcould have been introduced during the treatment.During the course of an infection, interrelation-ships develop between microbial species, based ontheir nutritional demands and nutritional interac-tions, and the pathogenicity of the polymicrobialroot flora is dependent on bacterial synergy. Bac-teriocins proteins produced by a microorganismenhance their ability to inhibit growth of somespecies competing for the same ecological niche.Additionally, they promote bacterial coaggregationand interactions establishing the ecology of theapical tissues.

The identification of the endodontic micro-biota in the apical third was reported in 1991 byBaumgartner (26) who employed both aerobicand anaerobic cultures in the same study, in orderto isolate and identify the microflora of the api-cal portion of root canals of teeth with cariouspulpal exposures and apical lesions. Ten freshlyextracted teeth with carious pulpal exposures andapical lesions contiguous with the root apex wereplaced inside an anaerobic chamber and the api-cal 5mm of the root canals cultured. In additionto anaerobic incubation, duplicate cultures wereincubated aerobically. Fifty strains of bacteria fromthe 10 root canals were isolated and identified.

The most prominent bacteria cultured from the10 root canals were Actinomyces, Lactobacillus,black-pigmented Bacteroides, Peptostreptococcus,nonpigmented Bacteroides, Veillonella, Enterococcusfaecalis, Fusobacterium nucleatum, and Streptococcusmutans. Of the 50 bacterial isolates, 34 (68%) werestrict anaerobes. Baumgartner’s study demon-strated the presence of predominantly anaerobicbacteria in the apical 5mm of infected root canalsin teeth with carious pulpal exposures and apicallesions.

Advancements in the identification of endodon-tic flora by Molander et al. in 1998 correlatedthe clinical outcome, refractory lesions, and thepresence of certain strains. Molander and cowork-ers examined the microbiological status of 100root-filled teeth with radiographically verifiedapical periodontitis and 20 teeth without signs ofapical pathoses. In teeth with apical periodontitis,117 strains of bacteria were recovered in 68 teeth.Facultative anaerobic species predominated amongthese isolates (69% of identified strains). Entero-cocci were the most frequently isolated genera,showing “heavy” or “very heavy” growth in 25out of 32 cases (78%). In 11 teeth without signs ofapical pathoses, no bacteria were recovered whilethe remaining 9 yielded 13 microbial strains. Eightof these grew “very sparsely.” It was concludedthat the microflora of the obturated canal differsfrom that found normally in the untreated necroticdental pulp, quantitatively as well as qualitatively.

In 1990, Sha and Collins reclassified the moder-ately saccharolytic, predominantly oral Bacteroidesspecies, which include B. melaninogenicus, Bac-teroides oralis, and related species. These bacteriaform a phenotypically and phylogenetically coher-ent group of species, which differ so significantlyfrom the emended description of the genus Bac-teroides that they should not be classified inthe same genus and proposed that these speciesbe reclassified in a new genus, Prevotella (27)(Figure 1.1).

By this time the advent of molecular biologytechniques advanced in leaps and bounds in thedetection and identification of microorganisms,uncovering the intimacy between genetics, bio-chemistry, and microbiology. Technique sensitiveanalyses of nucleic acids extracted from microor-ganisms directly, or from a sample containingthe microorganism in question, have identified

Root Canal Infection and Endodontic Apical Disease 7

New black-pigmentedclassification

Porphyromonas (Asaccharolytic)

Prevotella (Saccharolytic)

– Porphyromonas gingivalis

– Porphyromonas endodontalis

– Prevotella intermedius

– Prevotella melaninogenicus

– Prevotella denticola

– Prevotella loescheii

– Prevotella nigrescens

– Prevotella corporis

Figure 1.1 Current taxonomy and of black-pigmentedbacteria.

endodontic microorganisms of interest; as was thecase with Actinomyces. Polymerase chain reaction(PCR) is a biochemical technology in molecularbiology used to amplify a single or a few copies ofa piece of DNA across several orders of magnitude,

generating thousands to millions of copies of aparticular DNA sequence. Xia and Baumgartnerused PCR with a pair of universal primers for Acti-nomyces and species-specific primers to evaluatethe contents of infected root canals and aspiratesfrom abscesses or cellulitis for the presence ofActinomyces israelii, Actinomyces naeslundii, and

Actinomyces viscosus. DNA was extracted from131 clinical samples (28). DNA reacting with theuniversal primer for Actinomyces was detectedin 72 of 129 (55.8%) clinical samples. Of those, 41of 51 (80.4%) were from infected root canals, 22 of48 (45.8%) were from abscesses, and 9 of 30 (30%)were associated with cellulitis.

Since then, Siqueira and Rocas reported hun-dreds of species obtained from root canals usingPCR techniques. In a review article published in2008, they concluded that bacterial presence in theroot canal at the time of filling is a risk factor forposttreatment apical periodontitis (29). About 100species/phylotypes have already been detected

in postinstrumentation and/or postmedicationsamples, and Gram-positive bacteria are the mostdominant. However, it remains to be determinedby longitudinal studies if any species/phylotypespersisting after treatment procedures can influencethe outcome (29).

Waltimo et al. reported the current trend ofrecurrent yeast in endodontic infections, and theirantimicrobial response confirms that yeast may beisolated in about 5–20% of the infected root canals.Their article identifies multiple virulence factors ofCandida that allow it to infect the dentin–pulp com-plex and penetrate the dentinal tubules causing aninflammatory reaction and suggesting a pathogenicrole of this organism in apical periodontitis (30).

With the discovery and understanding thatfungus is present, the complexity of the micro-biota became clear. In 2003, Slots et al. (31) werethe first to report the presence of cytomegalovirusand Epstein–Barr virus (EBV) in more than 90%of granulomas of symptomatic and large apicallesions (31). Dual infection with cytomegalovirusand EBV is closely associated with symptomaticlesions. Herpes simplex virus’ (HSV) active infectionhas no apparent relationship to apical disease.

Another study aimed to identify herpes virus,including human cytomegalovirus (HCMV), EBV,HSV-1, and varicella zoster virus (VZV) in-vivo(32, 33). Patients with acute apical abscesses andcellulitis of endodontic origin were used for thestudy. The identification was carried out by theprimary PCR and nested PCR techniques. Theresults demonstrated the presence of HCMV, EBV,and HSV-1; however, they indicated that the pres-ence of herpes virus that were identified were verylow in their genetic copies, and therefore it wasconcluded that herpes viruses are present but donot have a direct relation to the development orestablishment of pathologies such as acute apicalabscess or cellulitis of endodontic origin.

The link between endodontic infectionand apical disease

The presence of bacteria within the root canalsystem is essential for the development of apicalinflammation (8). Apical periodontitis is a diseasecharacterized by inflammation and destructionof apical tissues caused by microbial agentsof endodontic origin. Initially, the tooth pulpbecomes infected and necrotic by an autogenousoral microflora. The microenvironment of rootcanal systems provides excellent conditions forthe establishment of a mixed, predominantlyanaerobic, flora. Collectively, this polymicrobial

8 Background

community residing in the root canal has severalbiological and pathogenic properties, such as anti-genicity, mitogenic activity, chemotaxis, enzymatichistolysis, and activation of host cells.

Microorganisms’ growth within the root canalis in the form of biofilm. The microbial com-munity develops as a biofilm and colonizes theenvironment. It occurs first by the depositionof a conditioning film; then there is adhesionand colonization of planktonic microorganismsin a polymeric matrix. The coadhesion of otherorganisms and the detachment of biofilm microor-ganisms into their surroundings happen at a laterstage (34). Bacterial biofilm has an open architec-ture with channels traversing from the biofilmsurface. The structure of biofilm affects the move-ment of molecules, and the gradients are keydeterminants in its development. Bacteria growingon a surface may display a novel phenotype that,consequently, may allow increased resistance toantimicrobial agents. Resistance can result fromrestricted inhibitor penetration, slower bacterialgrowth rates, transfer of resistance genes, subopti-mal environmental conditions for inhibitor activity,and the expression of a resistant phenotype (35).

This ecological view on the persisting infectionin endodontics suggests that the action of individ-ual species in refractory endodontic infections issecondary when compared to the adaptive changesof a polymicrobial biofilm community undergoingphysiological and genetic changes in response tochanges in the root canal environment (36). Theinvasion of root dentinal tubules by root canalbacteria is a multifactorial event in which a limitednumber of oral bacterial species have the requiredproperties to participate. Current literature hasdemonstrated that biofilms may remain viablein anatomical areas of the root canal system thatremain untouched by either mechanical or chem-ical disinfection (Figure 1.2) (37–39). Scanningelectronmicroscopy (SEM) examination of root tipsassociated with refractory apical periodontitis hassuggested the presence of bacterial biofilm at theapical portion of the root canal (Figure 1.3) (40–42).

SEM analysis revealed bacterial biofilm sur-rounding the apical foramen and external radicularsurface (Figures 1.4a and 1.2c). Careful observationof these structures under higher magnificationrevealed clumps of coaggregated bacterial cellsin a matrix of extracellular polymeric substance

(EPS). E. faecalis biofilms displayed a complexthree-dimensional structure that demonstratedspatial heterogeneity and a typical architectureshowing microcolonies with ramifying water chan-nels (Figure 1.4b). Fibrillar structures appeared tobe made up of twisted fibers. Larger structures ofwrapped sheets were also present and consistedof small numbers of bacteria cells embedded in amatrix of fibers (Figure 1.4d).

Bacterial endotoxins and by-products egressthrough portals of exit causing the destruction ofthe apical tissues. In response, the host has an arrayof defenses consisting of several classes of cells,intercellular messengers, antibodies, and effectormolecules. The microbial factors and host defenseforces encounter, clash with, and destroy much ofthe apical tissue, which result in the formation ofvarious categories of apical periodontitis lesions.In spite of the formidable defense, the body isunable to destroy the microbes well-entrenchedin the sanctuary of the necrotic root canal, whichis beyond the body’s immune system. Therefore,the major goal of the root canal debridement is toeliminate the biofilm and bacteria-harboring debris.

Contemporary endodontic microbiology hasbecome even more complex with the discoveryof fungi and viruses. Waltimo et al. (43) foundyeast in 7% of the culture-positive samples frompersistent root canal infections. Candida albicanswas the most frequently isolated yeast (80%). Thisfinding was later confirmed by several studies (44,45). Attachment to mucosal tissues and to abioticsurfaces and the formation of biofilms are crucialsteps for Candida survival and proliferation in theoral cavity. Candida species possess a wide arsenalof glycoproteins located at the exterior side ofthe cell wall, many of which play a determiningrole in these steps. In addition, C. albicans secretessignaling molecules that inhibit the yeast-to-hyphatransition and biofilm formation. In-vivo, Candidaspecies are members of mixed biofilms and subjectto various antagonistic and synergistic interactions(Figure 1.5) (46). Recently, Gomes et al. (47) con-firmed the presence of filamentous fungi, whichwere isolated in situ from 17 of 60 samples (28.3%).The genus Aspergillus was isolated from 7/17samples (41%).

The presence of HCMV, EBV, and HSV was firstreported by Sabeti et al. (48) in symptomatic apicallesions. In a different study, Slots et al. (49) detected

Root Canal Infection and Endodontic Apical Disease 9

(a)

(d)

(b)

(c) (f)

(e)

Figure 1.2 Composite figure ofphotomicrographs representative ofmicroscopic sections of Groups I(EndoVac—ANP), II (Ultrasound—PUI),and III (conventional irrigation—PP)stained by the Brown and Brenntechnique, revealing the presence andlocation of bacteria in the root canalsystem and apical tissues 180 days afterendodontic treatment: (a) Panoramicphotomicrograph, showingcontamination of the entire extension ofthe tooth (Zeiss, 1.25×). (b) Detail ofpanel a (rectangle) in which the cervicalroot canal third can be visualized withabundant presence of microorganismsinside the dentinal tubules (Zeiss, 20×).(c) Detail of panel a (square) showingthe middle root canal third with intensepresence of bacteria (Zeiss, 20×). (d)Detail of panel a (circle) revealing themiddle root canal third with presence ofbacteria in the cemental craters (arrow)(Zeiss, 10×). (e) Detail of panel a(triangle) showing abundant presence ofmicroorganisms in the apical root canalthird (Zeiss, 40×). (f) Detail of panel a(asterisk) in which bacteria can be seenin the apical lesion (Zeiss, 20×).

HCMV in 100% of the symptomatic and in 37%

of the asymptomatic study lesions (49). EBV was

identified only in HCMV-infected apical lesions.

The difference of HCMV and EBV between symp-

tomatic and asymptomatic lesions was found to be

statistically significant. Current studies confirmed

these findings by using primary and nested PCR as

well as reverse transcription PCR. (33) EBV DNA

and RNA were present in endodontic pathoses in

significantly higher percentages (43.9% and 25.6%,

respectively) compared with healthy pulp controls.

The DNA of HSV was found in low percentages in

endodontic patients (13.4%), and only one patient

showed the presence of VZV. In conclusion, the

presence of fungi and viruses had been associated

with irreversible pulpitis and periodontitis, con-

firming once again that endodontic micro flora is

complex and composed of well-organized biofilms

of microorganisms with synergetic interactions.

The host responses to root canal infection have

been the subject of much research in recent years.

There is great similarity among the pathogenic

10 Background

(a) (b)

(d)(c)

Figure 1.3 In-vivo E. faecalis biofilm. (a, c) Cocoidal structures attached to mature biofilm (6000×). (b) Extrapolymeric fibers(10,000×). (d) “Mushroom”-shaped structures (10,000×).

(a) (b)

(c) (d)

Figure 1.4 E. faecalis mature biofilm in-vivo. (a, c) Microcolonies wrapped in an extracellular matrix (8000×). (b) Networkconsisting of fibrillary structures (10,000×). (d) “Mushroom”-shaped structures (10,000×).

Root Canal Infection and Endodontic Apical Disease 11

Figure 1.5 Mature biofilm demonstrating the synergistic relationship between E. faecalis and Candida albicans.

processes in marginal and apical periodontitis, andmany of the findings in periodontal research havedirect relevance to apical periodontitis. A clearerconcept of the immunological processes involved inthe development of apical periodontitis is emerging(50, 51). Microbiological variability and virulencefactors in infected root canals have been demon-strated, and specific data indicate that the bacterialflora varies systematically with the clinical con-dition of the tooth involved (persistent infection,therapy-resistant infection). Different therapeu-tic strategies of antimicrobial control may haveto be applied, depending on the microbiologicaldiagnosis in a given case.

Apical periodontitis is not a self-healing disease(52). Untreated apical periodontitis may lead toa chronic infection of the oral tissues at locationscloser to more vital organs than many other oralinfections. Although these infections may remainquiescent for decades, they may also develop andspread with serious consequences for the individ-ual (53, 54). In the face of the risks of such chronicinfection from the infected teeth, their extraction

and replacement by implants has been put forwardand discussed as a viable alternative to endodon-tic treatment (55, 56). The variable success rates(by strict criteria) of treatment procedures for thecure of apical periodontitis are sometimes used asarguments for the implant “treatment” concept.However, what little evidence there is does notindicate a lower survival rate of endodonticallytreated teeth, and the superiority of tooth preser-vation compared to its replacement should beevident as a biological principle of preference.However, the challenge from other treatment con-cepts, to endodontics as a discipline, should act asa driving force to produce more and scientificallysolid evidence for the modalities of cure and pre-vention applied to our disease of interest, apicalperiodontitis.

Conclusion

Pulpal and apical inflammation, and the asso-ciated pain and the consequences of root canal

12 Background

infection, remain significant aspects of dentistrytoday. New knowledge and insights provide bet-ter treatment opportunities and stimulate furtherresearch activities. The prevention and control ofapical periodontitis has a solid scientific base butthe many variations in the clinical manifestationsof the disease still leave technical and biologicalproblems that need to be solved. Despite recenttechnological advances in treatment, evidence ofimproved outcome is still lacking. Alternativetreatment involving implants is promoted as beingbetter, but the criteria of evaluation of the outcomeof the two forms of treatment are dissimilar; there isno true evidence-based comparison. The advance-ment, and utilization, of our biologic principleswill allow a better understanding of the diseaseprocess and add to the fundamental truth thatthe biologic response to disease continues to bea biologic therapy; those therapies will and mustcontinue to advance with our understanding.

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